Rheumatology

PB0008: Serum IL-36γ levels are elevated in enthesitis related arthritis category of juvenile idiopathic arthitis

Sanjukta Majumder, Amita Aggarwal; Department of Clinical Immunology and Rheumatology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India

Background: IL-36, is a newly identified cytokine belonging to the IL-1 family and have been shown to be increased in serum and local tissue in Spondyloarthropathies (SpA) like psoriasis and IBD. IL-36R antagonist is being tried in psoriasis. JIA-ERA resembles SpA however, no data is available in JIA-ERA regarding IL-36.

Objective: To identify role of IL-36 in JIA-ERA by studying PBMC, serum and synovial fluid (SF) levels of IL-36.

Methods: JIA-ERA patients (ILAR criteria) were enrolled in the study. Forty-three SF samples and 37 paired serum samples from patients and 16 serum samples from healthy subjects were collected after obtaining informed consent. The study was approved by ethics committee. IL-36α and IL-36γ was measured by ELISA (R&D Systems) following manufacturers recommendations. In PBMCs of 11 patients mRNA levels of IL-36 were also measured. All Results: are denoted as Mean±SD.

Results: Among 43 patients 41 were boys. Their mean age was 15.7 ± 2.8 years and the mean duration of disease was 43.4 ± 37.8 months. All had active disease with mean ESR of 51.2 ± 32.9 mm. Out of 16 healthy control serum IL-36γ was detectable in 1 person while it was present in 15 JIA-ERA patients. The serum levels were higher in patients 64.51 ± 153.1 pg/μL as compared to controls (p<0.05). Among SF it was present in 19 of 43 samples and the mean level was 84.71 ± 271.21 pg/μL. In contrast IL-36α was detectable in only 1 patient's serum and synovial fluid and none in healthy control serum.

Real time qPCR analysis was done on 11 patients with JIA-ERA. Patient PBMCs had higher mRNA fold difference as compared to HC: IL-36αà2.9 ± 5.9 and IL-36γà9.54 ± 14.4 folds.

Conclusion: IL-36 may have a role in inflammation seen in JIA-ERA but its exact role needs to be studied.

PB0009: Circulatory histidine levels for evaluating disease activity in patients with takayasu arteritis: A targeted NMR based serum metabolomics study

Pankti Mehta, Umesh Kumar, Avinash Jain, Anupam Guleria, Durga P Misra, Ramnath Misra, Dinesh Kumar; Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India

Background: Objective assessment of disease activity is important for management in Takayasu arteritis (TA). The dominance of oxidative stress is the hallmark of active inflammation. Both preclinical and clinical data suggests that histidine has strong anti-oxidative and anti-inflammatory effect. Thus, we hypothesized that circulatory Histidine may serve as a biomarker of activity in TA.

Objective: To perform estimation of circulatory levels of histidine and evaluate its potential in diagnostic screening of active and inactive TA patients.

Methods: Serum samples were collected from 98 TA patients fulfilling American College of Rheumatology (ACR) criteria and 77 normal controls (NC). The 1D 1H CPMG NMR spectra was recorded on each serum sample at 800 MHz NMR spectrometer. Concentrations of Histidine were estimated (with formate as an internal reference) using NMR Suite of software program CHENOMX.

Results: According to Indian Takayasu Clinical Activity Score (ITAS) combined with acute phase reactant–erythrocyte sedimentation rate [ITAS-A (ESR)], 45 patients (46%) were clinically active, whereas 53 patients (54%) patients were inactive. Circulating levels of histidine were significantly decreased in active TA patients compared to both inactive TA patients and NC, whereas, there was no statistically significant difference between Inactive TA and NC. Further, the receiver operating characteristic (ROC) curve analysis was performed to assess the diagnostic potential of Histidine and yielded satisfactory sensitivity and specificity with AUROC equal to 0.65 [95% CI=0.54-0.76]. The circulatory levels of Histidine correlated well the erythrocyte sedimentation rate (r= - 0.19, p< 0.075) and with the C-reactive protein level (r= -0.26, p< 0.01).

Conclusion: The circulatory levels of histidine may serve as a useful biomarker for the assessment of disease activity and guiding treatment in TA patients. However, its use in clinical settings will require future studies on large patient cohorts in a longitudinal manner improve accuracy.

Figure 1: (a) Box plot showing comparison of circulatory histidine levels in active and inactive TA patients with respect to normal healthy control. (b) Receiver operating characteristic curve analysis performed form evaluating the diagnostic potential of histidine. The area under the receiver operating characteristic curve is shown in red to highlight the diagnostic value of circulatory histidine for differentiating active and inactive TA patients. (c and d) Graphs showing pearson-correlation form histidine with erythrocyte sedimentation rate and C-reactive protein levels Click here to view

PB0010: Altered systemic expression of micro RNAs miR-125a, miR-125b and miR-200a in patients with Rheumatoid arthritis

Sudipta Chatterjee, Dipanjan Bhattacharyya, Sanchaita Misra, Nitai Bhattacharyya¹, Alakendu Ghosh; Department of Rheumatology, Institute of Post Graduate Medical Education and Research, Crystallography and Molecular Biology Division, Saha Institute of Nuclear Physics, Kolkata, West Bengal, India

Background: Regulatory biomolecules, such as long non-coding RNA are being globally investigated to explain the pathogenesis of Rheumatoid arthritis (RA).

Objective: To study the systemic expression levels of micro RNAs miR-125a, miR-125b and miR-200a in patients with RA and comparative analysis of the same with healthy control group.

Methods: Thirty-eight patients with only RA and 15 healthy individuals were recruited. Blood samples were collected. Serum and PBMCs were separated. DAS28-CRP and HAQ-DI scorings were done at the time of recruitment. Laboratory markers for RA were measured from sera. RNA was extracted from the PBMCs, reverse transcribed to c-DNA using universal stem loop primer and qPCR was performed with specific primers for miR-125a, miR-125b and miR-200a using SYBR Green.

Results: Serum TNF- α and CRP was high inpatients (p<0.05). Increased expression of miR-125a was observed in the PBMCs of the patient group compared to healthy controls. On the contrary, miR-125b and miR-200a was found to be under expressed in RA patients. The fold change of the three miRNAs (calculated by 2^-ΔΔCt) were found to be mutually positively correlated.

Conclusion: The over expression of mir-125a suggests its excitatory function in RA pathogenesis. The under expression of mir-125b and mir-200a indicate their inhibitory role in RA pathogenic pathways. The mutual correlation of the micro RNAs indicate their common regulatory mechanism. The specific target molecules of these micro RNAs and their functional implication in context of RA needs to be investigated hence.

PB0011: Hypomethylation of the promoter region of TLR2 and TLR4 gene in patients with rheumatoid arthritis and periodontitis: a comparative study

Sudipta Chatterjee, Dipanjan Bhattacharyya, Ankur Rao¹, Arghya Chattopadhyay², Alakendu Ghosh; Department of Rheumatology, Institute of Post Graduate Medical Education and Research, St. Xaviers College, Kolkata, West Bengal, Department of Rheumatology, Postgraduate Institute of Medical Education and Research, Puducherry, India

Background: Association of Rheumatoid arthritis (RA) and Periodontitis (PD) has long been implicated in several studies. Epigenetic modifications are being recently explored to explain such associations, DNA methylation being one such mechanism that regulate gene expressions.

Objective: To study the effect chronic generalized periodontitis on systemic methylation of TLR2 and TLR4 genes and compare that with only RA and RA with PD patients.

Methods: Fifty-three RA patients, among which 21 patients had chronic generalized PD, 20 patients with only PD and 15 healthy individuals recruited. Blood samples were collected. Serum and PBMCs were separated. DNA was isolated from PBMCs, then were first bisulphite converted and then methylation specific PCR were performed using primers for methylated and um-methylated promoters of TLR2 and TLR4. The DNA amplifications were checked in horizontal gel electrophoresis. The methylation signatures were verified by DNA sequencing (Sanger) of the amplified products.

Results: The anti-CCP, DAS-CRP and HAQ DI were higher in patients with both RA and PD (220+40, 5.7+0.2, 1.5+0.1 respectively, p<0.05). Serum TNFα, IL8 and IL-17 were highest in patients with both RA and PD (p<0.05). Control samples had shown amplification bands for methylated primers TLR2 and TLR4 but not for un-methylated primers. However, only RA, only PD and RA with PD samples, had shown amplification for un-methylated primers and not for methylated primers. These Results: together with DNA sequencing indicated that 2 CpG islands in TLR2 promoter and six CpG sites in the promoter of TLR4 genes were hypo-methylated in the PBMCs of patients whereas those remain methylated in healthy individuals.

