Clinical relevance of nitrated beta 2-glycoprotein I in antiphospholipid syndrome: Implications for thrombosis risk.

Β2-Glycoprotein I (ß2GPI) is an important anti-thrombotic protein and is the major auto-antigen in the antiphospholipid syndrome (APS). The clinical relevance of nitrosative stress in post translational modification of ß2GPI was examined.The effects of nitrated (n)ß2GPI on its anti-thrombotic properties and its plasma levels in primary and secondary APS were determined with appropriate clinical control groups. ß2-glycoprotein I was nitrated at tyrosines 218, 275 and 309. ß2-glycoprotein I binds to lipid peroxidation modified products through Domains IV and V. Nitrated ß2GPI loses this binding (p < 0.05) and had diminished activity in inhibiting platelet adhesion to vWF under high shear flow (p < 0.01). Levels of nß2GPI were increased in patients with primary APS compared to patients with either secondary APS (p < 0.05), autoimmune disease without APS (p < 0.05) or non-autoimmune patients with arterial thrombosis (p < 0.01) and healthy individuals (p < 0.05).In conclusion tyrosine nitration of plasma ß2GPI is demonstrated and has important implications with regards to the pathophysiology of platelet mediated thrombosis in APS. Elevated plasma levels of nß2GPI in primary APS may be a risk factor for thrombosis warranting further investigation.

Outcome of refractory to conventional and/or biologic treatment adult Still's disease following canakinumab treatment: Countrywide data in 50 patients.

OBJECTIVE: To assess the efficacy and safety of the IL-1b inhibitor canakinumab in all adults with refractory Still's disease identified from the National Organization For Medicines for off-label drug use. METHODS: In a retrospective longitudinal multicenter cohort of 50 patients (median age 39 years) with active Still's disease despite treatment with corticosteroids (n = 11), conventional and synthetic (n = 34) and/or biologic disease modifying anti-rheumatic drugs (n = 30), we assessed the efficacy of canakinumab 150-300 mg administered every 4 (n = 47) or 8 weeks (n = 3) as combination therapy or monotherapy (n = 7) during a median follow-up of 27 (3-84) months. RESULTS: Α complete response was initially observed in 78% of patients within 3 months (median), irrespective of age at disease onset. A partial response was evident in 20%. One patient had resistant disease. Treatment de-escalation was attempted in 15 of 39 complete responders and a complete drug discontinuation in 21 patients for 8 months (median). Eleven patients (22%) relapsed during treatment, one during de-escalation process, and 11 after treatment discontinuation. Overall, 9 of 11 relapses were successfully treated with canakinumab treatment intensification or re-introduction. At last visit, 18% of patients were off treatment due to remission and 26% due to disease activity. Canakinumab had a significant corticosteroid sparing effect allowing weaning in 21 of 41 cases. Infections (20%, severe 4%) and leucopenia (6%) led to treatment cessation in one patient. CONCLUSION: High rates of sustained remission were observed in this, largest so far, real-life cohort of adult patients with refractory Still's disease treated with canakinumab.

The role of thrombospondin-1 in the pathogenesis of antiphospholipid syndrome.

OBJECTIVE: Antiphospholipid syndrome (APS) is an acquired thrombophilia characterized by recurrent thrombosis and/or pregnancy morbidity, in the presence of antibodies to ß2 glycoprotein-I (ß2GPI), prothrombin or Lupus anticoagulant (LA). Anti-ß2GPI antibodies recognize complexes of ß2GPI dimers with CXCL4 chemokine and activate platelets. Thrombospondin 1 (TSP-1) is secreted by platelets and exhibits prothrombotic and proinflammatory properties. Therefore, we investigated its implication in APS. METHODS: Plasma from APS patients (n = 100), Systemic Lupus Erythematosus (SLE) (n = 27) and healthy donors (HD) (n = 50) was analyzed for TSP-1, IL-1ß, IL-17A and free active TGF-ß1 by ELISA. Human Umbilical Vein Endothelial Cells (HUVECs) and HD monocytes were treated with total HD-IgG or anti-ß2GPI, ß2GPI and CXCL4 and CD4+ T-cells were stimulated by monocyte supernatants. TSP-1, IL-1ß, IL-17A TGF-ß1 levels were quantified by ELISA and Real-Time PCR. RESULTS: Higher plasma levels of TSP-1 and TGF-ß1, which positively correlated each other, were observed in APS but not HDs or SLE patients. Patients with arterial thrombotic events or those undergoing a clinical event had the highest TSP-1 levels. These patients also had detectable IL-1ß, IL-17A in their plasma. HD-derived monocytes and HUVECs stimulated with anti-ß2GPI-IgG-ß2GPI-CXCL4 secreted the highest TSP-1 and IL-1ß levels. Supernatants from anti-ß2GPI-ß2GPI-CXCL4 treated monocytes induced IL-17A expression from CD4+ T-cells. Transcript levels followed a similar pattern. CONCLUSIONS: TSP-1 is probably implicated in the pathogenesis of APS. In vitro cell treatments along with high TSP-1 levels in plasma of APS patients suggest that high TSP-1 levels could mark a prothrombotic state and an underlying inflammatory process.

