The prevalence and incidence of systemic lupus erythematosus in children and adults: a population-based study in a mountain community in northern Italy.

OBJECTIVES: To estimate prevalence and incidence of systemic lupus erythematosus (SLE) in paediatric and adult populations in Italy. METHODS: The study was carried out in Valtrompia, a valley in northern Italy, where a relatively close community lives, in 2009-2012. The only referral centre for SLE in the area is the Rheumatology Unit of the University Hospital of Brescia. The ascertainment of SLE cases was performed through the integration of three sources: 1) hospital database; 2) database of the Rheumatology laboratory; 3) database of general practitioners and general paediatricians practicing in the area. Each patient was evaluated by a rheumatologist for confirmation of SLE classification based on the presence of at least 4 criteria according to the American College of Rheumatology. RESULTS: Forty-four SLE patients (39 females, 89%) were identified. The prevalence of SLE at 31st December 2012 was 39.2 (95% C.I. 28.5-52.6) cases per 100,000 individuals in all subjects, and 42.3 (30.5-57.2) and 15.3 (1.8-55.1) in adults and children, respectively. Nine new cases of SLE were diagnosed over the 4 years of the study period, with an annual incidence rate of 2.0 (0.9-3.8) per 100,000 individuals. CONCLUSIONS: This is the first study estimating the prevalence and incidence of SLE in Italy in both adult and paediatric population. Prevalence and incidence rates in line with those reported in other Mediterranean European countries. The accurate assessment of the SLE frequency is supported by the choice of a well-defined area, the integration of multiple data sources and the revision of each case by a rheumatologist.

European registry of babies born to mothers with antiphospholipid syndrome.

OBJECTIVES: This study aimed to describe the long-term outcome and immunological status of children born to mothers with antiphospholipid syndrome, to determine the factors responsible for childhood abnormalities, and to correlate the child's immunological profile with their mothers. METHODS: A prospective follow-up of a European multicentre cohort was conducted. The follow-up consisted of clinical examination, growth data, neurodevelopmental milestones and antiphospholipid antibodies (APL) screening. Children were examined at 3, 9, 24 months and 5 years. RESULTS: 134 children were analysed (female sex in 65 cases, birth weight 3000±500 g, height 48±3 cm). Sixteen per cent had a preterm birth (<37 weeks; n=22), and 14% weighted less than 2500 g at birth (n=19). Neonatal complications were noted in 18 cases (13%), with five infections (4%). During the 5-year follow-up, no thrombosis or systemic lupus erythematosus (SLE) was noted. Four children displayed behavioural abnormalities, which consisted of autism, hyperactive behaviour, feeding disorder with language delay and axial hypotony with psychomotor delay. At birth lupus anticoagulant was present in four (4%), anticardiolipin antibodies (ACL) IgG in 18 (16%), anti-ß(2) glycoprotein-I (anti-ß2GPI) IgG/M in 16 (15%) and three (3%), respectively. ACL IgG and anti-ß2GPI disappeared at 6 months in nine (17%) and nine (18%), whereas APL persisted in 10% of children. ACL and anti-ß2GPI IgG were correlated with the same mother's antibodies before 6 months of age (p<0.05). CONCLUSION: Despite the presence of APL in children, thrombosis or SLE were not observed. The presence of neurodevelopmental abnormalities seems to be more important in these children, and could justify long-term follow-up.

Inhibiting insulin resistance mechanisms by DTS phytocompound: an experimental study on metabolic syndrome-prone adipocytes.

The present study was designed to determine whether DTS a phytocompound endowed with antioxidant properties, could beneficially modulate nitric oxide (NO) production stimulated by lipopolysaccharide (LPS) and tumor necrosis factor-alpha (TNF-alpha) in adipocytes. Combined stimulation (CS-treatment) exerted by using 5 microg/ml of LPS together with 100 ng/ml of TNF-alpha significantly enhanced NO production in 3T3-L1 adipocytes. Preincubation of the adipocytes with DTS (10-30 mM) inhibited such phenomenon in a dose-dependent fashion. The production of NO was decreased by 52% at the concentration of 30mM of DTS. The decrease in NO production by DTS was associated also with a decrease in inducible nitric oxide synthase (iNOS) protein and iNOS mRNA expression. Nuclear factor-kappa B (NF-kappaB) was significantly enhanced by CS-treatment, while the pretreatment with 30 mM of DTS prevented the activity by 27%. IL-6 production in 3T3-L1 adipocytes was markedly increased by CS stimulus, and the enhanced secretion of IL-6 was suppressed in a dose-dependent manner by DTS. These results suggest that DTS regulates iNOS expression and NO production in adipocytes through the modulating activation of NF-kappaB and may have a potential clinical application within protocols designed for treating metabolic syndrome. (www.actabiomedica.it).