Conclusion: The observations indicated systemic involvement of TLR mediated pathways in only PD patients which is similar to those in RA. However, further validation in larger cohort and down-stream signalling molecules in vitro needs to be studied.

PB0013: Increased expression of long non-coding RNAs NEAT1, MALAT1 and MEG3 in patients with lupus nephritis and their impact on disease activity

Sulagna Chatterjee, Dipanjan Bhattacharjee, Sudipta Chatterjee, Ayindrila Saha, Sanchaita Misra, Dipendranath Ghosh, Nitai P Bhattacharyya, Alakendu Ghosh; Department of Rheumatology, Institute of Post Graduate Medical Education and Research, Kolkata, West Bengal, India

Background: Deregulations of long non-coding RNAs (lncRNA) recently implicated in rheumatic diseases including Systemic Lupus Erythematosus (SLE). Although, their specific roles in pathogenesis of disease and clinical impact in lupus nephritis remain largely unknown.

Objectives:

1. Comparative quantification of expression of regulatory long non coding RNAs (NEAT1, MALAT1 and MEG3).
2. Association between the lncRNAs and the serological markers (C3, C4, Anti-dsDNA antibody) in patients with lupus nephritis.

Methods: 10 Lupus patients with nephritis (Class III & Class IV) and 9 healthy controls (age-sex matched) were recruited. Peripheral blood was collected from each study participants and the PBMCs were respectively isolated. RNA was isolated from the PBMCs of individual samples and expression of the lncRNAs was measured by Real time PCR. C3, C4 and Anti-dsDNA were quantified by Nephelometry and ELISA respectively for every participant.

Results: Expression of lncRNAs (NEAT1, MALAT1, MEG3) increased significantly (p=0.0002, p=0.0003, p=0.01 respectively) in Patients. Significant negative correlation found between NEAT1 and C3(r=-0.75, p=0.01) as well as with C4(r=-0.82, p=0.004). Positive correlation found between NEAT 1 and Anti-dsDNA antibody(r=0.42, p=0.2) and with SLEDAI score (r=0.63, p=0.05). No significant correlation found between MALAT1 and MEG3 with C3 {(r=-0.01, p=0.9), (r=-0.22,p=0.5) respectively}, C4{(r=0.07,p=0.83),( r=-0.29,p=0.4) respectively}. Negative correlation found between MALAT1 and Anti-dsDNA antibody (r=-0.7, p=0.02), while no correlation found between MEG3 and Anti-dsDNA antibody ( r=-0.006,p=1). No significant correlation was found between MALAT1 and SLEDAI score (r=-0.17, p=0.63) and between MEG3 with SLEDAI score (r=0.34, p=0.33).

Conclusion: Increased expression of NEAT1 and its correlation with the serological markers (C3, C4, Anti-dsDNA antibody) and SLEDAI score well reflect the disease activity compared to MALAT1 and MEG3 which indicates the potentiality of NEAT1 as a probable additional marker of disease activity in patients with lupus nephritis. However validation of this finding in large sample is necessary.

PB0014: Polymorphisms in genes involved in methotrexate pathway: Predictor of response to methotrexate therapy in Indian rheumatoid arthritis patients

Ankita Singh, G Harikrishnan, Vikas Gupta, Pradepto Sekhar Patro, Ramnath Misra, Amita Aggarwal; Department of Clinical Immunology and Rheumatology, Sanjay Gandhi Postgraduate Institute, Lucknow, Uttar Pradesh, India

Introduction: Methotrexate (MTX) is first line therapy to treat rheumatoid arthritis (RA) Variable response to MTX therapy(50-60%) is observed in patients with RA.The variability can be explained by single nucleotide polymorphism (SNP) of genes involved in MTX metabolic or adenosine pathway. Hence, we studied association of seven genes polymorphism [rs2236225 (MTHFD1 1958G>A), rs17602729 (AMPD1 C>T), rs1127354 (ITPA C>A), rs1431131 (TGFBR2 A>T), rs2372536 (ATIC C>G), rs11188513 (ENTPD1 C>T), rs5751876 (ADORA2A T>C)] with response to MTX therapy in RA patients.

Methods: Patients satisfying EULAR/ACR 2010 criteria,DMARD naïve with active RA (DAS28) >3.2 were enrolled. Genotyping was done by TaqMan 5' nuclease assay. Patients were treated with MTX monotherapy ,gradually escalating the dose to maximum of 25 mg/week or maximal tolerated dose.Based on EULAR response criteria, patients were classified into responder and non-responder groups at 4 months. Using Chi-squared test, SNPs were associated with response to MTX therapy.

Results: 207 patients (85.5% females; median-age 40 [17] years); median disease duration 24 (40) months; median DAS28-CRP 4.64 (1.39) were enrolled. At 4 months, based on EULAR response 172 patients were classified as responders and 35 as non-responders. The G allele(p=0.009) and GG genotype(p=0.006) of ATIC gene and C allele(p=0.00005) and CC genotype(p=0.006) of ITPA gene associated with response to MTX therapy.On binary logistic regression analysis including clinical measures:DAS28 ESR,TJC,SJC, age and ATIC, ENTPD1and ITPA genes polymorphism; CC genotype of ITPA and GG genotype of ATIC gene in additive(p=0.006,p=0.002respectively)and recessive model(p=0.019,p=0.002 respectively) were independent predictors of response to MTX

Conclusion: Two genes polymorphism i.e. ATIC C>G and ITPA C>T were associated with response to MTX therapy in Indian RA patients. In a model comprising of clinical variables along with three genes polymorphism, GG genotype of ATIC C>G and CC genotype of ITPA C>T gene had an independent association with response to MTX.

PB0015: Urinary renalase as a biomarker in lupus nephritis

Ranjan Gupta, Akhilesh Yadav¹, Amita Aggarwal¹; Department of Rheumatology, All India Institute of Medical Sciences, New Delhi¹Department of Clinical Immunology and Rheumatology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India

Background: Renalase is a renal hormone secreted chiefly by tubular epithelial cells and degrades catecholamines. Serum renalase levels have been shown to correlate with markers of renal disease activity in patients with proliferative Lupus Nephritis (LN).

Objective: To test urinary Renalase in a cross-sectional and longitudinal study as a biomarker of renal disease activity of LN

Methods: Forty-Seven SLE patients with active nephritis (AN), 20 with active disease without nephritis (active non-renal; ANR) and 20 with inactive disease (ID) were enrolled. Patients in AN group were treated according to the ACR 2012 guidelines and followed up every 6 months for 1 year. At baseline, urine samples were collected from all and serum samples from 10 AN patients. Follow-up samples were collected every 6 months only in AN group. Urine samples from 10 healthy subjects (HC) served as controls. All urinary values were normalized for creatinine excretion. Variables are expressed as median (range) and non-parametric tests were used. A p-value<0.05 was considered significant.

Results: Baseline normalized urinary renalase (mcg/mg of creatinine) was significantly higher (p<0.001) in AN group [100.03 (0.45 – 22178.34)] as compared to HC [2.36 (0 – 104.47)]. However, the levels were not different as compared to ID [113.46 (8.04 – 1494.56] and ANR [57.32 (5.36 – 615.25)] groups. Urinary renalase did not show any correlation with serum Renalase [78993.5 (40206 – 293915) mcg/ml] which was tested in 10 patients with AN.

On follow-up of AN patients, with reduction in disease activity, urinary Renalase also decreased significantly at 6 [15.55 (0.64 – 266.88)] and 12 [5.68 (0.16 – 235.2)] months visits as compared to baseline (p<0.001).

Conclusion: Urinary Renalase is a poor biomarker of LN. However, it may be useful in assessing the response to therapy as its levels decrease with the fall in disease activity with treatment.

PB0016: Urinary MRP8/14, an endogenous toll-like receptor 4 ligand, reflects renal disease activity in lupus nephritis: A cross-sectional and longitudinal assessment

Ranjan Gupta, Dipendra K Mitra¹, Sonam Rani

Departments of Rheumatology and¹Transplant Immunology and Immunogenetics, All India Institute of Medical Sciences, New Delhi, India

Background: In Lupus Nephritis (LN), monocytes/macrophages are the most abundant cells infiltrating the glomeruli and have a relative abundance of Toll-Like Receptor 4 (TLR4).

Objective: To test MRP8/14 (Calprotectin), an endogenous TLR4 ligand, as a urinary biomarker of renal disease activity of LN.

