Frequency Evaluation of the Interleukin-6 -174G>C Polymorphism and Homeostatic Iron Regulator (HFE) Mutations as Disease Modifiers in Patients Affected by Systemic Lupus Erythematosus and Rheumatoid Arthritis.

Autoimmune diseases are generally characterized by a multifactorial etiology and are often associated with a genetic predisposition. Both iron metabolism and the inflammatory cytokine system have been shown to play a pivotal role in the dysregulation of the immune response in many different autoimmune conditions, rheumatologic diseases included. The purpose of this work was to analyze the frequency of mutations altering the expression of IL-6 or influencing iron metabolism in patients affected by autoimmune diseases such as Rheumatoid Arthritis (RA) and Systemic Lupus Erythematosus (SLE). In this study, 144 patients were enrolled: 77 and 67 patients were affected by RA and SLE, respectively. In these cohorts, the frequency of the IL-6 polymorphism -174G>C located in the IL-6 gene promoter was tested. Moreover, the frequencies of the three HFE gene variations associated with iron overload were analyzed: p.His63Asp, p.Ser65Cys and p.Cys282Tyr. The two mutations p.His63Asp and p.Ser65Cys in the HFE gene did not reach statistical significance in any of the comparisons, regardless of the statistical model, cohorts of patients and control populations analyzed. The frequencies of the p.Cys282Tyr mutation and the IL-6 polymorphism -174G>C were found to be overall significantly decreased in RA and SLE patients when the Dominant model and Allele contrast were adopted with both the Odds Ratio and Chi-square. Although further investigation is needed, the examination of the frequencies of the -174G>C IL-6 promoter polymorphism and HFE mutations may add some valuable information on the interplay linking iron metabolism, inflammation and immunity in autoimmune diseases such as SLE and RA.

Systemic Sclerosis-Specific Antibodies: Novel and Classical Biomarkers.

Disease-specific autoantibodies are considered the most important biomarkers for systemic sclerosis (SSc), due to their ability to stratify patients with different severity and prognosis. Anti-nuclear antibodies (ANA), occurring in subjects with isolated Raynuad's phenomenon, are considered the strongest independent predictors of definite SSc and digital microvascular damage, as observed by nailfold videocapillaroscopy. ANA are present in more than 90% of SSc, but ANA negativity does not exclude SSc diagnosis: a little rate of SSc ANA negative exists and shows a distinct subtype of disease, with less vasculopathy, but more frequent lower gastrointestinal involvement and severe disease course. Anti-centromere, anti-Th/To, and anti-Topoisomerase I antibodies could be considered as classical biomarkers, covering about 60% of SSc and defining patients with well-described cardio-pulmonary complications. In particular, anti-Topoisomerase I represent a risk factor for development of diffuse cutaneous involvement and digital ulcers in the first 3 years of disease, as well as severe interstitial lung disease (ILD). Anti-RNA polymerase III is a biomarker with new clinical implications: very rapid skin thickness progression, gastric antral vascular ectasia, the occurrence of synchronous cancers, and possible association with silicone breast implants rupture. Moreover, novel SSc specific autoantibodies have been globally described in about 10% of "seronegative" SSc patients: anti-elF2B, anti-RuvBL1/2 complex, anti-U11/U12 RNP, and anti-BICD2 depict specific SSc subtypes with severe organ complications. Many autoantibodies could be considered markers of overlap syndromes, including SSc. Anti-Ku are found in 2-7% of SSc, strictly defining the PM/SSc overlap. They are associated with synovitis, joint contractures, myositis, and negatively associated with vascular manifestation of disease. Anti-U3RNP are associated with a well-defined clinical phenotype: Afro-Caribbean male patients, younger at diagnosis, and higher risk of pulmonary hypertension and gastrointestinal involvement. Anti-PM/Scl define SSc patients with high frequency of ILD, calcinosis, dermatomyositis skin changes, and severe myositis. The accurate detection of autoantibodies SSc specific and associated with overlap syndromes is crucial for patients' stratification. ANA should be correctly identified using indirect immunofluorescent assay and a standardized way of patterns' interpretation. The gold-standard technique for autoantibodies' identification in SSc is still considered immunoprecipitation, for its high sensitivity and specificity, but other assays have been widely used in routine practice. The identification of SSc autoantibodies with high diagnostic specificity and high predictive value is mandatory for early diagnosis, a specific follow-up and the possible definition of the best therapy for every SSc subsets. In addition, the validation of novel autoantibodies is mandatory in wider cohorts in order to restrict the gap of so-called seronegative SSc patients.

Similarities and differences between younger and older disease onset patients with newly diagnosed systemic lupus erythematosus.