Laboratory criteria of the obstetrical antiphospholipid syndrome. Data from a multicentric prospective European women cohort.

A debate on updating the laboratory criteria of antiphospholipid syndrome (APS) was recently opened in view to lower the risk of over diagnosis of the syndrome. Based on data related to thrombotic APS, it proposes the exclusion of anticardiolipin antibodies (aCL) and anti-beta2-glycoprotein 1 (a-beta2-GPI) IgM detection. Here, we examine this possibility in a study which focuses on obstetrical APS (OAPS). We report new data on a prospective multicenter European cohort of 109 pregnant women having APS. Among them, 73 had purely obstetrical APS, not associated to autoimmune diseases or thrombosis. Isolated antibodies and isolated aCL positivity were present in 50/109 (46%) and in 34/109 (31%) of the women, respectively. An isolated a-beta2-GPI IgM was present in three women. These results suggest that aCL and a-beta2-GPI IgM cannot be dropped for the diagnosis and classification of OAPS. The low level of some antibodies associated with severe obstetrical complications raise the issue of keeping or not the same laboratory criteria for OAPS and for thrombotic APS and whether additional criteria after large prospective studies could further improve diagnosis.

A Case-Crossover Study to Investigate the Effects of Atmospheric Particulate Matter Concentrations, Season, and Air Temperature on Accident and Emergency Presentations for Cardiovascular Events in Northern Italy.

Atmospheric particulate matter (PM) has multiple adverse effects on human health, high temperatures are also associated with adverse health outcomes, and the frequency of cardiovascular events (CVEs) varies with season. We investigated a hypothesized increase in PM-related accident and emergency (A&E) presentations for CVE with high temperature, warm season, days of high influenza incidence, and in people with a cancer diagnosis, using a time-stratified case-crossover study design. Outcomes were associations of A&E presentation for CVE with atmospheric PM ≤ 10 µm (PM10), season, and air temperature. PM10 levels in the municipality of residence (exposure variable) were estimated by modeling data from local monitoring stations. Conditional logistic regression models estimated odds ratios (OR) with 95% confidence intervals (CI) for presentations in relation to supposed influencers, adjusting for confounders. Study participants were all who presented at the A&E of a large hospital near Milan, Italy, for a CVE (ICD-9: 390-459) from 1st January 2014 to 31st December 2015. There were 1349 A&E presentations for CVE in 2014-2015 and 5390 control days. Risk of A&E presentation was significantly increased on hot days with OR 1.34 (95%CI 1.05-1.71) per 10 µg/m3 PM10 increment (as mean PM10 on day of presentation, and 1 and 2 days before (lags 0-2)), and (for lag 0) in autumn (OR 1.23, 95%CI 1.09-1.37) and winter (OR 1.18, 95%CI 1.01-1.38). Risks were also significantly increased when PM10 was on lag 1, in people with a cancer diagnosis in the spring and summer months (1.88, 95%CI 1.05-3.37), and on days (lags 0-2) of high influenza incidence (OR 2.34, 95%CI 1.01-5.43). PM10 levels exceeded the 50 µg/m3 "safe" threshold recommended by the WHO and Italian legislation for only 3.8% of days during the warm periods of 2014-2015. Greater risk of A&E presentation for CVE in periods of high PM10 and high temperature suggests that "safe" thresholds for PM10 should be temperature-dependent and that the adverse effects of PM10 will increase as temperatures increase due to climate change.

Are the current attempts at standardization of antiphospholipid antibodies still useful? Emerging technologies signal a shift in direction.

The pathogenic role of antiphospholipid antibodies (aPL) has been widely established over past years in several experimental models and clinical studies. Accordingly, the detection of aPL by immunoassays (anticardiolipin antibodies; anti-beta2 glycoprotein I antibodies) has become a routine practice in the clinical workup of patients with systemic autoimmune diseases. aPL are mostly assayed using commercial ELISA kits, whose performance has not been found to be sufficiently concordant among the different manufacturers. In the past years, collaborative groups have spent considerable effort to reach some form of standardization but this process is still ongoing. Such lack of standardization has recently become even more crucial, as manufacturers have had to face an increasing demand for fully automated tests for aPL, like those test systems that have been developed for other autoantibodies (e.g., antinuclear antibodies, anti-ENA antibodies). We therefore report our recent experience with two newly developed automated methods for anticardiolipin antibodies testing. In particular, we discuss the results obtained using routine samples, as we believe that these better reflect the "real-life" situation in which those automated methods will operate. We also mention other emerging technologies in the field of aPL detection.

Anti-DNA antibodies: a diagnostic and prognostic tool for systemic lupus erythematosus?