Methods: SLE patients with active nephritis (AN), active disease without nephritis (active non-renal; ANR) and inactive disease (ID) were enrolled. Patients in AN group were treated according to the ACR 2012 guidelines and followed up every 3 months for 1 year. Urine and serum samples were collected at baseline for all and every 3 months in AN group. Urine samples from 10 healthy subjects (HC) and 10 patients of rheumatoid arthritis (RA) served as controls. Variables are expressed as median (range) and non-parametric tests were used for analysis. A p-value<0.05 was considered significant.

Results: Baseline normalized uMRP8/14 (ng/mg of creatinine) was significantly higher in AN group [n=32; 4464 (569-128894)] as compared to ANR [n=10; 1167 (4-18357)], ID [n=10; 438 (147-4320)], HC [1348 (3-2726)] and RA [339 (6-11519)] (p<0.05) groups. It also showed good correlation with urinary protein:creatinine ratio (r=0.55; p<0.001), rSLEDAI (r=0.5, p<0.001) and SLEDAI (r=0.3, p<0.001) but not with sMRP8/14 (r=0.25). Baseline uMRP8/14 but not sMRP8/14 could differentiate between AN and ANR groups and performed better on Receiver Operator Curve (ROC) analysis to differentiate between the two groups.

On follow-up of AN patients, with reduction in disease activity, uMRP8/14 also decreased significantly at 6, 9 and 12 months visits as compared to baseline (p<0.05). uMRP8/14 also showed a rise before conventional markers in 6 patients who relapsed within 1 year.

Conclusion: uMRP8/14 is a potential biomarker of LN disease activity. In patients with active SLE, it helps differentiate between patients with and without renal involvement and has the potential to predict relapse of LN.

PB0017: CD-14 (C-159 T) variants are associated with SLE and lupus nephritis: Plasma levels of sCD14 is a promising biomarker of SLE disease activity

Saumya Ranjan Tripathy, Aditya Kumar Panda¹, Rina Tripathy, Manoj Kumar Parida, Bidyut Kumar Das; SCB Medical College, Cuttack,¹Khallikote University, Berhampur, Odisha, India

Background: Cluster of differentiation-14 (CD-14) is a co-receptor for TLRs (2, 4, 7 and 9) signaling pathways and believed to plays an important role in innate immune system. Host innate receptor TLR7 and 9 recognizes nucleic acids as their ligands and produce type-I interferon which has been demonstrated in pathogenesis of SLE. Since TLR7 and 9 require CD14 for effective signaling, we hypothesized that CD-14 would correlate with disease activity and pathogenesis of SLE.

Objectives: To evaluate association of C159-T polymorphism of CD14 with SLE and its disease activity; to evaluate association of soluble CD14 (sCD14) with SLE disease activity.

Methods: In the present hospital-based case-control study, SLE patients (diagnosed based on SLICC-criteria) (n=200 females), and 200 age and sex-matched healthy controls were enrolled. CD14 (C-159T) polymorphism was genotyped by PCR-RFLP. Based on availability of samples (78 SLE patients and 46 healthy controls), plasma sCD14, TNF-α, IFN-α levels were quantified by ELISA. Clinical, serological and other markers of disease activity (C3, C4 and anti-dsDNA) were measured by standard laboratory procedures.

Results: Mutant genotypes (CT and TT) and allele T for CD 14 (C-159T) were significantly associated with predisposition to development of SLE and lupus nephritis. Mutants (CT and TT) for CD14(C-159T) polymorphism was associated with higher plasma sCD14, IFN-α and TNF-α compared to wild type CC.

Plasma sCD14 levels were significantly high in SLE patients compared to healthy controls (P<0.001). Plasma levels of sCD14 correlated with SLEDAI-2K scores (P=0.002, r=0.34), proteinuria (P=0.001, r=0.34) and negatively correlated with C3 (P=0.003, r=-0.33) and C4 (P<0.0001, r=-0.50). Patients with lupus nephritis displayed higher plasma levels of sCD14, TNF-α and IFN-α.

Conclusions: CD14 (C-159T) polymorphism is associated with susceptibility to SLE and lupus nephritis. Plasma sCD14 is a promising marker in the assessment of SLE disease activity.

PB0018: Interferon gene signature in Indian SLE patients is not correlated with disease activity and clinical manifestations

Vineeta Shobha*, Anu Mohan*, Shailesh Dudhgaonkar#, Preethi Karunanithi#; *St. Johns Academy of Medical Sciences, Bengaluru, Karnataka, India

Background: SLE is an autoimmune disease characterized by inflammation and tissue damage. Despite the availability of effective therapies, resistance to treatment and premature mortality remain major concerns. Interferon (IFN) signature genes are highly expressed in peripheral blood of patients with systemic lupus erythematosus (SLE), especially in the presence of active disease. However, the alteration in the expression of this gene signature upon treatment has not been fully elucidated in Indian SLE patients.

Objective: To identify the IFN gene signature in Indian patients and understand its correlation with clinical manifestations of disease and its treatment.

Methods: We characterized 54 Indian SLE patients for the IFN signature, and its correlation / concordance with clinical manifestations and SLEDAI score. Patients with mild, moderate and severe disease with SLEDAI ≤ 4, 5 to 12 and ≥13 respectively were recruited. All patients were on treatment with steroids in combination with other immunosuppressants like hydroxycloroquin, cyclophosphamide, MMF, azathioprine and methotrexate.

Results: Multiple IFN related pathways were enriched with significant increase in IFN gene signature in SLE patients as compared to normal heathy volunteers. These gene signatures did not correlate with SLEDAI score or clinical manifestations. Based on these signatures, we were able to stratify patients into two groups - i.e. patients with high IFN signature and those with low IFN signature [ref [Figure 1]]. Furthermore, we noted that IFN-high and IFN-low groups were composed of patients from across disease severity.

Figure 1 Click here to view

Conclusion: These findings indicate that more studies are required to understand if specific immune cell types such as pDCs, monocytes, contribute to the IFN gene signature in different aspects of SLE disease biology and their clinical correlations. An an in-depth characterization of variants within the IFN pathway may provide guidance for personalized therapies.

PB0019: Lymphocyturia is a good and cheap biomarker for active lupus nephritis and is sensitive to change

Sarit Sekhar Pattanaik, Ankita Singh, Shilpa Venkataraman¹, Ramnath Misra, Vinita Agarwal¹, Amita Aggarwal; Departments of Clinical Immunology and Rheumatology and¹Pathology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India

Introduction: Multiple urinary biomarkers have been described for lupus nephritis, however none has reached the clinic. Recently, urinary CD4 and CD8 T cells have shown high specificity for active lupus nephritis(LN). Thus we studied whether urinary lymphocyte count by simple staining of sediment or T cell count by flowcytometry can serve as a simple and cheap biomarker.

Methods: Patients with active LN(ALN) defined as renal SLEDAI(rSLEDAI) >4, active lupus without nephritis(ANR) defined as SLEDAI >4, inactive nephritis(IN) defined as rSLEDAI ≤4 and SLEDAI <4, and age matched healthy controls(HC)were included. Lymphocytes in the urine were stained using Kovak stain and reported as number of cells/high power field. CD3 T cells were counted by FACS using anti-CD3 antibody and DAPI for live gate. Immunohistochemistry was done on renal biopsy for CD3 cells.

Results: Among 74 patients, 44 had ALN, 10 had ANR disease and 20 patients had IN. The median (IQR) urinary Lymphocyte count was 4(7) in ALN as compared to ANR[2(3.5),p=0.1], IN(0, p<0.001) and HC(0,p<0.001). The median CD3 count was 2793(8982) in ALN as compared to ANR[209(539.2), p=0.001],IN[86(303),p<0.001] .There was moderate correlation between lymphocyturia and urinary T cell number(r=0.59, p<0.001).

Lymphocyturia and urinary CD3 cell count had moderate correlation with SLEDAI(r=0.59, p<0.001;r=0.47,p<0.001) and rSLEDAI(r=0.58, p<0.001;r=0.54, p<0.001).The ROC curve for lymphocyturia to differentiate ALN from IN(AUC: 0.87) and for CD3(AUC: 0.874) [Figure 1].

Figure 1 Click here to view

In 15 patients of ALN on follow up, there was a excellent correlation between change in SLEDAI and change in lymphocyte count(r=0.845,p<0.001).In 31 patients with renal biopsy, there was a moderate correlation between the CD3 cells by IHC in tubulointerstitial compartment and urinary CD3 cells(r=0.55,p=0.001)

Conclusion: Lymphocyturia measured by simple staining of urinary sediment can be a cheap and effective marker of active LN and can be used to follow up patients with active nephritis in resource poor countries.