Evidence of epigenetic alterations in thrombosis and coagulation: A systematic review.

Thrombosis in the context of Cardiovascular disease (CVD) affects mainly the blood vessels supplying the heart, brain and peripheries and it is the leading cause of death worldwide. The pathophysiological thrombotic mechanisms are largely unknown. Heritability contributes to a 30% of the incidence of CVD. The remaining variation can be explained by life style factors such as smoking, dietary and exercise habits, environmental exposure to toxins, and drug usage and other comorbidities. Epigenetic variation can be acquired or inherited and constitutes an interaction between genes and the environment. Epigenetics have been implicated in atherosclerosis, ischemia/reperfusion damage and the cardiovascular response to hypoxia. Epigenetic regulators of gene expression are mainly the methylation of CpG islands, histone post translational modifications (PTMs) and microRNAs (miRNAs). These epigenetic regulators control gene expression either through activation or silencing. Epigenetic control is mostly dynamic and can potentially be manipulated to prevent or reverse the uncontrolled expression of genes, a trait that renders them putative therapeutic targets. In the current review, we systematically studied and present available data on epigenetic alterations implicated in thrombosis derived from human studies. Evidence of epigenetic alterations is observed in several thrombotic diseases such as Coronary Artery Disease and Cerebrovascular Disease, Preeclampsia and Antiphospholipid Syndrome. Differential CpG methylation and specific histone PTMs that control transcription of prothrombotic and proinflammatory genes have also been associated with predisposing factors of thrombosis and CVD, such us smoking, air pollution, hypertriglyceridemia, occupational exposure to particulate matter and comorbidities including cancer, Chronic Obstructive Pulmonary Disease and Chronic Kidney Disease. These clinical observations are further supported by in vitro experiments and indicate that epigenetic regulation affects the pathophysiology of thrombotic disorders with potential diagnostic or therapeutic utility.

Differential CpG methylation of the promoter of interleukin 8 and the first intron of tissue factor in Antiphospholipid syndrome.

BACKGROUND: Antiphospholipid syndrome (APS) is an autoimmune thrombophilia characterized by recurrent thromboembolism and/or pregnancy morbidity in the presence of Antiphospholipid antibodies, mainly anti-ß2 glycoprotein I (anti-ß2GPI). The autoantibodies lead to monocyte and endothelial cell activation and subsequent secretion of tissue factor (F3) and proinflammatory cytokines, like interleukins 6 (IL6) and 8 (IL8). The etiology of the syndrome remains largely unknown, with the contribution of environmental, genetic and epigenetic factors considered significant. PURPOSE: We aimed to identify epigenetic changes and factors potentially implicated in the pathophysiology of APS. To this end, we compared DNA methylation levels of the IL8 and F3 genes between healthy donors (HDs) and APS patients, using whole blood as a source. RESULTS: Methylation was significantly reduced in the IL8 promoter and significantly increased in the F3 gene body in APS patients compared to HDs and correlated with specific clinical parameters. In an ex vivo model partially mimicking APS, stimulation of monocytes with a mixture of ß2GPI, anti-ß2GPI and CXCL4 also induces DNA methylation changes in the above genes, along with increase of their expression. Stimulation of human umbilical vein endothelial cells (HUVECs) with the same mixture also results in transcriptional upregulation of epigenetic factors, including MΕCP2, DNMT3, TET1, HDAC9 and ARID5B. CONCLUSIONS: The above data support that epigenetic alterations could be implicated in the pathophysiology of APS and prompt further investigation of their potential diagnostic or therapeutic utility.