The clinical impact of anti-DNA antibodies lies on their diagnostic power for systemic lupus erythematosus (SLE), being a formal classification criterion. In spite of such a disease association, low-avidity anti-DNA antibodies might also be part of the natural autoantibody repertoire. Their switch to pathogenic high-avidity autoantibodies is the result of the autoimmune process leading to SLE.Anti-DNA antibodies were shown to play a role in SLE pathogenesis and particularly in kidney damage. Accordingly, antibody titres might fluctuate in relation to disease activity, but their prognostic value for flares is still debated.Several methods for anti-DNA detection were described and there is evidence that the assays identify different antibodies with different prognostic value. The results of a multicenter study on four different routine tests for anti-dsDNA antibody detection showed that: (i) Farr assay displays the best diagnostic specificity/sensitivity for SLE, followed by Crithidia luciliae method (CLIFT), (ii) the new generation of solid phase assay (EliA) shows an increased sensibility versus the classical enzyme linked immune assay (ELISA) but a decreased specificity. Antibody titre detected by EliA and Farr assay correlated with disease activity. These findings would suggest that more than one assay should be useful for SLE diagnosis and monitoring.

Clinical characterization of antiphospholipid syndrome by detection of IgG antibodies against ß2 -glycoprotein i domain 1 and domain 4/5: ratio of anti-domain 1 to anti-domain 4/5 as a useful new biomarker for antiphospholipid syndrome.

OBJECTIVE: It has been suggested that only antibodies against domain 1 (D1) of ß2 -glycoprotein I (ß2 GPI) are pathogenic and diagnostic. The role of antibodies against other ß2 GPI domains is still debated. This study was undertaken to evaluate the clinical relevance of domain specificity profiling of anti-ß2 GPI IgG antibodies in antiphospholipid syndrome (APS) patients and in control groups of patients with systemic autoimmune rheumatic diseases and in asymptomatic antiphospholipid antibody (aPL) carriers. METHODS: We evaluated 159 subjects with persistently positive, medium or high-titer anti-ß2 GPI IgG, including 56 patients with thrombotic (obstetric or nonobstetric) primary APS, 31 women with obstetric primary APS, 42 aPL-positive patients with systemic autoimmune rheumatic diseases, and 30 asymptomatic aPL carriers. One hundred healthy donors were included. Anti-ß2 GPI D1 and D4/5 IgG were tested on research enzyme-linked immunosorbent assays containing recombinant ß2 GPI domains. RESULTS: As compared to other groups, aPL carriers displayed higher frequency/titer of anti-D4/5 IgG. Unlike anti-D4/5, anti-D1 IgG antibodies were more frequent and at higher titer in triple than in single or double aPL-positive subjects. An anti-D1 to anti-D4/5 ratio of ≥1.5 was predictive of systemic autoimmunity (odds ratio 3.25 [95% confidence interval 1.45-7.49], P = 0.005). Neither anti-D1 nor anti-D4/5 antibodies were associated with APS clinical criteria. CONCLUSION: Anti-D1 IgG is the preferential specificity not only in vascular and obstetric primary APS, but also in patients with systemic autoimmune rheumatic disease with no clinical features of APS. Conversely, aPL carriers do not have a polarized profile toward D1. Combined testing for anti-ß2 GPI IgG with different domain specificity allows a more accurate aPL profiling, with polarization toward anti-D1 IgG as a possible fingerprint of systemic autoimmunity.

Minimal requirements for antiphospholipid antibodies ELISAs proposed by the European Forum on antiphospholipid antibodies.

Antiphospholipid ELISAs are part of the Antiphospholipid Antibodies Syndrome classification criteria, having the same diagnostic value as lupus anticoagulant. However, sometimes their results appear scarcely meaningful especially when wide metanalyses studies are performed, probably because of their well-known inter-laboratory variability. The application of a common protocol was shown to improve the test reproducibility, but this observation did not have any influence on the routine performances. After discussion among experts at the European level, we identified four conditions named "minimal requirements" considered useful to decrease the inter-laboratory variability: (1) to run the samples in duplicate; (2) to determine the cut off level in each laboratory analysing at least 50 samples from normal subjects, possibly age- and sex-matched with the patient population usually attending the Centre; (3) to calculate the cut-off level in percentiles; (4) to use stable external control in the tests. A collaborative study involving 36 European centres proved that the use of monoclonal anti-beta2 glycoprotein I antibodies, HCAL (IgG) and EY2C9 (IgM) as standards, can help to reduce the inter-laboratory coefficient of variation both in anticardiolipin (aCL) and anti-beta2GPI (anti-beta2 glycoprotein I) ELISA. Therefore, we propose HCAL and EY2C9 as external controls, but other monoclonal or polyclonal preparations may be considered. During an interactive workshop held last May in Italy, 16 companies producing these tests agreed to consider the introduction of the "requirements" in their products. We suggest to adopt these "requirements" particularly in clinical studies, in order to compare more easily the literature data.

