Background Lupus nephritis (LN) is one of the most severe forms of Systemic Lupus Erythematosus (SLE). Given that the kidney is the main site of inflammation in LN, biomarkers in urine may reflect this inflammation more closely than those in the blood. Many groups have studied messenger RNA (mRNA) expression of urinary biomarkers in patients with SLE, investigating which is likely to be most helpful for monitoring renal disease activity. Endostatin (END) is a natural proteolytic fragment of collagen XVIII and has been known to have modulatory function in angiogenesis and inflammation. In contrast to the general angiogenic factors, the expression profiles and activity of angiogenic inhibitors like END in LN are not well defined. Objectives We proposed to study gene expression level of END in urine from LN patients. Methods A total of 45 patients, 36 with renal involvement and 9 non-renal were included. Urine samples from active patients were divided according Protein Creatinin ratio (P/C) as: Group 1, P/C <1 (n=18) and Group II, P/C>1 (n=18), and non renal patients: Group III (n=9). Levels of gene expression of END were measured using Quantitative Real Time PCR (QPCR). All amplifications were carried out in duplicate and threshold cycle (Ct) scores were averaged for calculations of relative expression values. The Ct scores were normalized by subtracting the corresponding β2Microglobuline (β2M) control, or ΔCt=Ct,gene − Ct,B2M. To test for differential gene expression between groups a variance analysis (ANOVA) and t test was performed. Results ΔCt is inversely proportional to the gene expression level. Urinary END was significantly decreased in active renal SLE patients compared with no-renal SLE patients (Table 1). Among active renal patients, END gene expression was elevated in Group I compared with Group II (test t, p=0.0192). Table 1. Levels of END gene expression between groups (ANOVA, p=0.0327) END (mean ΔCt) SLE patients p=0.0327 Active renal No-renal Group I (P/C <1) Group II (P/C >1) Group III 6,521 9,572 5,414 Conclusions Urinary END had the capacity to discriminate patients with active renal SLE from those with no-renal disease and exhibited the lowest urinary level in patients with P/C >1. This study provides evidence that measuring urinary END could be an important new biomarker in patients with SLE without renal activity. References Angiogenesis and hypoxia in the kidney. Tanaka T et al. Nat. Rev. Nephrol. 9, 211-222 (2013). Can measuring urinary biomarkers improve the management of Lupus Nephritis? Rahman A. Arthritis Research & Theraphy. 14, 127 (2012). Disclosure of Interest : None declared DOI 10.1136/annrheumdis-2014-eular.5600

Background The B Lymphocyte Stimulator (BLyS) and A PRoliferation-Inducing Ligand (APRIL) signaling pathway has an important role in the selection, maturation and survival of B cells. Dysregulation of BLyS/APRIL is involved in the pathogenesis of B-cell related autoimmune diseases including Rheumatoid Arthritis (RA). Synovial fluid cells are highly activated in patients with active RA inducing lymphocyte proliferation, expression of cell surface molecules, cytokine and auto antibody secretion. Objectives The purpose of this work is to evaluate Synovial Fluid (SF) and Peripheral Blood (PB) mRNA expression of BLyS and APRIL in RA patients. Methods SF and PB were obtained and classified in two groups, Group I: active RA patients with DAS 28 score >5.1 (n=11, 8F/3M, age: 56,2±20,9; range: 17-84) and Group II, control: Osteoarthritis (OA, n=20, 13F/7M, age: 69,8±9,5, range: 47-84). Levels of BLyS and APRIL expression were evaluated using Quantitative Real Time PCR (QPCR). All amplifications were carried out in duplicate and threshold cycle (Ct) scores were averaged for calculations of relative expression values. The Ct scores were normalized against Ct scores by subtracting the corresponding β2Microglobuline (β2M) control, or ΔCt=Ct,gene- Ct,B2M. To test for differential gene expression between groups, a two sample t-test was performed to compare the DCt in the two groups. Results BLyS and APRIL gene expression is shown in Table 1. Table 1.Levels of BLyS and APRIL gene expression in the two