OBJECTIVES: Several studies show that age at onset has an impact on the clinical-serological presentation, comorbidities and disease course of patients with systemic lupus erythematosus (SLE). We evaluated whether, in patients with recent onset SLE, the age at onset correlates with clinical-serological manifestations and with comorbidities. METHODS: We analysed 171 patients with a SLE diagnosis obtained within 12 months of diagnosis enrolled in the Early Lupus project. Based on the age of onset of the first disease symptom, they were stratified into 2 groups: early onset (18-45 years) and late onset (>45 years). The analysis was replicated by stratifying patients based on age at diagnosis (fulfillment of ACR classification criteria). Each comparison was made at baseline and at 36 months of follow-up. RESULTS: Baseline: patients with late onset displayed comorbidities (hypertension, dyslipidemia and osteoporosis) more frequently than early onset group. 11.4% of late onset patients had a malignancy in medical history, not recorded in the early onset cohort. The two groups differed neither in organ involvement (domain BILAG) nor in disease activity (ECLAM). Patients with early onset showed a disease with signs of higher serologic activity (higher frequency of anti-dsDNA positivity and lower mean C3 and C4 levels) and had malar rash more frequently than the late onset group (36.2% vs. 18.2%, p=0.042). Similar results were obtained by stratifying patients by age of diagnosis (18-45 years and >45 years), except for the higher frequency of discoid rash in the group with age at diagnosis >45 years (18% vs. 6.6%, p=0.045). 36 months: the 2 groups of patients independently of the stratification applied did not differ in the accumulation of damage, but showed a different pattern of 8 organ involvement. Musculoskeletal involvement was more frequent both in the late onset group (18.6% vs. 7.3%, p=0.043) and in the group with age at diagnosis >45 years (20.4% vs. 5.9%, p=0.009) compared to their counterparts, while renal involvement was more frequent in the group with age at diagnosis 18-45 years (21.4% vs. 6.1%, p=0.03).A sub analysis at 36 months on patients without hypertension and osteoporosis at enrollment showed that patients with older age at onset had a higher frequency of these comorbidities, compared to their counterparts. CONCLUSIONS: In our cohort, younger disease SLE onset seems to correlate with a more active immunological profile, while late onset with a higher incidence of comorbidities.

An Italian Multicenter Study on Anti-NXP2 Antibodies: Clinical and Serological Associations.

The identification of anti-NXP2 antibodies is considered a serological marker of dermatomyositis (DM), with calcinosis, severe myositis and, in some reports, with cancer. Historically, these associations with anti-NXP2 antibodies have been detected by immunoprecipitation (IP), but in the last few years commercial immunoblotting assays have been released. The aim of this collaborative project was to analyse the clinical features associated to anti-NXP2 antibodies, both with commercial line blot (LB) and IP. Myositis-specific and myositis-associated autoantibodies were detected in single centres by commercial line blot (LB); available sera were evaluated in a single centre by protein and RNA immunoprecipitation (IP), and IP-Western blot. Sixty patients anti-NXP2+ (NXP2+) positive by LB were compared with 211 patients anti-NXP2 negative with idiopathic inflammatory myositis (IIM). NXP2+ showed a younger age at IIM onset (p = 0.0014), more frequent diagnosis of dermatomyositis (p = 0.026) and inclusion-body myositis (p = 0.009), and lower rate of anti-synthetase syndrome (p < 0.0001). As for clinical features, NXP2+ more frequently develop specific skin manifestations and less frequently features related with overlap myositis and anti-synthetase syndrome. IP confirmed NXP2 positivity in 31 of 52 available sera (62%). Most clinical associations were confirmed comparing NXP2 LB+/IP+ versus NXP2-negative myositis, with the following exceptions: inclusion-body myositis diagnosis was not detected, whilst dysphagia and myositis were found more frequently in NXP2 LB+/IP+ patients. The 21 LB+ /IP-myositis patients did not show differences in clinical features when compared with the NXP2-myositis patients and more frequently displayed multiple positivity at LB. Risk of developing cancer-associated myositis was similar between NXP2-positive and NXP2-negative myositis patients, either when detected by LB or IP. Protein-IP confirmed NXP2 antibodies in nearly 60% of sera positive for the same specificity with commercial assay. Double-positive cases rarely occurred in myositis patients with a clinical diagnosis other than dermatomyositis. Patients only positive by LB (LB+/IP-) did not display clinical features typical of NXP2. NXP2 positivity by LB should be confirmed by other methods in order to correctly diagnose and characterize patients affected by idiopathic inflammatory myositis.

Immunosuppressive therapy withdrawal after remission achievement in patients with lupus nephritis.