PB0020: Clinical utility of Antibodies to C1q sub-component as a biomarker of anti-phospholipid syndrome

G Arvind, J Kabeerdoss, A Nair, A Mathew, R Goel, SKumar, J Mathew, D Danda ; Department of Clinical Immunology and Rheumatology, Christian Medical College Hospital, Vellore, Tamil Nadu, India

Background:

Several clinical and experimental animal model studies have emphasised the role of complement activation in anti-phospholipid syndrome (APS). Recently, in a 2016 study by Oku et al published in Oxford rheumatology, antibodies to C1q (Anti C1q) sub-component were present in significantly higher number of primary APS patients compared to controls ( healthy subjects, non-lupus connective tissue disease (CTD) patients and lupus patients negative for anti-phospholipid antibodies (APLs).

Aim: To evaluate the utility of Anti C1q as a biomarker of APS.

Methods: We enrolled 49 APS patients and 341 age-matched controls in a retrospective design, recruited from June 2016 to June 2019. Anti C1q ELISA was performed by commercial ELISA kit (Human Diagnostics, Germany).

Results: Median age of APS patients was 37 years in comparison to 35 years in controls. Among the APS patients,13 were primary and 36 were secondary. Among the controls, 85 were non-lupus CTD patients, 44 were lupus patients with positive APLs and 211 were lupus patients negative for APLs. Anti C1q antibody was positive in 12/49(24%) APS cases in comparison to 114/ 341 (33.4%) controls (p value- 0.1, chi-square test). Anti C1 q was present only 1 out 13 (7.6%) primary APS patients and was positive in 11/36 (30.5%) secondary APS patients. Anti C1q positivity in APS secondary to lupus was 11/36 (30.5%) in comparison in 87/ 211 (41.2%) in lupus controls (p value-0.3, chi-square test)

Conclusion: Anti C1q was not helpful as a biomarker of APS in our study, in both primary as well as secondary APS clinical settings.

PB0021: NLR reflects the clinical activity in AS and corresponds to BASDAI than other inflammatory parameters

Shivputra Ghanti, S Chandrashekara, C Renuka¹, K R Anupama; ChanRe Rheumatology and Immunology Centre and Research,¹ChanRe Diagnostic Centre, Bengaluru, Karnataka, India

Background: Neutrophil-to-lymphocyte ratio (NLR) has emerged as one of the important markers in inflammatory rheumatic diseases. The present study is intended to analyze the relationship of the NLR with various measurable parameters of AS.

Materiala and Methods: The demographic, clinical and laboratory parameters obtained at the recruitment phase were, age, gender, clinical symptoms for scoring the BASDAI, BASFI and BASMI, total leucocyte count, ESR and CRP. NLR was calculated. The BASDAI was considered as the outcome variable. Patients were classified as those with low (<4) and high disease (≥4) activities. The NLR was considered as a categorized variable with sub groups <2, 2-4 and >4.

Statistics: Mann Whitney test for no normal distribution, chi square test for categorized variables and Spearman's correlation for bivariate linear association were performed. Mountain plots were used to verify agreement of clinical and inflammatory parameters of BASDAI, BASFI, BASMI, NLR and CRP.

Results: The study involved a total of 162 AS patients (112 males and 50 females) with a median age of 39.5 (18-73) yrs. The corresponding values noted were: BASDAI 2.2 (0-8.5), BASMI 1 (0-10), BASFI 2.2 (0-7.7), ESR 29 (2-126), CRP 8.45 (0.3-120) and NLR 2.37 (0.99-12.39). BASDAI <4 was noted in 124 subjects and 38 had ≥4.

NLR was high in BASDAI ≥4 group. BASDAI strongly correlated with BASFI and moderately with BASMI. NLR showed weak correlation with BASFI and BASMI. Mountain plots revealed BASFI had least bias 0.1 (2.5th and 97.5th percentiles were -3.43 and 2.99) with BASDAI, followed by NLR -0.22 (-4.35 and 3.86), BASMI 0.8 (-6 and 4.99) and CRP -7.25 (-91.41 and 2.85).

Conclusion: NLR corresponds with BASDAI and serves as a better marker of inflammation than CRP in AS. NLR correlates weakly with BASFI and BASMI in lower BASDAI score.

PB0022: Adenosine deaminase-genetic polymorphism and baseline serum level as a biomarker of treatment response to methotrexate in rheumatoid arthritis

G Harikrishnan, A Singh, A Aggarwal; Department of Clinical Immunology and Rheumatology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India

Background: Despite methotrexate (MTX) being the first line therapy for RA,30% of patients do not respond to MTX.Methotrexate acts by increasing adenosine levels leading to anti-inflammatory effects.Adenosine deaminase (ADA) enzyme converts adenosine to inosine thus reduces levels of adenosine.Thus,ADA levels and single nucleotide polymorphism(SNP) in ADA gene may affect the treatment effect of MTX.

Objective: To study if genetic polymorphism in ADA gene and baseline serum ADA levels in RA patients is associated with MTX response

Methods: 207 DMARD naïve active RA(DAS28 ESR ≥3.2)patients satisfying EULAR/ACR 2010 criteria were enrolled. Genotyping was done in all patients(n=207) by TaqMan 5' nuclease assay.In a subset of patients(n=59) blood sample was collected at baseline and 2 months for ADA estimation by ELISA. MTX was initiated at dose of 15mg/week, escalated 5mg/month till the patients had DAS28 < 2.6 or a maximum dose of 25 mg/week was reached. At 4 months based on EULAR response patients were classified as responders or non-responders.Association of SNPs in ADA gene, baseline clinical parameters and serum ADA levels with EULAR response was studied.

Results: Among 207 patients(177females)mean age was 41.49± 12.14 years and median disease duration 24(40) months.172 patients(83.1%) were responders and 35(16.9%) non responders.47.8% were AA,42.5% AG and 9.7% were GG genotype.Mean DAS 28 CRP was 4.76 ± 1.05 and median DAS 28 ESR was 6.00(1.38). Mean serum baseline ADA was 10.82 ±5.33 pg/dl.The baseline parameters like age, disease duration, ESR,CRP,DAS 28 ESR,DAS 28 CRP were similar between responders and non-responders. SNPs in ADA gene were not associated with EULAR response(p=0.475).Baseline ADA levels(10.52 ± 5.37 vs 12.28 ± 5.14;p=0.34)2month ADA levels(p=0.34) and delta ADA(p=0.55) didnot show any association with MTX response. Mean ADA level was similar between the 3 genotypes of ADA(p=0.736).

Conclusion: ADA gene polymorphism and serum ADA levels do not affect response to MTX

PB0023: Ficolin H assay in pediatric-onset systemic lupus erythematosus

Pratap Kumar Patra, B N Anil Kumar, Amit Rawat, Surjit Singh; Post Graduate Institute of Medical Education and Research, Chandigarh, India

Background: Systemic lupus erythematosus (SLE) is a complex autoimmune disease of childhood with a myriad of presentation. Though the complement pathway plays a key role in the pathogenesis of SLE, the role of ficolin pathway in its pathogenesis is not well studied.

Objectives: To study the role of ficolin pathway by estimating ficolin H levels in children with SLE and to establish a correlation of ficolin H level with the disease activity.

Methods: A cross-sectional study was carried out in the Pediatric Rheumatology Clinic, Advanced Pediatrics Centre, Post Graduate Institute of Medical Education and Research, Chandigarh, India. Thirty-three cases of SLE and 31 controls were included in the study. Ficolin level in both groups was measured by enzyme-linked immunosorbent assay (ELISA) and compared.

Results: There were 21 girls and 12 boys in the study group with a ratio of 1.75:1, whereas the control group had 18 girls and 13 boys, a ratio of 1.8:1 ( p = 0.64 ). A significant difference in the mean ficolin concentration between the groups (cases: 248.35±93.29); (control: 184.71±57.02; p =0.002) was found. A negative correlation between Anti-dsDNA and ficolin level was observed (r -3.72). However, there was no significant difference between the mean ficolin concentration in patients with active disease; 266.76μg/dl as compared to inactive disease; 234.79/μg/dl, (p 0.339).

Conclusion: We observed an increased ficolin level in the study group as compared to control suggesting involvement of ficolin pathway in SLE. A negative correlation was also observed between the disease activity and ficolin H level. However, we did not find any significant difference in ficolin level between active and inactive disease group. Further studies with a larger sample size required to confirm any association between anti-dsDNA titers and ficolin H level in SLE.