groups Group BLyS (Mean ΔCt) p value APRIL (Mean ΔCt) p value SF Group I 5,883 p=0,002 7,195 p=0,024 SF Group II 7,878 9,829 PB Group I 9,315 p=0,830 9,119 p=0,514 PB Group II 8,926 8,549 ΔCt is inversely proportional to the gene expression level. Analysis of PB showed no significant difference in gene expression between RA and OA. In SF, we observed a significant difference for BLyS and APRIL expression between Group I vs. Group II (p=0,002 and p=0,024 respectively). After t test, we evaluated data from $Δ $Ct analysis observing that in SF, mRNA of BLyS and APRIL in Group I was higher than those from Group II. Conclusions Gene expression of BLyS/APRIL in PB does not correlate with expression in SF. Increased BLyS and APRIL expression in active RA SF can be linked to B cell activation and maintenance in RA synovium. References Moura RA, et al. BAFF and TACI gene expression are increased in patients with untreated very early Rheumatoid Arthritis. The journal of Rheumatology 2013. La Cava A. Targeting the BLyS-APRIL signaling pathway in SLE. Clin Immunol 2013. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.2971

BackgroundLupus nephritis (LN) is a severe complication of Systemic Lupus Erythematosus (SLE). Non-invasive biomarkers are needed for diagnosis of LN and to identify patients at risk of a renal flare (1). Thus the presence of biomarkers associated with inflammation, tissue damage or cell activation in the urine of patients with LN may be a useful tool in the evaluation of LN patients.The glomerular filtration rate (GFR) is considered the best overall index of renal function in health and disease. Because GFR is difficult to measure in clinical practice, most clinicians estimate the GFR (eGFR) from the serum creatinine concentration (2).B Lymphocyte Stimulator (BLyS) is a cytokine that fosters B cell activation, antibody production, B cell - T cell interaction and plasma cell survival. These events have been demonstrated to play a role in patients with LN (3).ObjectivesWe evaluated urinary levels of BLyS as biomarker for LN and their relationship with eGFR.MethodsUrine samples (n=86) were obtained from LN patients and classified in two groups: patients with eGFR >60 (GFRhigh, n=68, 62F/6M, age: 34.07±13.24) and patients with eGFR ≤60 (GFRlow, n=18, 14F/4M, age: 35.22±13.76). RNA from urine samples was isolated using TRIzol-Chloroform technique and then reverse-transcribed using random primers. Levels of BLyS expression were evaluated using Quantitative Real Time PCR (QPCR). All amplifications were carried out in duplicate and threshold cycle (Ct) scores were averaged for calculations of relative expression values. The Ct scores were normalized against Ct scores by subtracting the corresponding β2Microglobuline (β2M) control, or DCt=Ct,gene- Ct,B2M.To test for differential gene expression between groups, a two sample t-test was performed to compare the DCt in the two groups.ResultsDCt is inversely proportional to BLyS's expression. We evaluated data from ΔCt analysis observing that mRNA levels of BLyS in eGFRlow (6.193±1.787) were higher than those from eGFRhigh (7.564±2.326), with a statistically significant difference between groups (p=0.0288).eGFRloweGFRhigh BLyS (ΔCt)6.193±1.7877.564±2.326p=0.0288ConclusionsIn the present cross-sectional study, increased levels of BLyS were observed in patients with eGFR ≤60. These gene expression results might be linked to B cell activation and proliferation in kidney and thus in urine samples. Combination of eGFR and BLyS appears to be a good biomarker.References Rahman A. Can measuring urinary biomarkers improve the management of lupus nephritis? Arthritis Research & Therapy 2012, 14:127.Levey AS, Bosch JP, Lewis JB, Greene T, Rogers N, Roth D. A more accurate method to estimate glomerular filtration rate from serum creatinine: a new prediction equation. Modification of Diet in Renal Disease Study Group. Ann Intern Med. 1999 Mar 16; 130 (6):461–70.Phatak S, Chaurasia S, Mishra SK, Gupta R, Agrawal V, Aggarwal A, Misra R. Urinary B cell activating factor (BAFF) and a proliferation inducing ligand (APRIL): potential biomarkers of active lupus nephritis. Clin Exp Immunol. 2016....