Osteoporosis and vascular calcification in postmenopausal women: a cross-sectional study.

Objectives To estimate the correlation between osteoporosis and vascular calcification in postmenopausal women and the influence of calcium/vitamin D supplements on vascular calcification. Methods A cross-sectional study was performed including 29 women with osteoporosis (15 not taking supplements) and 18 age-matched, non-osteoporotic women. They were evaluated for cardiovascular risk factors and blood tests, lateral X-ray of lumbar spine (assessment of abdominal aorta calcification, AAC) and carotid ultrasound (increased intima media thickness (iIMT) or calcified plaques) were performed. Results In univariate analysis, osteoporotic women were 16 times more likely to develop AAC (odds ratio (OR) 15.8, 95% confidence interval (CI) 1.9-135.4) and seven times more likely to develop iIMT (OR 6.8, 95% CI 1.8-25.4) compared to normal individuals. The odds of developing AAC and iIMT were increased each year after menopause (OR 1.11, 95% CI 1.01-1.2 and OR 1.18, 95% CI 1.05-1.3, respectively) and with aging (OR 1.27, 95% CI 1.1-1.47 and OR = 1.17, 95% CI 1.04-1.3, respectively). Calcified plaques were significantly correlated with osteoporosis (p = 0.014). In multivariate analysis, osteoporosis was an independent risk factor for AAC (OR 13.3, 95% CI 1.3-134.4) and iIMT (OR 4.7, 95% CI 1.1-19.9). Low doses of supplements did not appear to affect vascular calcification (p = 0.6). Conclusions Osteoporosis is associated with increased calcification of the abdominal aorta and carotids. Low doses of supplements do not appear to cause any increase in vascular calcification in osteoporotic women.

TGFß receptor gene variants in systemic sclerosis-related pulmonary arterial hypertension: results from a multicentre EUSTAR study of European Caucasian patients.

INTRODUCTION: Systemic sclerosis (SSc)-related pulmonary arterial hypertension (PAH) has emerged as a major mortality prognostic factor. Mutations of transforming growth factor beta (TGFß) receptor genes strongly contribute to idiopathic and familial PAH. OBJECTIVE: To explore the genetic bases of SSc-PAH, we combined direct sequencing and genotyping of candidate genes encoding TGFß receptor family members. MATERIALS AND METHODS: TGFß receptor genes, BMPR2, ALK1, TGFR2 and ENG, were sequenced in 10 SSc-PAH patients, nine SSc and seven controls. In addition, 22 single-nucleotide polymorphisms (SNP) of these four candidate genes were tested for association in a first set of 824 French Caucasian SSc patients (including 54 SSc-PAH) and 939 controls. The replication set consisted of 1516 European SSc (including 219 SSc-PAH) and 3129 controls from the European League Against Rheumatism Scleroderma Trials and Research group network. RESULTS: No mutation was identified by direct sequencing. However, two repertoried SNP, ENG rs35400405 and ALK1 rs2277382, were found in SSc-PAH patients only. The genotyping of 22 SNP including the latter showed that only rs2277382 was associated with SSc-PAH (p=0.0066, OR 2.13, 95% CI 1.24 to 3.65). Nevertheless, this was not replicated with the following result in combined analysis: p=0.123, OR 0.79, 95% CI 0.59 to 1.07. CONCLUSIONS: This study demonstrates the lack of association between these TGFß receptor gene polymorphisms and SSc-PAH using both sequencing and genotyping methods.

Sjogren's syndrome: an update on clinical, basic and diagnostic therapeutic aspects.

The 11th International Symposium for Sjogren's syndrome was held in Athens, Greece in September 2011. This symposia is part of a long series of meetings that have attempted to meet the needs of both scientists and physicians in improving the healthcare of their patients with Sjogren's syndrome. Sjogren's syndrome affects almost 0.5% of the general population and is second only to rheumatoid arthritis amongst the systemic autoimmune diseases. More importantly, it has daily implications for the millions of sufferers around the world. The goal of this meeting, which included nearly 200 abstracts and invited lectures, was to address the critical needs in the clinical practice of Sjogren's syndrome. This volume is a composite of select papers that were presented at this meeting and attempts to provide a critical overview of clinical and basic science. The volume includes a variety of themes and, importantly, raises issues that are still unresolved but which are important in our future diagnostic and therapeutic efforts.