PB0024: Significantly decreased histidine/tyrosine ratio in ANCA†associated vasculitis compared to takayasu's arteritis: A marker for inflammation

Avinash Jain, Umesh Kumar¹,Gayatri G Ekbote², Latika Gupta, Ritu Raj¹, Anupam Guleria, Rajiva Gupta², Ramnath Misra1, Dinesh Kumar¹; Department of Clinical Immunology and Rheumatology, SGPGIMS,¹Centre of Biomedical Research, Lucknow, Uttar Pradesh,²Medanta-The Medicity, Gurgaon, Haryana, India

Introduction: Takayasu arteritis (TA) and Anti-neutrophil cytoplasmic autoantibody (ANCA)-associated vasculitis (AAV) predominantly affects large and small vessel respectively. Biomarkers are invaluable tools in clinical medicine that aid in disease diagnosis, understanding disease pathogenesis, predicting prognosis, and response to therapy. The oxidative stress is the hallmark of inflammatory state and studies suggest that histidine has strong anti-oxidative and anti-inflammatory effects whereas tyrosine has a limited role. Based on this, we hypothesized that the circulatory Histidine/Tyrosine ratio (HTR) will be lower in AAV (considering the extent of inflammation) compared to TA.

Objective: The aim is to compare the circulatory HTR values in TA and AAV patients.

Methods: The serum metabolic profiles of 53 AAV and 98 TA patients (satisfying their respective American College of Rheumatology (ACR) classification criteria) and 77 healthy controls (NC) were measured using 1D 1H-CPMG NMR experiments at 800 MHz NMR spectrometer and analyzed using CHENOMX (w.r.t formate as an internal reference) and compared.

Results: The median age in AAV and TA groups was 46.5 years and 27 years respectively. Female to male ratio was 1.79 in the AAV group and was 4.16 in the TA group [Figure 1]a. Circulating HTR levels were significantly decreased in AAV and TA patients compared to NC [Figure 1]b but were lower in AAV when compared with TA. Further, the receiver operating characteristic (ROC) curve analysis showed good sensitivity and specificity with AUROC equal to 0.8 [95% CI=0.72-0.87] [Figure 1]c.

Figure 1: (a) Clinical and demographic characteristics of AAV, TA and NC subjects. (b) Box NC. (c) Receiver operating characteristic curve analysis performed for evaluating the diagnostic potential of circulatory HTR. Highlighting the diagnostic value of circulatory HTR for differenctiating Anca and TA patients Click here to view

Conclusion: This proof of the concept study demonstrates that circulatory HTR level has the potential to serve as a surrogate biomarker for differentiating TA from AAV and might aid in screening TA patients with small vessel involvement, understand the pathogenesis and guiding the disease treatment. Limitation – Needs validation and larger cohort.

PB0025: Correlation of serum calprotectin level with C – reactive protein and jadas – 27 score in children with juvenile idiopathic arthritis

F Remthangpuii, Anu Maheshwari, Jagdish Chandra, Sunita Sharma, Deonath Mahto; Lady Hardinge Medical College and Associated SSK and KSC Hospitals, New Delhi, India

Background: Serum Calprotectin is a new inflammatory marker that can be used as a marker for disease activity in patients with JIA. We hypothesized that there is a correlation between Disease Activity Score (JADAS-27) and serum levels of Calprotectin and CRP in children with JIA.

Objectives: To correlate Disease Activity Score (JADAS-27) with serum levels of Calprotectin and quantitative C-Reactive Protein (CRP) in children with Juvenile idiopathic arthritis(JIA).

Methods: This cross-sectional observational study was conducted in Department of Pediatrics, Pediatric Rheumatology and immunology clinic and Department of Pathology, Lady Hardinge Medical College, New Delhi from November 2017–March 2019. Fifty patients with Juvenile Idiopathic Arthritis (JIA) attended in rheumatology and immunology clinic were enrolled. BioVendor Human S100A8/A9 ELISA was used for determination of serum Calprotectin level. CRP Ultra EIA is used for quantitative determination of CRP.

Results: The mean serum Calprotectin level was 42,284.5±14,129.98 ng/ml with minimum and maximum values being 8,560ng/ml and 63,160ng/ml, respectively. The mean Q–CRP was 40.14±37.14 mg/l, and the minimum and maximum values being 0.02 mg/l and 107.4 mg/l, respectively. The mean JADAS-27 score was 13.42±8.12, 0 and 32 being minimum and maximum values, respectively. Spearman correlation demonstrated a positive correlation between serum Calprotectin and JADAS-27 score (r=0.418), and p value was 0.003, which indicated a statically significant association between the two. There was also a positive correlation between Q-CRP and JADAS-27 score (r = 0.619), and the correlation was statistically significant (p=0.000). We also found a positive correlation between serum Calprotectin and Q – CRP (r=0.489), and the association was statistically significant (p=0.000).

Conclusions: Disease Activity Score (JADAS-27) correlate with serum levels of Calprotectin and Q-CRP, and the correlations were statistically significant.

PB0026: TLR7/9 expression and humoral response against Epstein barr virus in systemic sclerosis

Sanghamitra Machhua, Shefali Khanna Sharma², Yashwant Kumar, Sanjeev Handa², Surjit Singh³, Shashi Anand, Ranjana W Minz; Departments of Immunopathology,¹Internal Medicine,²Dermatology, Venerology and Leprolosy and³Pediatrics, Post Graduate Institute of Medical Education and Research, Chandigarh, India

Objective: To analyze TLR7/9 expression and Humoral response against Epstein Barr virus in Systemic sclerosis

Materials and Methods: EBV viral load detection was done by absolute quantification using Taqman probe based real time PCR. Antibodies of class IgG and IgM against EBV was detected by Immunoblotting (Euroimmun, Germany), With the EUROLINE Anti-EBV Profile 2 (IgG and IgM –Anti VCA gp125,VCA p19, EBNA-1, p22, EA-D) and the Results: were interpreted using EUROLINE Scan software. Gene expression of TLR-7, TLR-9 was analyzed by PCR array based on SYBR green chemistry using real time PCR (Qiagen).

Results: EBV was not detected in peripheral blood of 49 SSc patients out of 50 patients, whereas in 50 age matched controls, 10 were positive. Out of 48 SSc patients 33 had antibody profile suggestive of late phase infection whereas 14 were suggestive of reactivation phase, while in controls 23 had late phase infection but none of them were in reactivation phase. Significant down regulation of EBV receptors, TLR 7 and TLR 9 was observed in SSc patients as compared to controls [Figure 1].

Figure 1: TLR 7 and 9 gene expression Click here to view

Conclusion: EBV and systemic sclerosis have a unique relationship. Humoral response to EBV antigens was strong in patients detected with re-activation phase, but the virus was not detected in peripheral blood by Real time PCR .There are however foot prints of the virus in terms of down –regulation of EBV receptor TLR 7 and TLR 9 in peripheral blood cells .We postulate that the virus hides in the niche of skin and endothelial cells where it triggers pro-fibrogenic environment, which is needs to be further explored in near future.

PB0027: Identifying endothelial dysfunction through circulating endothelial cells in patients with ankylosing spondylitis: a tertiary centre pilot study

Ayindrila Saha, Aniruddha Bagchi, Sumantro Mondal, Sanchaita Misra, Dipanjan Bhattacharjee, Sulagna Chatterjee, Sudipta Chatterjee, Parasar Ghosh, Alakendu Ghosh; Department of Rheumatology, Institute of Post Graduate Medical Education and Research, Kolkata, West Bengal, India

Background: Ankylosing spondylitis(AS) is characterised by inflammatory back pain with evidence of radiographic sacroiliitis. Impaired endothelial function is a harbinger of cardiovascular complications in AS patients. The endothelial cells, lining the entire vascular system, are detached from the vessel wall as an immediate consequence of compromised endothelial integrity and can be detected as circulating endothelial cells (CEC) in the peripheral blood.

Objectives: To investigate the relationship between endothelial function and CECs in AS patients in a cross-sectional study.

Methods: CECs were characterized and quantified sourced from peripheral blood of patients with AS (n=15) and age and sex matched healthy controls (n=10). Flow-mediated dilatation (FMD) was assessed for all subjects. Disease activity was evaluated using Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) and functional ability was monitored using Bath Ankylosing Spondylitis Functional Index (BASFI). All subjects were free of any other known traditional CV risk factors.

Results: Frequency of CECs in AS patients was upregulated compared to healthy controls being 2.54(1.640-4.210) vs 0.33(0.07-0.56), p<0.001, along with reduced FMD% being 14(5.9-17.50) vs 18.26(11.86-25.25), p<0.05). Frequency of circulating endothelial cell population was negatively correlated with FMD%, r=-0.56, p<0.05, but positively correlated with disease duration, r=0.7, p<0.05 and & to some extent with disease activity, BASDAI.