OBJECTIVES: Whether immunosuppressive therapy may be safely withdrawn in lupus nephritis (LN) is still unclear. We assessed rate and predictors of flare after IS withdrawal in patients with LN in remission. METHODS: Patients with biopsy-proven LN treated with immunosuppressants (IS) between 1980 and 2020 were considered. Remission was defined as normal serum creatinine, proteinuria <0.5 g/24 h, inactive urine sediment, and no extra-renal SLE activity on stable immunosuppressive and/or antimalarial therapy and/or prednisone ≤5mg/day. IS discontinuation was defined as the complete withdrawal of immunosuppressive therapy, flares according to SLEDAI Flare Index. Predictors of flare were analysed by multivariate logistic regression analysis. RESULTS: Among 513 SLE patients included in our database, 270 had LN. Of them, 238 underwent renal biopsy and were treated with IS. Eighty-three patients (34.8%) discontinued IS, 46 (30) months after remission achievement. During a mean (s.d.) follow-up of 116.5 (78) months, 19 patients (22.9%) developed a flare (8/19 renal) and were re-treated; 14/19 (73.7%) re-achieved remission after restarting therapy. Patients treated with IS therapy for at least 3 years after remission achievement had the lowest risk of relapse (OR 0.284, 95% CI: 0.093, 0.867; P = 0.023). At multivariate analysis, antimalarial maintenance therapy (OR 0.194, 95% CI: 0.038, 0.978; P = 0.047), age at IS discontinuation (OR 0.93, 95% CI: 0.868, 0.997; P = 0.040), remission duration >3 years before IS discontinuation (OR 0.231, 95% CI: 0.058, 0.920; P = 0.038) were protective against disease flares. CONCLUSIONS: Withdrawal of IS is feasible in LN patients in remission for at least 3 years and on antimalarial therapy. Patients who experience flares can re-achieve remission with an appropriate treatment.

Durable renal response and safety with add-on belimumab in patients with lupus nephritis in real-life setting (BeRLiSS-LN). Results from a large, nationwide, multicentric cohort.

BACKGROUND: Belimumab was recently approved for treatment of lupus glomerulonephritis (LN). AIM: To evaluate renal response and its predictors in LN patients receiving belimumab in real-life. PATIENTS AND METHODS: We considered all patients fulfilling the SLEDAI-2K renal items and/or having estimated glomerular filtration rate (eGFR)≤60 ml/min/1.73 m2, with positive anti-dsDNA and/or low C3/C4 enrolled in the multicentre Italian lupus cohort BeRLiSS (BElimumab in Real LIfe Setting Study), treated with monthly IV Belimumab 10 mg/kg over standard treatment. Primary efficacy renal response (PERR), defined as proteinuria ≤0.7 g/24 h, eGFR≥60 ml/min/1.73 m2 without rescue therapy, was considered as primary outcome. Complete renal response (CRR; proteinuria <0.5 g/24 h, eGFR≥90 ml/min/1.73 m2) was considered as secondary outcome. Prevalence and predictors of PERR were evaluated at 6, 12, 24 months by multivariate logistic regression. RESULTS: Among the 466 SLE patients of BeRLiSS, 91 fulfilled the inclusion criteria, 79 females, median age 41.0 (33.0-47.0) years, median follow-up 22.0 (12.0-36.0) months. Sixty-four (70.3%) achieved PERR, of whom 38.4% reached CRR. Among patients achieving PERR at 6 months, 86.7% maintained response throughout the follow-up. At multivariable analysis, hypertension (OR [95%CI]: 0.28 [0.09-0.89], p = 0.032), high baseline serum creatinine (0.97 [0.95-0.99], p = 0.01) and high baseline proteinuria (0.37, [0.19-0.74], p = 0.005) negatively predicted PERR. Positive predictors of PERR at 12 and 24 months were baseline anti-Sm positivity (OR [95%CI]: 6.2 [1.21-31.7], p = 0.029; 19.8 [2.01-186.7], p = 0.009, respectively) and having achieved PERR at 6 months (14.4 [3.28-63.6]; 11.7 [2.7-48.7], p = 0.001 for both). CONCLUSIONS: Add-on therapy with belimumab led to durable renal response in patients with LN in a real-life setting.

Serum IgG2 antibody multicomposition in systemic lupus erythematosus and lupus nephritis (Part 1): cross-sectional analysis.

