V. S. T. Viana

Ovarian reserve in adult patients with childhood-onset lupus: a possible deleterious effect of methotrexate?

Objectives: To assess ovarian reserve markers and anti-corpus luteum antibodies (anti-CoL) in adult patients with childhood-onset systemic lupus erythematosus (c-SLE). Method: Fifty-seven adult c-SLE female patients and 21 healthy controls were evaluated for anti-CoL. Ovarian reserve was assessed by: follicle stimulating hormone (FSH), luteinizing hormone (LH), oestradiol, anti-Müllerian hormone (AMH), and antral follicle count (AFC). Demographic data, menstrual abnormalities, disease activity, damage, and treatment were also analysed. Results: The median current age was similar in adult c-SLE patients and controls (27.7 vs. 27.7 years, p = 0.414). The medians of AMH (1.1 vs. 1.5 ng/mL, p = 0.037) and AFC (6 vs. 16, p < 0.001) were significantly reduced in SLE patients compared to controls without significant menstrual abnormalities. Anti-CoL were solely observed in c-SLE patients (16% vs. 0%, p = 0.103) and were not associated with demographic data, ovarian reserve parameters, disease activity/damage, and treatment. Further evaluation of c-SLE patients treated with cyclophosphamide revealed a higher median of FSH levels compared to c-SLE patients not treated with cyclophosphamide and controls (8.8 vs. 5.7 vs. 5.6 IU/L, p = 0.032) and lower median AMH (0.4 vs. 1.5 vs. 1.5 ng/mL, p = 0.004) and AFC (4.0 vs. 6.5 vs. 16 IU/L, p = 0.001) levels. Nineteen patients treated exclusively with methotrexate demonstrated a negative correlation between the cumulative dose and AMH levels (p = 0.027, r = –0.507). Conclusions: The present study demonstrated for the first time that a high cumulative methotrexate dose is a possible cause of subclinical ovarian dysfunction in adult c-SLE patients. Further studies are required to confirm this deleterious effect in other rheumatic diseases, particularly juvenile idiopathic arthritis and idiopathic inflammatory myopathy.

Podocyte injury in pure membranous and proliferative lupus nephritis: distinct underlying mechanisms of proteinuria?

Proteinuria is a major feature of lupus nephritis (LN) and reflects podocyte injury. Analysis of podocyte biomarkers was performed attempting to identify if podocyte phenotype is distinct in pure membranous and proliferative LN. Expression of synaptopodin, Wilms tumor protein 1 (WT1), glomerular epithelial protein 1 (GLEPP1) and nephrin was evaluated in 52 LN biopsies by immunohistochemistry. Preserved synaptopodin expression was observed in only 10 (19.2%) of all biopsies while 42 (80.8%) had reduced expression. Both groups had comparable proteinuria at the time of biopsy (p = 0.22); however, in the mean follow-up of four years there was a tendency toward lower mean levels of proteinuria in patients with preserved synaptopodin staining (0.26±0.23 vs. 0.84±0.90 g/24 h, p = 0.05) compared with those with diminished expression. Thirty-nine (75%) biopsies were classified as proliferative and 13 (25%) as pure membranous. Comparison of podocyte biomarkers demonstrated a predominance of preserved staining of synaptopodin (69.2%), WT1 (69.2%), GLEPP1 (53.9%) and nephrin (60%) in the pure membranous group whereas only <10% of the proliferative showed preserved expression. Our data suggest that in proliferative forms there seems to occur structural podocyte damage, whereas in the pure membranous the predominant preserved pattern suggests a dysfunctional podocyte lesion that may account for the better long-term prognosis of proteinuria outcome.