BackgroundLupus Nephritis (LN) is one of the most frequent manifestations of SLE and can be present in 60% of SLE patients. It has been shown that structural changes and inflammatory infiltrate associated with LN contribute to a hypoxic state which induces angiogenesis [1]. Angiogenesis is a complex process that requires interaction between different cell types, the extracellular matrix, several cytokines and growth factors.ObjectivesWe proposed to study gene expression levels of angiogenic factors such as VEGF-A (Vascular Endothelial Growth Factor), VCAM-1 (Vascular cell adhesion molecule 1), TGF-β (Transforming Growth Factor), SEL-E (Selectin-E), ANGPT1 (Angiopoietin 1), AGT (Angiotensinogen) and END (Endostatin) in kidney biopsies from LN patients and their relationship with LN severity.MethodsThirty two kidney biopsies samples were obtained from 32 LN patients and then classified according to ISN/RPS scoring system [3] in two groups, Class I/Class II biopsies (n=11, 10F/1M, age: 34.64±15.10, range: 21-72) and Class IV biopsies (n=21, 16F/5M, age: 30.95±11.36, range: 17-64). RNA was isolated using TRIzol-Chloroform technique and then was reverse-transcribed using random primers.Gene expression level of pro-angiogenic factors: VEGF, VCAM-1, TGF-β, SEL-E, ANGPT-1 and anti-angiogenic factors: AGT and END were evaluated using Quantitative Real Time PCR (QPCR). The threshold cycle (Ct) scores were averaged for calculations of relative expression values. The Ct scores were normalized against Ct scores by subtracting β2Microglobuline (β2M) control, or ΔCt=Ct,gene- Ct,B2M. To test for differential gene expression between groups, a two sample T-test was performed to compare the ΔCt in the two groups.ResultsΔCt is inversely proportional to the gene expression level. Significant differences between groups were found in pro-angiogenic genes VEGF-A (p=0,05), VCAM-1 (p=0,05) and in anti-angiogenic gene END (p=0,05) (Table 1). There were not statistically significant differences in the expression of AGT, ANGPT-1, SEL-E and TGF-β between groups. After T test, we evaluated data from ΔCt analysis observing that the levels of mRNA of VEGF-A and VCAM-1 in Class I/II group were higher than those from Class IV group. Conversely, END gene expression was decreased in Class I/II group.Table 1.Gene expression levels of angiogenic factors between groupsGeneClass I/IIClass IVp valueVEGF-A1,68±1,723,13±2,020,05VCAM-15,62±3,458,09±2,950,05TGF-β6,36±1,806,58±1,570,73SEL-E14,24±3,4415,63±4,640,45ANGPT-19,64±2,7010,53±2,120,50AGT4,48±1,854,45±1,010,92END6,48±6,133,19±3,210,05ConclusionsIn the present cross-sectional study, increased levels of VEGF-A and VCAM-1 and decreased levels of END were observed in biopsies classified as Class I and II. These findings could be associated with angiogenesis process in early stages of the disease. Angiogenesis in progression to LN deserves further evaluation.ReferencesFeliers D. Vascular Endothelial Growth Factor as a prognostic marker of Lupus Nephritis. Kidney Int; 75:1251-1253. 2009.Weening JJ. et al. Th...

Background: Gastrointestinal manifestations are frequent in patients with common variable immunodeficiency (CVID), and present with celiac-like features in a subgroup of these patients. On the other hand diagnosing bona fide/authentic celiac disease (CD) in CVID is challenging, as serology and small intestine histopathology cannot reliably discriminate between true CD and a celiac-like disease phenotype in these individuals. In CD a special HLA haplotype involving the loci DQA1* and DQB1* and encoding two different HLA DQ heterodimers is the prerequisite for disease development. Objectives: We aim to determine the frequency of these haplotypes in CVID patients with suspected CD. Furthermore, we report on autoimmune manifestations and the lymphocyte phenotype in these patients. Methods: By retrospective analysis data on gastrointestinal symptoms, concurrent autoimmune diseases, and routine laboratory values were collected. CVID patients were classified by phenotyping of their peripheral B-lymphocytes. HLA-DQA1* and HLA-DQB1* genes were analyzed to determine the frequency of CD associated heterodimers. Results: 20 out of 250 CVID patients presented with a clinical phenotype resembling celiac disease. 5 (25%) out of these CVID patients carried the CDassociated HLA DQ2.5 or DQ8 heterodimer, while HLA DQ2.5 was present in 100% of a CD control cohort. Gluten-free diet (GFD) resulted in a clinical and histological response in 2 out of 5 patients with HLA high-risk alleles for CD. The response could not be assessed in the remaining 3 patients, as these patients did not adhere sufficiently long to GFD. The percentage of autoimmune manifestations other then CD was high (50%) in CVID patients presenting with a CD-like enteropathy, and most of these patients had an expansion of B-cells with low expression of CD21 (CD21low B-cells). Conclusions: In CVID patients with suspected celiac disease typing of the HLA loci DQA1 and DQB1 can help to identify those that have a genetic susceptibility for CD. In CVID patients with a celiac-like phenotype but negativity for the HLA-DQ markers predisposing to CD, an alternative pathogenesis of gastrointestinal manifestations should be favoured. This has important implications for further diagnostics and therapy of these patients.