OBJECTIVES: Serum anti-dsDNA and anti-nucleosome IgGs have been proposed as signatures for SLE and LN in limited numbers of patients. We sought to show higher sensitivity and specificity of the same antibodies with the IgG2 isotype and included IgG2 antibodies vs specific intracellular antigens in the analysis. METHODS: A total of 1052 SLE patients with (n = 479) and without (n = 573) LN, recruited at different times from the beginning of symptoms, were included in the study. Patients with primary APS (PAPS, n = 24), RA (RA, n = 24) and UCTD (UCTD, n = 96) were analysed for comparison. Anti-nucleosome (dsDNA, Histone2A, Histone3), anti-intracellular antigens (ENO1), anti-annexin A1 and anti-C1q IgG2 were determined by non-commercial techniques. RESULTS: The presence in the serum of the IgG2 panel was highly discriminatory for SLE/LN vs healthy subjects. Serum levels of anti-dsDNA and anti-C1q IgG2 were more sensitive than those of IgGs (Farr radioimmunoassay/commercial assays) in identifying SLE patients at low-medium increments. Of more importance, serum positivity for anti-ENO1 and anti-H2A IgG2 discriminated between LN and SLE (ROC T0-12 months), and high levels at T0-1 month were detected in 63% and 67%, respectively, of LN, vs 3% and 3%, respectively, of SLE patients; serum positivity for each of these was correlated with high SLEDAI values. Minor differences existed between LN/SLE and the other rheumatologic conditions. CONCLUSION: Nephritogenic IgG2 antibodies represent a specific signature of SLE/LN, with a few overlaps with other rheumatologic conditions. High levels of anti-ENO1 and anti-H2A IgG2 correlated with SLE activity indexes and were discriminatory between SLE patients limited to the renal complication and other SLE patients. TRIAL REGISTRATION: The Zeus study was registered at https://clinicaltrials.gov, NCT02403115.

Serum IgG2 antibody multi-composition in systemic lupus erythematosus and in lupus nephritis (Part 2): prospective study.

OBJECTIVES: Circulating anti-ENO1 and anti-H2A IgG2 have been identified as specific signatures of LN in a cross-over approach. We sought to show whether the same antibodies identify selected population of patients with LN with potentially different clinical outcomes. METHODS: Here we report the prospective analysis over 36 months of circulating IgG2 levels in patients with newly diagnosed LN (n=91) and SLE (n=31) and in other patients with SLE recruited within 2 years from diagnosis (n=99). Anti-podocyte (ENO1), anti-nucleosome (DNA, histone 2 A, histone 3) and anti-circulating proteins (C1q, AnnexinA1-ANXA1) IgG2 antibodies were determined by home-made techniques. RESULTS: LN patients were the main focus of the study. Anti-ENO1, anti-H2A and anti-ANXA1 IgG2 decreased in parallel to proteinuria and normalized within 12 months in the majority of patients while anti-dsDNA IgG2 remained high over the 36 months. Anti-ENO1 and anti-H2A had the highest association with proteinuria (Heat Map) and identified the highest number of patients with high proteinuria (68% and 71% respectively) and/or with reduced estimated glomerula filtration rate (eGFR) (58% for both antibodies) compared with 23% and 17% of anti-dsDNA (agreement analysis). Anti-ENO1 positive LN patients had higher proteinuria than negative patients at T0 and presented the maximal decrement within 12 months. CONCLUSIONS: Anti-ENO1, anti-H2A and anti-ANXA1 antibodies were associated with high proteinuria in LN patients and Anti-ENO1 also presented the maximal reduction within 12 months that paralleled the decrease of proteinuria. Anti-dsDNA were not associated with renal outcome parameters. New IgG2 antibody signatures should be utilized as tracers of personalized therapies in LN. TRIAL REGISTRATION: The Zeus study was registered at https://clinicaltrials.gov (study number: NCT02403115).

Systematic review and meta-analysis of epidemiological studies on the association of occupational exposure to free crystalline silica and systemic lupus erythematosus.

OBJECTIVES: Some evidence suggests that exposure to free crystalline silica may contribute to the risk of developing SLE. A systematic search was carried out for all published epidemiological studies concerning this association. A meta-analysis was conducted on relevant studies. METHODS: We searched PubMed and EMBASE databases for original articles published from 1960 to November 2019 in any language. In addition, we also searched the reference lists of included studies manually for additional relevant articles. Finally, seven studies were included in the systematic review and six studies in the meta-analysis (four case-control and two cohort studies). The odds ratio and 95% CI were calculated using a random effect meta-analysis. RESULTS: The meta-analysis of the studies, applying a random effect model, yielded an overall odds ratio of 3.49 (95% CI, 1.24, 9.83), with I2 = 92.36% (pronounced heterogeneity). We also stratified the meta-analysis by study design; case-control studies: odds ratio 1.85 (95% CI, 0.96, 3.59) with I2 = 75.92%; and cohort studies (cases with silicosis): odds ratio 9.71 (95% CI, 1.13, 83.58) with I2 = 72.65%. CONCLUSIONS: The obtained results support the hypothesis of a possible association between occupational exposure to free crystalline silica and SLE, in particular at higher exposure levels, known to induce silicosis. The studies that have investigated this association are still scarce and the heterogeneity between the studies remains high. New studies are deemed necessary to confirm the association.

Severe muscle damage with myofiber necrosis and macrophage infiltrates characterize anti-Mi2 positive dermatomyositis.