C-ANCA-positive IgG fraction from patients with Wegener's granulomatosis induces lung vasculitis in rats

The aim of the present study was to analyse in rats the ability of C‐ANCA‐positive IgG fraction in triggering inflammatory response on pulmonary tissue. Wistar rats (n = 18) were injected via the the internal jugular vein with 20 mg of total C‐ANCA‐positive IgG fraction isolated from serum of three different Wegeners granulomatosis patients obtained before therapy. Similarly, control rats were treated with IgG fraction from two rheumatoid arthritis patients (n = 7), IgG from six normal human sera (n = 15) or saline (n = 18), respectively. Animals were sacrificed after 24h of injection for histological analysis of the lungs. Vasculitis and inflammatory infiltrate were consistently absent in rats injected with rheumatoid arthritis IgG or saline and in 14/15 of normal IgG treated animals. In contrast, marked vasculitis was observed in all 18 animals injected with C‐ANCA‐positive IgG fraction. The histological features were characterized by the presence of a perivascular pleomorphic cellular sheath, particularly around small vessels, endothelial adherence and diapedesis of polymorphonuclear leucocytes and presence of granuloma‐like lesions. A dose–response relationship was observed between protein concentration of C‐ANCA IgG sample and the intensity of the inflammatory response in the animals. In addition, IgG fraction with undetectable C‐ANCA, obtained from one patient in remission after treatment, was not able to reproduce the pulmonary tissue alterations induced by its paired IgG that was positive for C‐ANCA taken before therapy. The experimental model described herein may be useful to characterize more effectively the pathogenic mechanism of C‐ANCA in Wegeners disease.

Electrophysiological dysfunction induced by anti-ribosomal P protein antibodies injection into the lateral ventricle of the rat brain

Objective Anti-ribosomal P antibodies (anti-P) are strongly associated with neuropsychiatric lupus. This study was designed to determine whether these antibodies are capable of causing electro-oscillogram (EOSG) and behavior alterations in rats. Methods IgG fraction anti-P positive and affinity-purified anti-P antibodies were injected intraventricularly in rats. Sequential cortical and subcortical EOSGs were analyzed during 30 days. IgG anti-Ro/SS-A and normal IgG were used as controls. Results All 13 animals injected with IgG anti-P demonstrated a high prevalence of polyspikes, diffusely distributed in hippocampal fields and cerebral cortex. These abnormalities persisted approximately a month. Remarkably, an identical electrical disturbance was observed with the inoculation of affinity-purified anti-P antibodies. The EOSG alterations were associated with behavioral disorders with varying degrees of severity in every animal injected with anti-P. In contrast, no changes in EOSG or behavioral disturbances were observed in the control group. Conclusion Our study indicates that anti-P antibodies can directly induce electrophysiological dysfunction in central nervous system particularly in hippocampus and cortex associated with behavior disturbances.

FRI0493 Clinical and Serological Comparative Analysis of Systemic Sclerosis with or without Overlap Syndromes in A Large Brazilian Cohort

Background There are scarce data comparing clinical and serological features in patients with systemic sclerosis (SSc) and SSc overlap syndromes (SSc-OS) due to the rarity of these associations. Objectives To analyze clinical and SSc-associated serological profiles including a panel of novel described antinucleolar antibodies (ANoA) in a large cohort of Brazilian patients with SSc and SSc overlap syndromes. Methods Three-hundred twenty-eight SSc patients classified according to the ACR/EULAR 2013 SSc criteria, attended at the Scleroderma Outpatient Clinic of a tertiary referral university hospital from 2000 to 2011 were enrolled in the study; 32% had diffuse cutaneous SSc and 68% limited cutaneous SSc. Clinical and demographic data were obtained from an electronic register database. Serum samples were analyzed for the presence of antinuclear antibodies (indirect immunofluorescence on HEp-2 cells), antibodies to Scl-70, PM-Scl, RNA-Pol III, CENP-A/CENP-B, and Ro/SS-A (52 and 60 kDa) (ELISA), and antinucleolar antibodies (ANoA) fibrillarin, Ku, Th/To and NOR90 (line blot immunoassay) using commercial available standardized kits. Results Two-hundred sixty eigth patients were classified as SSc and sixty patients as SSc-OS (14 with systemic lupus erythematosus, 11 with polymyositis, 7 with rheumatoid arthritis, and 35 with Sjogren Syndrome, isolated or combined). Both groups (SSc-O and SSc) were similar regarding mean age at onset, female predominance, disease duration and ethnicity (p>0.05). Concerning clinical features, there were no significant differences related to the occurrence of pitting scars, digital amputation, calcinosis, telangiectasia, interstitial lung disease, pulmonary hypertension, cardiovascular disease, renal crisis (p>0.05). Conversely, SSc-OS patients had lower mean modified Rodnan skin score (5.6±4.24 vs. 10±11.7, p<0,001),but higher percentage of joint (33 vs. 21%, p=0.04) and muscle involvement (22 vs. 8%, p=0.003) compared to the SSc group. There was no difference regarding the frequency of all autoantibodies tested in the two groups (p>0.05), except for the negative association among anti-RNA-Pol III with SSc-OS (p=0.011). Conclusions This study identified a comparable occurrence of major organ involvement and ischemic lesions in SSc-OS and SSc. This finding is strengthened by the observation of a similar frequency of all scleroderma autoantibodies in both groups and suggests that SSc determines the clinical manifestation in overlap syndromes in spite of a less extensive skin involvement in these patients. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.5863