Conclusion: A proportionate amount of CECs were found to be present in patients with AS compared to healthy controls, denoted a high degree of endothelial damage which was further supported by reduced FMD in this population. Frequency of CECs were directly proportional with disease duration and BASDAI and inversely proportional with FMD%. These circulating endothelial cells may be sloughed from the vascular lining under conditions of systemic inflammation. Thus elevated frequency of CECs can be a potential bio-marker for early stage of endothelial damage in patients with ankylosing spondylitis.

PB0028: Urinary IL-36 γ in urine: A new potential marker of active lupus

Pratibha Singh, Sanjukta Majumder, Amita Aggarwal; Department of Clinical Immunology and Rheumatology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, India

Background: IL-36 is a new member of the IL-1 family and has inflammatory properties. Recent studies have shown elevated serum levels of IL-36α and IL-36γ in patients with Systemic Lupus Erythematosus (SLE). As no data is available on urinary levels of IL-36α and IL-36γ in SLE, we studied these in patients with SLE including lupus nephritis (LN).

Objective: To identify role of IL- 36 by studying urinary levels of IL-36 in SLE patients by ELISA.

Methods: Urine samples were collected from 106 patients with SLE (active LN (ALN)=68, inactive LN (ILN)=20, active non-renal (ANR)=18) and 10 healthy subjects (HC) after informed consent. Urinary IL-36α and IL-36γ levels were measured by ELISA (R&D Systems) and are expressed as pg/ml and as median (IQR).

Results: Out of 106 patients 94 were females. Their mean (SD) age was (28.3(9.7)] years, duration of disease 26.3(44.1) months and SLE disease activity index (SLEDAI) score 10.3(7.5).

None of the healthy control subjects had detectable IL-36γ levels in urine. Median IL-36γ levels in patient's urine were significantly higher than healthy controls (P<0.01). IL-36γ levels in urine of ALN [4.1 (42.1)] patients were also significantly higher than ILN [0(8.3), P<0.05] and ANR [0(7.7), P<0.05] patients. Urinary IL-36γ level had moderate positive correlation with SLEDAI score (r= 0.31, P<0.002).

Two healthy controls had IL-36α detectable in the urine. Median IL-36α levels in patient's urine were significantly higher than healthy controls (P <0.01). The median IL-36α levels in ALN [3.6 (35.8)] were no different than ILN [10.5(28.4)] and ANR [30.2(75)] patients.

Conclusion: Both IL-36α and IL-36γ levels were higher in SLE, however only IL-36γ correlates with lupus activity. In addition, IL-36γ levels were higher in active nephritis as compared to inactive nephritis patients.

PB0029: Identification of disease-associated specific protein to understand RA pathogenesis and diagnosis development

Monu, Prachi Agnihotri, Uma Kumar¹, Sagarika Biswas; CSIR-Institute of Genomics and Integrative Biology,¹All India Institute of Medical Sciences, New Delhi, India

Background: Rheumatoid arthritis is a chronic, autoimmune and systemic rheumatic disease with advanced inflammation in or around of one or more joints. RA diagnosis at early stage have potential to increase chance for successful treatment, whereas, delays can alter treatment process at late stage of disease presentation. In spite of availability of diagnostic markers for RA, the seropositive and seronegative patient have been ascended resulting into difficulties in diagnosis conditions



Objectives: Identification of a disease-associated specific differential protein/marker to improve RA diagnosis.

Methods: The plasma proteins profiling of RA was performed using combination of two-dimensional electrophoresis (2-DE) and LC-MS/MS techniques. The plasmatic expression of identified proteins was validated using Western blot and biochemical assay.

Results: Preliminary, 11 protein spots were identified using 2-DE and LC MS/MS analysis. Amongst differentially expressed proteins, a transthyretin (TTR) protein was selected and validated in RA plasma. The Western blot densitometric analysis reveals upregulation of TTR in RA (n=10) plasma compared to healthy control (n=10) with a significant difference of p=0.0112. Furthermore, to determine expression consistency of TTR, ELISA of RA patient's plasma (n=72) and healthy control (n=37) was analysed, indicating the up-regulation of TTR compared to control with an odd ratio of 1.44 at significant difference (p<0.0014).

Conclusions: The distinctive identification and differential expression inflammatory proteins/markers can reflect a late stage of disease, so measuring disease activities at the early stage would be promising to diagnose RA. In our understanding the expression of TTR increased with disease severity and their systematic investigation of these altered proteins may act as a complementing therapeutic tool for RA management.

Table 1: List of few identified plasma proteins from rheumatoid arthritis patients Click here to view

PB0031: Does gut inflammation correlate with disease activity in non-IBD spondyloarthritis?

Sakshi Mittal, A K Jain¹, Varun Dhir, Sanjay Jain, Shefali K Sharma; Department of Medicine, Clinical Immunology and Rheumatology Wing, PGIMER,¹Department of Gastroenterology, PGIMER, Chandigarh, India

Background: Studies have demonstrated subclinical gut inflammation in more than half of Spondyloarthritis (SpA) patients(1). It has been hypothesized that gut inflammation has a role in disease pathogenesis(2). Moreover, gut inflammation seems to co-relate with SpA disease activity and sacro-iliac joint marrow oedema(1,3). Calprotectin is the most abundant protein in neutrophils(4) and faecal calprotectin is a well-established marker of gut inflammation(5,6).

Objectives: To evaluate faecal calprotectin as a marker of disease activity in Spondyloarthritis.

Methods: Axial or peripheral SpA patients without known IBD or loose stools/ blood in stools for last 3 months were enrolled. Faecal calprotectin estimation was done by ELISA (cut off – 43 m/g). BASDAI was calculated at the same visit and ESR and CRP were estimated. NSAID index score was calculated for the last 3 months of NSAID intake as per ASAS recommendations(7).

Results: Faecal calprotectin was tested in 301 patients of axial and peripheral SpA. Disease was active (BASDAI > 4) in 50 (16.6%) patients. Faecal calprotectin was raised in 68 (22.59%) patients. We found a significant association between elevated faecal calprotectin and BASDAI, ESR and CRP. Faecal calprotectin was not affected by age, sex, disease duration, HLA B-27 positivity, previous NSAID intake or sulfasalazine use. Through the ROC analysis faecal calprotectin level of 43 m/g could predict active spondyloarthritis (BASDAI > 4) with a sensitivity of 32% and specificity of 83.5%.

Conclusion: There is a significant correlation between gut inflammation and disease activity in axial and peripheral spondyloarthritis.

Table 1: Association of elevated faecal calprotectin with spondyloarthritis parameters Click here to view

PB0032: In vitro characterization of treg cells isolated from peripheral blood of rheumatoid arthritis patients

Vallayyachari Kommoju, K G Chengappa, V S Negi; Department of Clinical Immunology, Jawaharlal Institute of Postgraduate Medical Education and Research, Pondicherry, India

Background: Present study was designed to understand the influence of synovial inflammatory milieu on peripheral blood Treg cells in patients with Rheumatoid Arthritis (RA).

Methods: The Peripheral Blood (PB) and synovial fluid (SF) of RA (n=80) and OA (n=30) patients were analyzed for CD4+T-cell subset frequencies and phenotypes by flow cytometry. Cytokine concentrations in plasma and SF were measured by cytometric bead array. Tregs from 5 RA-PB were isolated and cultured in autologous synovial fluid for 24 hrs. Phenotypic expression of Th1 and Th17 chemokines on the cell surface were analyzed by flow cytometry and expression levels of T-bet, RORγ and FOXP3 in the cultured Treg cells were measured with quantitative real-time PCR (RT-qPCR).

Results: The PB and SF frequencies of Th1, Th17 and Tregs. The pro-inflammatory cytokines were high in the plasma and SF of RA but the anti-Inflammatory cytokines were similar [Figure 1]a and [Figure 1]b. Treg cells isolated from PB and cultured in autologous SF of RA showed increased cell surface expression of CXCR3+ and CCR6+ [Figure 1]c. Gene expression studies showed an increased expression of T-bet, RORγ and decreased expression of Foxp3 after in-vitro culture [Figure 1]d.