OBJECTIVE: The aim of our study was to investigate clinical and histopathological findings in adult DM patients positive for anti-Mi2 (anti-Mi2+) antibodies compared with DM patients negative for anti-Mi2 (anti-Mi2-). METHODS: Clinical data of adult DM patients, who fulfilled EULAR/ACR 2017 classification criteria, were gathered from electronic medical records of three tertiary Rheumatology Units. Histopathological study was carried out on 12 anti-Mi2+ and 14 anti-Mi2- muscle biopsies performed for diagnostic purpose. Nine biopsies from immune mediated necrotizing myopathy (IMNM) patients were used as control group. RESULTS: Twenty-two anti-Mi2+ DM [90.9% female, mean age 56.5 (15.7) years] were compared with 69 anti-Mi2- DM patients [71% female, mean age 52.4 (17) years]. Anti-Mi2+ patients presented higher levels of serum muscle enzymes than anti-Mi2- patients [median (IQR) creatine-kinase fold increment: 16 (7-37)vs 3.5 (1-9.9), P <0.001] before treatment initiation. Moreover, a trend towards less pulmonary involvement was detected in anti-Mi2+ DM (9.1% vs 30.4%, P =0.05), without any case of rapidly progressive interstitial lung disease. At muscle histology, anti-Mi2+ patients showed more necrotic/degenerative fibres than anti-Mi2- patients [mean 5.3% (5) vs 0.8% (1), P <0.01], but similar to IMNM [5.9% (6), P >0.05]. In addition, the endomysial macrophage score was similar between anti-Mi2+ and IMNM patients [mean 1.2 (0.9) vs 1.3 (0.5), P >0.05], whereas lower macrophage infiltration was found in anti-Mi2- DM [mean 0.4 (0.5), <0.01]. CONCLUSIONS: Anti-Mi2+ patients represent a specific DM subset with high muscle damage. Histological hallmarks were a higher prevalence of myofiber necrosis, endomysial involvement and macrophage infiltrates at muscle biopsy.

Predictive Features and Clinical Presentation of Interstitial Lung Disease in Inflammatory Myositis.

Interstitial lung disease (ILD) represents one of the most severe extra-muscular features of idiopathic inflammatory myositis (IIM). We aimed to identify any clinical and serological predictors of ILD in a monocentric cohort of 165 IIM patients.ILD+ patients were defined as having restrictive impairment in lung function tests and signs of ILD at chest high-resolution computed tomography (HRCT). Available HRCT images were centralized and classified in different ILD patterns: non-specific interstitial pneumonia (NSIP), organizing pneumonia (OP), usual interstitial pneumonia-like (UIP), indeterminate for UIP, and interstitial lung abnormalities (ILA). Lung function test data were recorded at onset, at 1 and 5 years after ILD diagnosis.ILD was found in 52 IIM patients (31.5%): 46.2% was affected by anti-synthetase syndrome (ARS), 21% by polymyositis (PM), 19% by dermatomyositis (DM), and 13.5% by overlap myositis. Most of ILD+ showed NSIP (31.9%), OP (19%), indeterminate for UIP (19%), and UIP (12.8%) patterns. At multivariate analysis, ILD was predicted by anti-Ro52 (p: 0.0026) and dyspnea (p: 0.015) at IIM onset. Most of ILD onset within is 12 months after IIM. In five cases, ILD occurs after 12 months since IIM diagnosis: these patients more frequently show dry cough and anti-Ku antibodies. Anti-Ro52 + ILD patients showed a significant increase of DLCO at 1 and 5 years of follow-up, compared with anti-Ro52 negative cases.ILD occurs in about one third of IIM and was predicted by dyspnea at onset and anti-Ro52 antibodies. Anti-Ro52 defines a subgroup of ILD showing a significant improvement of DLCO during follow-up. This retrospective study has been approved by local ethic committee (ASST-Spedali Civili of Brescia, Italy); protocol number: NP3511.

Celiac Disease Prevalence is Increased in Primary Sjögren's Syndrome and Diffuse Systemic Sclerosis: Lessons from a Large Multi-Center Study.