Diagnostic value of anti-mutated citrullinated vimentin in comparison to anti-cyclic citrullinated peptide and anti-viral citrullinated peptide 2 antibodies in rheumatoid arthritis: an Italian multicentric study and review of the literature.

In the last years, the detection of antibodies (Abs) against citrullinated peptides (ACPA) has largely replaced rheumatoid factor (RF) as the most helpful biomarker in the diagnosis of rheumatoid arthritis (RA). Current assays detect ACPA reactivity with epitopes on various different citrullinated proteins. Among these, anti-cyclic citrullinated peptide (CCP) Abs have been widely demonstrated to be an important diagnostic and prognostic tool because of their high specificity. Recently, citrullinated vimentin, a protein highly released in synovial microenvironment, has been identified as potential autoantigen in the pathophysiology of RA and an enzyme-linked immunosorbent assay (ELISA) for the detection of Abs directed against a mutated citrullinated vimentin (anti-MCV) was developed. Several recent studies evaluating the characteristics of anti-MCV in comparison to anti-CCP Abs, have given conflicting results. Anti-MCV have been demonstrated to perform better than anti-CCP as predictor of radiographic damage. Conversely, its additional diagnostic and prognostic role in comparison to anti-CCP in both early and established RA is controversial. Aim of this study was to evaluate the diagnostic performance of anti-MCV in RA and to compare it to anti-CCP and the recently developed assay targeting viral citrullinated peptide 2 (VCP2) in a large cohort of RA patients (n=285), healthy subjects and other disease controls (n=227). Anti-MCV resulted to have a sensitivity of 59% and a specificity of 92%. In comparison, anti-CCP and anti-VCP2 displayed a sensitivity of 77% and 61% and a specificity of 96% and 95%, respectively. Of interest, at the manufacturer recommended cutoff value of 20U/mL, a high percentage of healthy subjects as well as Epstein Barr (EBV) and hepatitis C (HCV) virus infected patients resulted anti-MCV positive. In our large cohort of RA patients, anti-MCV demonstrated lower sensitivity than anti-CCP and VCP2 test, thus not allowing to confirm previously published data. Moreover, the high rate of detection in infectious diseases limits its diagnostic value in undifferentiated arthritis.

Antinucleosome antibodies in primary antiphospholipid syndrome: a hint at systemic autoimmunity?

BACKGROUND: Antinucleosome antibodies (anti-NCS) are reported to be highly sensitive and specific for systemic lupus erythematosus (SLE) and to correlate with disease activity. They may appear in early stages of the disease, in particular before anti-dsDNA antibodies, being a potential marker for identifying patients susceptible to SLE. Patients with primary antiphospholipid syndrome (PAPS) may develop full-blown SLE but there is no evidence for markers predictive for that. AIM: To evaluate whether anti-NCS may be predictors for full-blown or lupus like disease (LL) in a cohort of PAPS patients. METHODS: A multicentric cohort of 105 PAPS patients was tested for IgG/IgM anti-NCS by using a home made assay with H1-stripped chromatin as antigen. RESULTS: Eighty-one out of 105 (77%) of the patients were positive for anti-NCS; medium-high titre results were present only in 49/105 (46%). Anti-NCS were more frequently detected in PAPS+LL, but no relationship with clinical/serological features was found, except for a weak correlation with anti-dsDNA antibodies. Two PAPS patients evolved into full-blown SLE during the follow-up and displayed high titre anti-NCS many years before. CONCLUSIONS: Our findings suggest that anti-NCS might be added to the mosaic of autoimmune phenomena characterizing PAPS patients and in particular those with more chance to evolve to SLE.

Anticardiolipin and anti-beta2 glycoprotein I antibodies in infants born to mothers with antiphospholipid antibody-positive autoimmune disease: a follow-up study.

Infants born from mothers with antiphospholipid antibody (aPL) -positive autoimmune disease were prospectively evaluated for anticardiolipin and anti-beta2 glycoprotein I antibodies (group 1) and for growth and neurological development. The results were compared with those obtained from two age-matched control groups (group 2 and 3). All infants were negative for anticardiolipin at 12 months of life, whereas 14 (63.6%), eight (33.3%), and 10 (55.5%) of infants from group 1, 2, and 3, respectively, were positive for anti-beta2 glycoprotein I. At follow-up, all infants had normal growth and neurological development. No thrombotic complication was observed. The negativity of anticardiolipin in all infants at 12 months suggests that anticardiolipin detection is the best assay to evaluate the disappearance of maternal aPL and to estimate the potential risk of thrombosis associated with these antibodies. The high rate of anti-beta2 glycoprotein I positivity in all three groups of infants may indicate that the synthesis of this antibody is stimulated by aspecific factors.