Figure 1: (a) comparison of plasma cytokine levels between of RA and OA patients; (b) comparison of synovial fluid between RA and OA patients; (c) surface characerzation of in vitro cultured peripheral blood treg cells of RA patients. C1 and C2 CXCR3 (Th1) snf VVR6 (Th17) chemokines expressing treg cells isoloated from peripheral compartment of RA patients; C3 abd C4 CXCR3 (Th1) and CCR6 (Th 1) and CCR6 (Th17) chemoking expressing treg cells cultured in autologous synovial fluid. Expression of (D1) T-bet, (D2) ROR-g, and (D3) Foxp3. Mamm–Whitney U-test was used to analyze cytokine data, unpaired Student's t-test was used to analyze treg frequency data, paired t-test was used to analyze gene expression data; *P < 0.05 considered significant Click here to view

Conclusion: Tregs in RA are converted to Th17 phenotype on exposure to inflammatory cytokine in the synovial fluid, thus losing their regulatory functions. Understanding factors influencing stability of Treg cells may help improve future therapeutics.

https://drive.google.com/file/d/13BD0-E4wRVRcRCw3PvITMd4t250RqVH1/view?usp=sharing

PC0001: Validation of Hindi version of the Pittsburg sleep quality index

Anil Kumar, Rohini Handa, Sundeep Kumar Upadhyaya, Sirinder Jit Gupta, Bindhyachal Kumar Gupta; Department of Rheumatology, Indraprastha Apollo Hospital, New Delhi, India

Objectives: Pittsburg sleep quality index (PSQI) is used to assess subjective sleep quality in different population groups over the last one month. There is lack of suitable instrument to assess sleep quality in Hindi speaking population. We aimed to translate and assess the reliability and validity of the Hindi translated version of the Pittsburgh Sleep Quality Index (PSQI-H).

Methods: We translated and assessed the reliability and validity of the Hindi translated version of the Pittsburgh Sleep Quality Index (PSQI-H). The PSQI-H was developed from PSQI according to the following steps: (a) translation, (b) back-translation, (c) comparison between translation and back-translation, and (d) pre-pilot test in intended population. The PSQI-H was applied to 105 bilingual individuals knowing Hindi and English. The internal consistency of PSQI-H was assessed by Cronbach's alpha. For test-retest reliability assessment, intraclass correlation coefficient (ICC) was measured between PSQI-H at baseline and PSQI after 4 weeks. Pearson's coefficient was used to assess the correlation between the score of the questions and the PSQI-H scores.

Results: The seven components of PSQI-H shows an acceptable level of internal consistency with Cronbach's alpha of 0.776. There is good test-retest reliability between PSQI-H and PSQI as measured by ICC of 0.979. The score of individual items and global scores of PSQI-H were highly correlated with each other (p< 0.001). The mean of the seven individual components score and global scores of PSQI-H at baseline and original PSQI after 4 weeks did not differ significantly.

Conclusion: This study Results demonstrate that PSQI-H is a valid and reliable instrument for the assessment of sleep quality in Hindi speaking population.

PC0002: A study on estrogen level in systemic lupus erythematosus patients and it's co-relation with the disease activity

U Faruque, P Dihingia, A K Jha; Assam Medical College and Hospital, Dibrugarh, Assam, India

Introduction/Background: Systemic Lupus Erythematosus (SLE) is an auto-immune disease. There are evidences of hormone effect like estrogen, genes on X chromosome and epigenetic differences between the genders playing a role. Supporting to the hormonal factor there are studies showing that women using estrogen containing OCPs or hormone replacement therapy have increased risk of developing SLE.

Objectives: This study was undertaken to know the co-relation between serum estrogen level and the disease activity.

Materials and Methods: A total of 123 patients visiting the OPD of AMCH, Dibrugarh were taken up for study after applying the inclusion and exclusion criteria. Their SLEDAI score was calculated and simultaneously estrogen level was also taken. A proforma was filled which also had the menstrual history of the patient.

Results: Male:Female was 1:24. 52% had duration of disease less than 1 year. Most common symptom was malaise seen in 47% patients. 49% patients were in follicular phase of their cycle. 60% patients belonged to moderate disease activity group (SLEDAI 4-12). On looking the overall co-relation between SLEDAI score and estrogen level, with increasing disease activity the estrogen level increased but on taking the mean of each SLEDAI group, there was a dip in the estrogen level in the severe disease activity group.

Conclusion: With increase in estrogen level the disease activity increases but only till the disease activity is of moderate nature. Once severe disease activity sets in there is a decline in the estrogen level. It may be due to the suppression of the hypothalamic-pituitary-gonadal axis suppression during severe disease activity. Also, the aromatase enzyme levels are supressed leading to decreased estrogen synthesis.

Table 1: Mean value of estrogen in various groups Click here to view

PC0003: Development of an Indian brief international classification of functioning, disability and health core set for rheumatoid arthritis: From patients and health care professionals' perspective

M Suja Sathya Dharshini, Samuel Kamalesh Kumar, R Aruna, Ronald Thomvic Maradona, Raji Thomas, John Mathew; Christian Medical College, Vellore, Tamil Nadu, India

Background and Objective: An ICF Core Set (ICF-CS) is the assemblage of necessary categories which are relevant to elucidate the functioning of a person with a specific health condition. The comprehensive core set is vast and time consuming to be performed in an outpatient setting. The brief core set can be used in variety of settings but geographic and socio-cultural specific core sets need to be developed for accuracy in assessment and treatment. Therefore, our study was designed to develop a brief “Indian ICF Core Set for RA” from the perspective of patients and health professionals.

Methods: Participants include 106 patients with RA and 46 health care professionals who were providing services to patients with RA. A cross sectional survey was done in our tertiary care teaching hospital. An open questionnaire and ICF-CS based questionnaire were given to patients and health care professionals to identify the most important problems that patients with RA experience.

Results: 32 categories from the open questionnaire and 32 categories from the ICF-CS based questionnaire were obtained. The final brief “Indian ICF core set” was formulated by combining both the approaches which resulted in 41 categories. The majority of categories were from the ICF-CS component - activities and participation (22 categories, 54%).There were dissimilarities observed between the response of patients and health care professionals. Only 15 (37%) categories were reflected in our Indian brief ICF-CS compared to the original brief ICF core set demonstrating the geographic and socio-cultural differences in our population.

Conclusion: A brief ICF core set for patients with RA from the perspectives of patients and health care professionals was developed for Indian population.

PC0004: Lupus is associated with worse outcomes after hip arthroplasty

Jasvinder Singh, John Cleveland; University of Alabama at Birmingham, Birmingham, Alabama, USA

Background/Objective: Hip osteonecrosis and hip osteoarthritis are common causes of severe hip disease in lupus, both treated successfully with a total hip arthroplasty (THA). To our knowledge, comprehensive analyses for specific outcomes such as infection, revision or associated health care utilization after THA are lacking. Therefore, our objective was to assess the risk of specific post-THA outcomes, i.e., infection, transfusion, revision and mortality and associated health care utilization, associated with lupus.

Methods: We used the 1998-2014 U.S. National Inpatient Sample data. Multivariable-adjusted separate Cox proportional hazard regression models assessed the association of lupus with post-operative complications (infection, transfusion, THA revision and mortality) and healthcare utilization outcomes (total hospital charges, discharge to inpatient facility, length of hospital stay) post-THA, adjusting for demographics, underlying diagnosis, comorbidity, insurance payer, and hospital characteristics, using hazard ratios (HR) and 95% confidence intervals (CI).

Results: Among 4, 116, 485 primary THA hospitalizations, 22,557 (0.5%) were in lupus patients. Patients with lupus were younger, more likely to be female, African-American or Hispanic and, have higher comorbidity, Medicaid insurance payer, lower income, or living in the South. In multivariable-adjusted analyses, lupus was associated with a significantly higher risk of infection, transfusion, hospital charges above the median ($37,658) and discharge to inpatient facility, with respective HRs of 1.95 (95% CI, 1.28, 2.97), 1.34 (95% CI, 1.25, 1.43), 1.21 (95% CI, 1.01, 1.44) and 1.38 (95% CI, 1.30, 1.47). Lupus was not significantly associated with the risk of revision, mortality or hospital stay above the median (>3 days), the HRs were 1.10 (95% CI, 0.68, 1.78) and 0.95 (95% CI, 0.61, 1.47) and 1.06 (95% CI, 0.99, 1.13).

Conclusions: Lupus was associated with a higher risk of infection and transfusion and higher hospital charges post-primary THA. Insight into modifiable factors associated with these outcomes may improve outcomes in lupus patients undergoing THA.

PC0005: Introduction of mini clinical evaluation exercise as a mode of assessment for postgraduate students in medicine for examination of sacroiliac joints

Anuj Singhal; AFMC, Pune, Maharashtra, India

Background: Formative and subjective assessment of medical education is the need of the hour today. Mini clinical evaluation exercise (mini- CEX) and directly observed procedural skills (DOPS) are the commonly used workplace-based assessment (WPBA) tools and have been extensively studied in both undergraduate and post graduate setting. In this study we tried to assess the feasibility of mini-CEX and also its acceptability amongst postgraduate students of Internal Medicine for examination of Sacroiliac joints and teaching faculty.