Association of celiac disease (CD) with systemic autoimmune diseases (ADs) remains controversial. Awareness of CD in these patients is important to prevent complications, including lymphoproliferative disorders. We evaluated previously diagnosed CD prevalence in systemic lupus erythematosus (SLE), primary Sjögren's syndrome (pSS) and systemic sclerosis (SSc) patients in comparison to 14,298 matched controls. All patients were screened for subclinical CD. Data from 1458 unselected consecutive SLE (580), pSS (354) and SSc (524) patients were collected. Previously biopsy-proven CD diagnosis and both CD- and AD-specific features were registered. All patients without previous CD were tested for IgA transglutaminase (TG). Anti-endomysium were tested in positive/borderline IgA TG. Duodenal biopsy was performed in IgA TG/endomysium+ to confirm CD. CD prevalence in AD was compared to that observed in 14,298 unselected sex- and age-matched adults who acted as controls. CD was more prevalent in pSS vs controls (6.78% vs 0.64%, p < 0.0001). A trend towards higher prevalence was observed in SLE (1.38%, p = 0.058) and SSc (1.34%, p = 0.096). Higher CD prevalence was observed in diffuse cutaneous SSc (4.5%, p ≤ 0.002 vs controls). Subclinical CD was found in two SLE patients and one pSS patient. CD diagnosis usually preceded that of AD. Primary SS and SSc-CD patients were younger at AD diagnosis in comparison to non-celiac patients. Autoimmune thyroiditis was associated with pSS and CD. CD prevalence is clearly increased in pSS and diffuse SSc in comparison to the general population. The association of CD with diffuse but not limited SSc may suggest different immunopathogenic mechanisms characterizing the two subsets. CD screening may be considered in pSS and diffuse SSc in young patients, particularly at the time of diagnosis.

Classification of primary antiphospholipid syndrome as systemic lupus erythematosus: Analysis of a cohort of 214 patients.

OBJECTIVES: To assess the limitations of the SLICC (Systemic Lupus International Collaborating Clinics) classification criteria for systemic lupus erythematosus (SLE), in patients with primary antiphospholipid syndrome (PAPS). METHODS: Retrospective study of a cohort of APS patients (Sydney criteria). We successively excluded patients with (1) at least one "SLE-specific" manifestation (biopsy-proven SLE nephropathy, arthritis, cutaneous, or neurologic SLE manifestations, pericarditis, autoimmune haemolytic anaemia, oral and nasal ulcers, non-scarring alopecia, anti-dsDNA, and anti-Sm antibodies), (2) any other autoimmune connective tissue disease, and/or (3) antinuclear antibodies >1/320. Careful file review confirmed PAPS among the remaining patients. We then assessed the number of SLICC criteria each patient met. RESULTS: Among these 214 APS patients, we excluded 85 with at least one SLE-specific manifestation, 8 with another connective tissue disease, and 21 with antinuclear antibodies >1/320, leaving 100 patients with primary APS. Among them, 28% met at least 4 SLICC classification criteria including one clinical and one immunological criterion (antiphospholipid antibodies, aPL, by definition) and could thus theoretically be classified with SLE. Fourteen had an arterial phenotype (50%), 9 a history of catastrophic APS (32%), and 18 a triple-positive profile for aPL (64%). None had developed SLE during a median follow-up of 12 [6.5-17] years. CONCLUSION: Because 28% of our patients with longstanding and strictly defined PAPS could be mistakenly classified as SLE, they were at risk of deleterious therapeutic management. We therefore suggest that any future classification for SLE should specifically require at least one SLE-specific criterion for patients with aPL.

Longterm Outcome of Patients with Primary Antiphospholipid Syndrome: A Retrospective Multicenter Study.

OBJECTIVE: To assess the longterm frequency of thrombotic recurrences, obstetrical complications, organ damage, severe comorbidities, and evolution toward connective tissue disease (CTD) in primary antiphospholipid syndrome (PAPS). METHODS: Medical records of patients with PAPS followed in 6 centers for ≥ 15 years were retrospectively reviewed. RESULTS: One hundred fifteen patients were studied: 88% women, followed between 1983 and 2014 with a mean (± SD) age at diagnosis of 33 (± 10) years. During a median followup of 18 years (range 15-30), 50 patients (44%) had at least a thrombotic event for a total of 75 events and an annual incidence of 3.5%. Thromboses were more frequent in patients with previous thrombotic history (p = 0.002). A catastrophic antiphospholipid syndrome occurred in 6 patients (5%). The use of oral anticoagulants in patients with thrombotic onset did not appear to be protective against recurrences (p = 0.26). Fifty-two women had 87 pregnancies, successful in 78%. Twenty-nine percent of patients accrued functional damage. Damage was significantly associated with a thrombotic history (p = 0.004) and with arterial events (p < 0.001), especially stroke, but not with demographics, serology, or treatment. Twenty-four major bleeding episodes were recorded in 18 patients, all receiving anticoagulants. Severe infections affected 6 patients (5%), with 1 fatality. A solid cancer was diagnosed in 8 patients (7%). Altogether, 16 patients (14%) developed an autoimmune disease and 13 (11%) a full-blown picture of CTD. CONCLUSION: Despite therapy, a high proportion of patients experienced new thrombotic events and organ damage, while evolution toward CTD was infrequent.

The Clinical and Histological Spectrum of Idiopathic Inflammatory Myopathies.