FRI0461 Subclinical Ovarian Dysfunction in Takayasu Arteritis Patients

Background Takayasu arteritis (TA) is a rare idiopathic systemic chronic vasculitis that involves large arteries. This disease occurs mainly in female gender during the reproductive age and ovarian reserve and future fertility are major topic of interest. Aging, surgery and hypothalamic-pituitary-gonad axis dysfunction have been found to influence the quantity and quality of primordial follicles in ovaries and, ultimately, the ovarian reserve. Other conditions such as autoimmune oophoritis and immunosuppressive drugs, particularly cyclophosphamide, may also result in diminished ovarian reserve. Female TA patients are susceptible to these factors but there is no systematic study assessing this ovary abnormality in these patients. Objectives To assess ovarian reserve markers and anti-corpus luteum antibodies (anti-CoL) in TA patients. Methods We have screened 52 consecutive female patients with TA. All patients aged between 18 and 45 years and fulfilled the American College of Rheumatology classification criteria. Exclusion criteria were: current pregnancy, hypothalamus-pituitary-gonadal axis dysfunction, use of hormonal contraceptive in the last six months, gynecological surgery, gynecological cancer, presence of an additional autoimmune disease, did not agree to participate in this study and incomplete gonadal evaluation. Thirty-two were excluded: use of hormonal contraceptive (n=15), did not agree to participate (n=7), incomplete assessment (n=4), presence of granulomatosis with polyangiitis (n=2), current pregnancy (n=2), hyperprolactinemia (n=1) and current treatment for gynecological cancer (n=1). Therefore, a cross sectional study was conducted in 20 patients with TA and 24 healthy controls according to the same exclusion criteria. Anti-CoL (immunoblot) and ovarian reserve were assessed by: follicle stimulating hormone (FSH), luteinizing hormone (LH), estradiol, anti-Müllerian hormone (AMH), and antral follicle count (AFC). Demographical data, menstrual abnormalities, disease parameters and treatment were also analyzed. Results The median current age was similar in TA patients and controls (31.2±6.1 vs. 30.4±6.9 years, p=0.69). The frequency of decreased levels of AMH (50% vs. 17%, p=0.02) and the median of AMH (0.7 vs. 2.7ng/mL, p=0.008) were significantly reduced in TA patients compared to controls without significant menstrual abnormalities with regard to the median of flow duration (p=0.25) and cycle length (p=0.85). The other hormones and AFC were similar to controls (p>0.05). Anti-CoL was solely observed in TA patients (5% vs. 0%, p=0.45). Further evaluation of TA patients with low AMH levels (<1.0 ng/mL) versus normal AMH levels (>1.0 ng/mL) revealed that the frequency of disease activity (p=1.0) and the median of ESR (p=0.6), CRP (p=0.4), prednisone cumulative dose (p=0.8) and methotrexate cumulative dose (p=0.8) were comparable in both groups. Cyclophosphamide use was reported in only one patient with reduced ovarian reserve, whereas none of the remaining patients received gonadotoxic drug. Conclusions Therefore, the present study demonstrated for the first time a high prevalence of diminished ovarian reserve in TA patients reinforcing fertility counseling. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.1484