Increased prevalence of anti-thyroid antibodies in patients with limited scleroderma.

OBJECTIVES: Thyroid dysfunction in the setting of systemic sclerosis (SSc) has been described previously. We aimed to determine the prevalence of anti-thyroid antibodies (ATA) in a large SSc cohort and to ascertain whether they are associated with distinct clinical phenotypes. METHODS: A total of 138 patients with SSc [46 with diffuse (dSSc) and 92 with limited scleroderma (lSSc)] and 100 healthy controls (HC) were tested for the presence of ATA [anti-thyroglobulin (anti-Tg) and anti-thyroid peroxidase (anti-TPO) antibodies] using a commercial enzyme-linked immunosorbent assay (ELISA). Clinical and serological data were recorded. RESULTS: An increased prevalence of anti-TPO but not anti-Tg antibodies was detected in patients with SSc compared to HC (27.5% vs. 14%, p = 0.016). Of note, a statistically significant increase of anti-TPO was detected only in patients with lSSc compared to HC (32.6% vs. 14%, p = 0.003). No correlations with other clinical features were detected. CONCLUSIONS: An increased prevalence of anti-TPO antibodies was identified in patients with lSSc. We propose that ATA testing should be offered to this subgroup of patients.

Characteristics of joint involvement and relationships with systemic inflammation in systemic sclerosis: results from the EULAR Scleroderma Trial and Research Group (EUSTAR) database.

OBJECTIVE: To determine the prevalence of and independent factors associated with joint involvement in a large population of patients with systemic sclerosis (SSc). METHODS: This study was cross-sectional, based on data collected on patients included in the European League Against Rheumatism (EULAR) Scleroderma Trials and Research (EUSTAR) registry. We queried this database to extract data regarding global evaluation of patients with SSc and the presence of any clinical articular involvement: synovitis (tender and swollen joints), tendon friction rubs (rubbing sensation detected as the tendon was moved), and joint contracture (stiffness of the joints that decreased their range of motion). Overall joint involvement was defined by the occurrence of synovitis and/or joint contracture and/or tendon friction rubs. RESULTS: We recruited 7286 patients with SSc; their mean age was 56 +/- 14 years, disease duration 10 +/- 9 years, and 4210 (58%) had a limited cutaneous disease subset. Frequencies of synovitis, tendon friction rubs, and joint contractures were 16%, 11%, and 31%, respectively. Synovitis, tendon friction rubs, and joint contracture were more prevalent in patients with the diffuse cutaneous subset and were associated together and with severe vascular, muscular, renal, and interstitial lung involvement. Moreover, synovitis had the highest strength of association with elevated acute-phase reactants taken as the dependent variable. CONCLUSION: Our results highlight the striking level of articular involvement in SSc, as evaluated by systematic examination in a large cohort of patients with SSc. Our data also show that synovitis, joint contracture, and tendon friction rubs are associated with a more severe disease and with systemic inflammation.

Disease activity and damage accrual during the early disease course in a multinational inception cohort of patients with systemic lupus erythematosus.

An inception cohort of patients with systemic lupus erythematosus from 14 European centres was followed for up to 5 years in order to describe the current early disease course. At inclusion patients (n = 200, 89% female, mean age 35 years, 97% Caucasian, mean SLEDAI 12.2) fulfilled a mean of 6.5 ACR classification criteria. The most prevalent criteria were antinuclear Ab presence (97%) followed by anti-dsDNA Ab (74%), arthritis (69%), leukocytopenia (54%) and malar rash (53%), antiphospholipid Ab (48%) and anti-synovial membrane Ab (21.6%). Clinical signs of lupus nephritis (LN) were present in 39% with biopsy-confirmed LN seen in 25%. Frequent additional findings were hypocomplementaemia (54%), anti-SSA Ab (49%), alopecia (26%) and Raynaud's phenomenon (31%). There were few regional differences in disease presentation and management. One and 5-year survival rates were 99% and 97% respectively. During the mean follow-up of 4.1 years 25% entered a state of early disease quiescence by global physician assessment, but the overall risk of subsequent flare was 60%. Maximum SLEDAI scores decreased over time, but 45% of patients accrued damage (SDI >or=1) for which baseline presence of proteinuria and persistent disease activity were independent predictors. The results indicate minor differences in SLE presentation and treatment within various regions of Europe and a high diagnostic reliance on anti-dsDNA Ab. Despite early reductions in disease activity and improved mortality, the risk for disease flare and damage development is, however, still substantial, especially in patients not entering an early remission.