Idiopathic inflammatory myopathies (IIMs) are a heterogeneous group of myositis, characterised by chronic muscle weakness, cutaneous features, different extra-muscular manifestations and circulating autoantibodies. IIMs included classical polymyositis (PM), dermatomyositis (DM) and other different types of myositis with a wide range of muscle involvement. A complete autoantibody profile and a muscle biopsy are mandatory to correctly diagnose different clinical entities and to define their different prognosis. Bohan and Peter's criteria included five items to diagnose adult onset PM and DM. The sensitivity was 74-100 %, while the specificity is low, due to a poor ability to differentiate PM from neuromuscular diseases. Other criteria included a more accurate histological definition of PM, DM or amyopathic DM, obtaining a higher specificity. Autoantibodies' association, interstitial lung disease and clinical cardiac involvement represent the main items that could define the prognosis of these patients. On the other hand, inclusion body myositis is a different myopathy characterised by a peculiar muscle mass involvement, muscle atrophy and progressive loss of function, due to complete failure to all immunosuppressive drugs used. Treatment of IIMs is based on corticosteroids (CS), which show rapid clinical response and functional improvement. Different immunosuppressant drugs are given to obtain a better control of the disease during CS tapering dose. No controlled double blind trials demonstrated the superiority of one immunesuppressant on another. The occurrence of interstitial lung involvement requires the immediate introduction of immunosuppressants in addiction to CS. Severe dysphagia seems to improve with intravenous immunoglobulins (Ig). Physical therapy could be started after the acute phase of diseases and seems to have a beneficial role in muscle strength recovery.

Calcinosis in poly-dermatomyositis: clinical and laboratory predictors and treatment options.

OBJECTIVES: We aimed to identify the possible clinical and laboratory predictors of calcinosis in a cohort of patients with a diagnosis of polymyositis (PM) and dermatomyositis (DM). METHODS: We carried out a retrospective analysis of a cohort of myositis patients attending our clinic between January 2013 and May 2014. RESULTS: 74 patients (58 females, 16 males) with PM (30 cases), DM (30 cases), overlap syndrome (13 cases) and inclusion body myositis (1 case) were enrolled. Sixteen patients (21.6%) had calcinosis that occurred a mean of 43.7 months after diagnosis of PDM. At multivariate analysis, patients with calcinosis experienced longer follow-up duration (p=0.006), anti-PM/Scl (p=0.033) and anti-NXP2 (p=0.024) positivity compared to patients without calcinosis. Furthermore, anti-NXP-2 positive C+ showed a diffuse form of calcinosis from the beginning and lower frequency of respiratory tract involvement. No single drug or associations of drugs was found effective in the treatment of calcinosis. CONCLUSIONS: A longer follow-up period of time, DM diagnosis and positivity for PM/Scl and NXP-2 could all be considered risk factors which foresee the development of calcinosis. Moreover, the positivity for antibodies to NXP-2 depicts a distinct phenotype of calcinosis with an early onset and quick widespread dissemination.

Myositis-specific autoantibodies and their association with malignancy in Italian patients with polymyositis and dermatomyositis.

This study aims to characterize myositis-specific antibodies in a well-defined cohort of patients with idiopathic inflammatory myopathy and to determine their association with cancer. Sera from 40 patients with polymyositis, dermatomyositis, and controls were tested by protein and RNA immunoprecipitation to detect autoantibodies, and immunoprecipitation-Western blot was used for anti-MJ/NXP-2, anti-MDA5, and anti-TIF1γ/α identification. Medical records were re-evaluated with specific focus on cancer. Anti-MJ/NXP-2 and anti-TIF1γ/α were the most common antibodies in dermatomyositis. In six dermatomyositis cases, we found five solid forms of cancer and one Hodgkin's lymphoma in long-term remission. Among patients with cancer-associated dermatomyositis, three were positive for anti-TIF1γ/α, two for anti-Mi-2, and one for anti-MJ/NXP-2. The strongest positivity of anti-TIF1γ was seen in two active forms of cancer, and this antibody was either negative or positive at low titers in the absence of cancer or in the 7-year remission Hodgkin's lymphoma. Four out of twenty (20 %) patients with polymyositis had solid cancer, but no specific association with autoantibodies was identified; further, none of the four cases of antisynthetase syndrome had a history of cancer. No serum myositis-associated autoantibody was observed in control sera, resulting in positive predictive value 75 %, negative predictive value 78.5 %, sensitivity 50 %, specificity 92 %, and area under the ROC curve 0.7083 for the risk of paraneoplastic DM in anti-TIF1γ/α (+) patients. Myositis-specific autoantibodies can be identified thanks to the use of immunoprecipitation, and their association with cancer is particularly clear for anti-TIF1γ/α in dermatomyositis. This association should be evaluated in a prospective study by immunoprecipitation in clinical practice.

Anti-HMGCR antibodies as a biomarker for immune-mediated necrotizing myopathies: A history of statins and experience from a large international multi-center study.