AB0637 Systemic Sclerosis-Related Auto-Antibodies Are Markers of New Clinical Associations in A Cohort of 328 Brazilian Patients

Background Systemic Sclerosis (SSc) shows a heterogeneous clinical presentation, characterized by marked skin and internal organ fibrosis and vascular dysfunction, associated with immunological abnormalities. A varied panel of SSc-related auto-antibodies has been described, and there is a growing interest to establish their prevalence and clinical associations in populations of different ethnicities. Objectives To evaluate the frequency and the putative associations of a panel of SSc-related auto-antibodies with demographic and clinical features in a large SSc cohort. Methods We analyzed serum of 328 consecutive SSc adult patients attended at the Scleroderma Outpatient Clinic of a tertiary referral university hospital in Brazil, classified according to the ACR/EULAR 2013 SSc criteria: 32% had diffuse cutaneous SSc and 68% limited cutaneous SSc. Clinical and demographic data were obtained through a review of the electronic register database. Serum samples were analyzed for the presence of autoantibodies using commercially available standardized kits. Results ANA positivity was 88%, in the following patterns: centromeric (26%), nuclear homogeneous/nucleolar (18%), nucleolar (16%), nuclear homogeneous (12%), nuclear speckled (12%), dense fine speckled (2%), nucleolar speckled (<1%), and cytoplasmatic (1%). Anti-Scl70 was present in 92 (28%), anticentromere (ACA) in 83 (25%), anti-fibrillarin in 39 (12%), anti-RNA Pol III in 23 (7%), anti-Th/To in 17 (5%), anti-PM-Scl in 16 (5%), anti-Ku in 12 (4%), and anti-NOR in 6 (2%), and anti-Ro/SSA was positive in 96 patients (29%). Anti-Scl70 was associated with the nuclear homogeneous/nucleolar and nuclear homogeneous patterns, while symptomatic ILD was associated to both ANA patterns (p<0.001 and 0.005), digital ulcers were associated with the nuclear homogeneous/nucleolar patterns (p=0,042). ACA was associated with female gender (p<0.001), white ethnicity (trend; p=0.076), limited cutaneous SSc (p<0.001), calcinosis (p=0.002), asymptomatic ILD (p<0.001), and isolated PAH (p<0.001). Otherwise, anti-Scl70 was associated with male gender (p=0.034), diffuse cutaneous SSc (p<0.001), digital ulcers (p<0.001), symptomatic ILD (p<0.001), and use of immunosuppressive drugs (cyclophosphamide, azathioprine, mycophenolate mofetil (p<0.001). Three patients were diagnosed as scleroderma renal crisis and two of then presented anti-RNA pol III, that was associated with diffuse cutaneous SSc (p=0.011). Anti-fibrillarin was associated with symptomatic ILD (trend; p=0.072) and heart involvement (p=0,043). Anti-Ku was associated with finger amputation (p=0.019). Anti-PM-Scl, anti-Th/To and anti-NOR90 did not show any specific association. Conclusions Among the different SSc-related auto-antibodies, anti-Scl70 and ACA clearly characterize distinct demographic and clinical presentations. Our study also identified new clinical associations, among anti-fibrillarin and heart involvement and anti-Ku with digital amputation, as well as the concomitance of anti-Ro/SSA with anti-Scl70 being associated with poor prognosis. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.5641

PReS-FINAL-2289: Ovarian dysfunction in adult childhood-onset lupus patients: a possible role of methotrexate?

Reduction of ovarian reserve has been observed in childhood-onset SLE (c-SLE) and adult SLE populations, and most of them were limited to follicle stimulating hormone (FSH) levels and few recent reports included antral follicle count (AFC) and/or anti-Mullerian hormone (AMH) levels. In addition, the contribution of diminished follicle ovarian pool using anti-corpus luteum antibodies (anti-CoL) was not available in pediatric lupus population.