Antithyroid antibodies in antiphospholipid syndrome: prevalence and clinical associations.

Autoimmune thyroid disease has been associated with several systemic autoimmune disorders. However, limited data are available regarding the prevalence and clinical associations of thyroid autoimmunity in antiphospholipid syndrome (APS). Seventy-five patients with APS, 75 patients with systemic lupus erythematosus (SLE) and 75 healthy controls were tested for the presence of antithyroid antibodies (ATAs) (anti-thyroglobulin and anti-thyroid peroxidase [anti-TPO]) using commercial ELISA. Clinical data were also recorded. Although no significant differences in the prevalence of ATAs were detected among APS, SLE patient groups and healthy controls, a significant increase of anti-TPO antibodies in patients with APS-SLE was found. An increased prevalence of ATAs in APS population with ischemic central nervous system (CNS) clinical manifestations was also detected. We present novel associations between thyroid autoimmunity and ischemic CNS clinical manifestations in the setting of APS.

Determinants of pulmonary function improvement in patients with scleroderma and interstitial lung disease.

BACKGROUND: In patients with scleroderma-related interstitial lung disease (ILD), improvements of pulmonary function have been reported after treatment with cyclophosphamide (CYC) alone or CYC and high-dose steroids. The study objective was to identify therapeutic regimen that alone or in combination with laboratory or clinical characteristics were associated with pulmonary function improvement in these patients. METHODS: Scleroderma patients with ILD and serial pulmonary function measurements were retrospectively analyzed. We recorded forced vital capacity (FVC, % predicted), diffusion capacity (DLCO, % predicted), type of therapy, and various clinical and laboratory parameters. Treatment with IV CYC was recorded as cumulative dose (grams) and treatment with steroids as high or low dose; outcome was defined as a sustained increase in FVC (% predicted) >or= 10 points. RESULTS: Of the 59 patients who were included in the study, 29 (49 %) patients received IV CYC (cumulative dose 13.9 +/-6.2, range 5.2-26.2 gr) for 3.3 +/- 2.4 years (range 5-60 months). Eighteen out of 59 (30 %) patients received high-dose prednisolone and 41 (70 %) received low-dose prednisolone. In an ordinal logistic model, patients receiving > 12 gr of CYC were 6 times more likely to improve FVC than to decrease or maintain FVC, compared to those who did not receive CYC (p = 0.02). In multivariate analysis, the effect of high dosage CYC on FVC persisted (OR 10.82, p = 0.02). Steroid dosage (high or low) was not associated with FVC improvement (p < 0.05). CONCLUSION: In patients with scleroderma and ILD, treatment with CYC is the only variable that is independently associated with pulmonary function improvement and that prolonged (> 1 year) CYC therapy increases the probability of pulmonary function improvement more than shorter CYC courses.

Current causes of death in systemic lupus erythematosus in Europe, 2000--2004: relation to disease activity and damage accrual.

Current therapeutic and diagnostic resources have turned systemic lupus erythematosus (SLE) into a chronic disease by reducing mortality rates. The exact contribution of disease activity and disease related damage to mortality is not well studied. The aim of this study was to describe the current causes of death (COD) in a multinational European cohort of patients with SLE in relation to quantified measures of disease activity and damage. Prospective five-year observational study of case fatalities in SLE patients at 12 European centres was performed. Demographics, disease manifestations, interventions and quantified disease activity (by ECLAM and SLEDAI) and damage (by SLICC-DI) at the time of death were related to the various COD. Ninety-one case fatalities (89% females) occurred after median disease duration of 10.2 years (range 0.2-40) corresponding to a annual case fatality of one for each of the participating cohorts. Cumulative mortality correlated linearly with disease duration with nearly 10% of fatalities occurring in the first year and 40% after more than 10 years of disease. Death occurred during SLE remission in one third of cases. In the remaining cases a mixture of disease activity (median ECLAM 5.5, median SLEDAI 15) and accrued damage (median SLICC-DI 5.0) with opposing relationships to disease duration contributed to death. Infections and cardiovascular events were the most frequent COD in both early and late fatalities with no gender differences for type of COD, disease activity, damage or comorbidity. In Europe, case fatalities have become uncommon events in dedicated SLE cohorts. The bimodal mortality curve has flattened out and deaths now occur evenly throughout the disease course with infectious and cardiovascular complications as the main direct COD in both early and late fatalities. Accrued damage supplants disease activity over time as the main SLE specific contributor to death over time.