In an effort to find naturally occurring substances that reduce cholesterol by inhibiting 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR), statins were first discovered by Endo in 1972. With the widespread prescription and use of statins to decrease morbidity from myocardial infarction and stroke, it was noted that approximately 5% of all statin users experienced muscle pain and weakness during treatment. In a smaller proportion of patients, the myopathy progressed to severe morbidity marked by proximal weakness and severe muscle wasting. Remarkably, Mammen and colleagues were the first to discover that the molecular target of statins, 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR), is an autoantibody target in patients that develop an immune-mediated necrotizing myopathy (IMNM). These observations have been confirmed in a number of studies but, until today, a multi-center, international study of IMNM, related idiopathic inflammatory myopathies (IIM), other auto-inflammatory conditions and controls has not been published. Accordingly, an international, multi-center study investigated the utility of anti-HMGCR antibodies in the diagnosis of statin-associated IMNM in comparison to different forms of IIM and controls. This study included samples from patients with different forms of IIM (n=1250) and patients with other diseases (n=656) that were collected from twelve sites and tested for anti-HMGCR antibodies by ELISA. This study confirmed that anti-HMGCR autoantibodies, when found in conjunction with statin use, characterize a subset of IIM who are older and have necrosis on muscle biopsy. Taken together, the data to date indicates that testing for anti-HMGCR antibodies is important in the differential diagnosis of IIM and might be considered for future classification criteria.

Testing for myositis specific autoantibodies: Comparison between line blot and immunoprecipitation assays in 57 myositis sera.

OBJECTIVE: To analyze the performance of a line blot assay for the identification of autoantibodies in sera of patients affected by myositis, compared with immunoprecipitation (IP) as gold standard. METHODS: 66 sera of patients with myositis (23 polymyositis, 8 anti-synthetase syndromes, 29 dermatomyositis and 6 overlap syndromes) were tested by commercial LB (Euroimmun, Lubeck, Germany); 57 sera were analyzed also by IP of K562 cell extract radiolabeled with (35)S-methionine. Inter-rater agreement was calculated with Cohen's k coefficient. RESULTS: Myositis-specific antibodies (MSA) were detected in 36/57 sera (63%) by IP and in 39/66 sera (59%) by LB. The most frequent MSA found by LB were anti-Jo1 and anti-Mi2 found in 15% (10/66) of sera, followed by anti-NXP2 and anti-SRP detected in 106% (7/66) of sera. Anti-TIF1gamma and anti-MDA5 were found in 6 (9%) and 5 sera (7.6%), respectively. A good agreement between methods was found only for anti-TIF1γ, anti-MDA5 and anti-NXP-2 antibodies, while a moderate agreement was estimated for anti-Mi2 and anti-EJ. By contrast, a high discordance rate for the detection of anti-Jo1 antibodies was evident (k: 0.3). Multiple positivity for MSA were found in 11/66 (17%) by LB and 0/57 by IP (p: 0001). Comparing the clinical features of these 11 sera, we found total discrepancies between assays in 3 sera (27.3%), a relative discrepancy due to the occurrence of one discordant autoantibody (not confirmed by IP) in 5 cases (45.5%) and a total discrepancy between LB and IP results, but with a relative concordance with clinical features were found in other 3 sera (27.3%). The semiquantitative results do not support the interpretation of the data. CONCLUSIONS: The use of LB assay allowed the detection of new MSA, such as anti-MDA5, anti-MJ and anti-TIF1gamma antibodies, previously not found with routine methods. However, the high prevalence of multiple positivities and the high discondant rate of anti-Jo1 antibodies could create some misinterpretation of the results from the clinical point of view. These data should be confirmed by enlarging the number of myositis cases.

Treatment and functional outcome of patients with cystoid macular edema: a single-center experience.

The aim of this study was to describe a single-center experience in the treatment and follow-up of cystoid macular edema patients. Clinical records of all patients with cystoid macular edema followed up in the Rheumatologic and Ophthalmological Unit of our center between 1993 and 2013 were retrospectively evaluated. The outcome was assessed by visual acuity and optical coherence tomography status during follow-up. Comparisons were made by Fisher's exact test (p < 0.05 significant). In this study 16 eyes in 9 patients were analyzed. Our study includes mainly post-uveitic (78 %) cases with a high prevalence of human leukocyte antigen B51 (67 %). Systemic immunosuppressive therapy was prescribed in 87 % of cases. The most frequently used drugs were cyclosporine, interferon-α, and infliximab. The first two molecules appeared respectively the most used as the first option and the one with the longest survival on treatment. Interferon-α was the most effective drug in contrasting visual acuity loss compared to the majority of drugs, but significantly more effective than mycophenolate (p = 0.01) in reducing macular edema. At the end of follow-up, 50 % of patients showed a significant visual loss, while 88 % did not present macular edema. In our small cohort, interferon-α is the most promising drug in contrasting visual acuity loss in cystoid macular edema. Visual prognosis remains severe in these patients.