Antiphospholipid antibodies: laboratory and pathogenetic aspects.

Antiphospholipid antibodies (aPL) constitute a heterogeneous group of autoantibodies that share the ability to bind phospholipids (PL) alone, protein-PL complexes, or PL-binding proteins. They have been detected in isolation, in association with autoimmune diseases such as systemic lupus erythematosus (SLE), and during the course of different infections. aPL have been associated with an array of clinical manifestations in virtually every organ, although deep vein and arterial thrombosis as well as pregnancy morbidity are predominant. The co-occurrence of these clinical findings with aPL constitutes the so-called antiphospholipid syndrome (APS). aPL can be detected by immunological methods [e.g., anticardiolipin antibodies (aCL)] or by functional methods that exploit the effect of aPL on blood coagulation [lupus anticoagulant (LA)]. Since aPL are heterogeneous, numerous immunological and coagulation assays have been developed. These assays have not been fully standardized, and, therefore, problems such as high interlaboratory variation are relatively frequent. Recently, recommendations have been published regarding LA and aCL testing. Not all aPL are pathogenic. However, when they are not associated with infections, they have a role in the pathogenesis of APS. Clinical and experimental data have shown that aPL exert their pathogenic activity by interfering with the function of coagulation factors, such as thrombin and factors X, XI and XII, and with the function of anticoagulant proteins of the protein C system. In addition, aPL interaction with platelets and endothelial cells induces a pro-adhesive activated phenotype.

International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS).

New clinical, laboratory and experimental insights, since the 1999 publication of the Sapporo preliminary classification criteria for antiphospholipid syndrome (APS), had been addressed at a workshop in Sydney, Australia, before the Eleventh International Congress on antiphospholipid antibodies. In this document, we appraise the existing evidence on clinical and laboratory features of APS addressed during the forum. Based on this, we propose amendments to the Sapporo criteria. We also provide definitions on features of APS that were not included in the updated criteria.

Estrogen receptor alpha gene polymorphism and systemic lupus erythematosus: a possible risk?

Estrogens and their receptors may play a role in the pathogenesis of systemic lupus erythematosus. Genetic alterations in the exon 8-coding region of the estrogen receptor alpha alter the intracellular signalling of estrogens, leading in enhanced or diminished activity. We investigated whether genetic alterations in exon 8 of ERalpha gene are associated with the occurrence and clinical features of lupus disease. The coding region of ERalpha exon 8 was subjected to mutation analysis using the polymerase chain reaction, denaturing gradient gel electrophoresis and sequence analysis, using DNA isolated from whole blood of 36 female patients and 38 healthy females. Clinical and laboratory parameters were available from the patients' files. We identified the codon 594 polymorphism either in homozygous for the wild type gene (ACG/ACG) or heterozygous (ACG/ACA), both in patients and healthy females. Statistical analysis of the genotype and allele distribution revealed that there was a significant difference (chi2 test, P = 0.02 and P = 0.04, respectively) between patients and healthy women. Odds ratio estimate revealed that carriers of ACG/ACA genotype have three-fold higher risk of developing lupus disease (OR = 3.129, 95% CI 1.181-8.292). Moreover, in patients the heterozygous genotype was associated with rash, mouth ulcers and serositis (Fisher's exact test, P = 0.055, P = 0.083, P = 0.065, respectively). The heterozygous patients were associated significantly with an early age at disease onset (ANOVA test, P < 0.05). We conclude that estrogen receptor alpha codon 594 genotype may influence the development of systemic lupus erythematosus at a younger age, as well as a certain disease clinical pattern.