Methodology: This is an interventional study which was carried out in a Government Medical college in Pune, Maharashtra from Jan to Jun 2019. A total of 18 first- and second-year postgraduate students and 9 teaching faculty from the Department of Internal Medicine participated in this study. Each student underwent 5 Mini-CEX evaluation over a period of 6 months under different teaching faculty. Feedback was taken from both the teaching faculty and students regarding the feasibility of Mini-CEX as an assessment tool.

Results: A total of 90 mini-CEX exposures involving 18 post graduate students and 9 faculty were analysed. We found a statistically significant improvement in the domain of medical interviewing (p value< 0.001), physical examination (p value= 0.003), professionalism (p value =0.001), clinical judgement (p value = 0.003), counselling skills (p value<0.001) and organizing efficiency (p value< 0.001). Overall clinical competence improved from a scale of 5(1.7) to 6.7 (0.8).

Conclusion: The Results: of this study proved that overall Mini-CEX is an acceptable and effective assessment tool. However, regular training of assessors through workshops on provision of effective feedback is required. Modification of the assessment form based on the feedbacks provided by teachers and students will further facilitate the implementation of this teaching tool in the curriculum.

Keywords: Medical education, mini clinical evaluation exercise, qualitative study, sacroilitis, workplace based assessment

Figure 1 Click here to view

PC0006: Correlation of sex hormones with disease activity in premenopausal females with rheumatoid arthritis

Pulin Kumar Gupta, Pankaj Gupta, Saurabh Tyagi, Manita Khatak, Lokesh Sharma; Department of Medicine Subdivision Rheumatology, PGIMER Dr RML Hospital, New Delhi, India

Introduction: Rheumatoid arthritis (RA) is an inflammatory disease involving multiple organ systems and the gonadal system is no exception. The relationship of sex hormones with the pathogenesis of RA is well understood however the same with disease activity is still not clear and hence the present study was planned.

Methods: It was a cross-sectional observational study done in 100 premenopausal females with definite RA. All cases with history of cardiac, renal, hepatic, metabolic disorders, biological or hormonal therapy anytime in the past or corticosteroids intake within last three months were excluded. Cases were subjected to detailed evaluation in early follicular phase of menstrual cycle.

Results: The mean age of the cases was 35.2 ± 5.35 years with mean duration of disease 4.6 ± 1.8 years. The mean DAS-28 was 3.53 ± 1.6. The proportion of cases with mild, moderate and high disease activity were 25%, 29% and 11% respectively and 33% were in remission. 12.5% of cases had RA associated deformities. 36% of females had symptoms of hypogonadism out of which nearly 50% had definite ovarian failure. A significant association was seen between female hypogonadism and exposure to methotrexate for more than three years.

A statistically significant direct correlation was seen between DAS-28 (as a marker of disease activity) as a dependant variable and serum prolactin (<0.002), estradiol (P<0.013), progesterone (P<0.005), FSH (P<0.02), LH (P<0.005) and anti ccp (P<0.005) as independent variables, while serum testosterone (p<0.016) and DHEAS (p<0.008) showed a significant inverse correlation with DAS-28. However higher VAS was numerically seen more in cases with lower levels of serum estradiol and progesterone.

Conclusion: Female hypogonadism is common in patients with RA and disease activity is directly associated with higher levels of gonadotropins and female sex hormones and lower levels of serum testosterone and DHEAS.

PC0007: Axial spondyloarthritis: MRI inflammation and disease activity in relation to smoking in an Egyptian cohort

Fatma Fayed, Mona Helmy, Mohamed Barakat, Abeer Abdelati; Department of Rheumatology and Immunology, Alexandria University, Alexandria, Egypt

Background: literature suggests that smoking is one of the crucial triggering factors of rheumatological diseases. (1) In axial Spondyloarthritis (axSpA), classified into radiographic SpA (AS) and non-radiographic SpA (nrSpA), smoking associated with disease activity and extra-articular manifestation. (2) The relationship between smoking and HLAB-27 as well as MRI inflammation in axSpA patients and the difference between nrSpA and AS regarding smoking have not been studied to date in details.

Objective: to investigate the influence of smoking on disease activity and MRI inflammation in axSpA patients (AS and nrSpA).

Methods: sixty Egyptian patients (42 males and 18 females) with the mean age (31.33 ± 7.02), with early active axial spondyloarthritis (49 AS and 11 non-radiographic SpA) within two years disease duration, diagnosed based on ASAS classification criteria. All clinical indices (BASDI, BASFI, BASMI, ASDAS-CRP) were applied to all patients. HLA-B27 and the inflammatory markers (ESR, CRP) was done. MRI of sacroiliac joints was performed in a standard protocol using short tau inversion recovery and T1 sequences (slice thickness 3-4mm, both semi-coronal and semi-axial orientations), and scored by the Berlin method. Smoking use assessed by smoking pack-year index.

Results: of all 60 patients, 38 smokers and 22 non-smokers. No significant difference regarding smoking packs index between nrSpA and AS. (p=0.822) There was a robust correlation between smoking packing index and the Berlin score of MRI in all axSpA patients (rs=0.631) (p=<0.001). Moreover, there was a significant correlation between smoking and C-reactive protein (rs=0.952) as well as HLA-B27. (rs=0.340) (p<0.001) Furthermore, a significant relationship between smoking and activity indices (BASDI (rs=0.961) and ASDAS-CRP (rs=0.938)). Otherwise, no significant correlation among smoking, BASMI, and BASFI as well as ESR.

Conclusion: Smoking has a significant association with MRI inflammation, disease activity scores as well as HLA b27 postivity

PC0008: The early effect of non-steroidal anti-inflammatory drugs on progression of newly diagnosed axial spondyloarthritis

Fatma Fayed, Mona Helmy, Mohamed Barakat, Abeer Abdelati; Department of Rheumatology and Immunology, Alexandria University, Alexandria, Egypt

Objectives: We aimed to evaluate the early effect of continuous treatment with optimum dose of NSAIDs on disease activity and radiographic progression of newly diagnosed axial spondyloarthritis (axSpA).

Methods: A six-week prospective study on thirty consecutive newly diagnosed active axSpA patients. All patients were assessed at baseline visit, a follow-up visit after 2 weeks, and after 6 weeks of treatment with a continuous optimal dose of NSAID. Assessment included calculation of ASAS NSAIDs index, inflammatory markers, patient's global assessment, physician's global assessment, quality of life index, as well as pain scale. Disease activity was assessed by determining BASDAI and ASDAS, while functional assessment was evaluated by using BASFI. Spinal mobility was assessed by the mean improvement in BASMI. Magnetic Resonance Imaging of sacroiliac joints was taken at baseline and at the end of the study and was evaluated according to Berlin scoring method.

Results: We observed improvement in Berlin MRI score, laboratory markers, as well as other clinical parameters including disease activity, spinal mobility, and pain scale. Furthermore, at week 6, ASDAS clinically important improvement and ASAS40 were achieved in 73.3% and 63.3% of patients, respectively. Additionally, BASDI50 was achieved in 30% and 56.7% of patients at week 2 and week 6, respectively.

Conclusion: A continuous intake of optimal dose of NSAIDs improves not only all clinical indices and inflammatory markers, but also the radiographic signs of activity in axial spondyloarthritis. Moreover, the higher the NSAID index the lower the radiographic progression.

PC0009: Beliefs about medications in very early rheumatoid arthritis: Indian patients are indifferent about their medication

Ankan Patel, Arup Mahapatra, Prasanta Pradhan, Sakir Ahmed; Kalinga Institute of Medical Sciences, KIIT University, Bhubaneswar, Odisha, India

Background Drug compliance is a major determinant of disease control in rheumatoid arthritis(RA). Various studies have shown that drug compliance depends on patients' beliefs towards medicine. But all of these studies are in established disease where the disease itself could have modified the patients' beliefs!

Table 1: Maximum possible scores, medians (quartiles) and percentage of high scorers obtained from the Belief about Medicines Questionnaire Click here to view

Objective: To determine the attitude of patients towards medicines in very early RA(<3 months).

Methods: 250 patients who had developed arthritis within last 3 months and who fulfilled the ACR/EULAR2010 criteria for RA were included. Demographic and clinical data were collected. Patients filled the Belief about Medicines Questionnaire(BMQ). The BMQ has 4 subsets: Specific necessity, Specific concern, General overuse and General harm. For these scores, more than scale mid-point is considered high. Depending on the Specific necessity and Specific concern scores patients can be classified as indifferent, accepting, sceptical or ambivalent.

Results: Mean (±SD) age was 47.6(±13.4) with 88.4%(221) females. 86 were graduates or above, 64 completed higher secondary school, 20 read till high school, 76 had primary education while one had no formal education.

12 (4.8%) had high specific necessity while 31 (12.4%) had high specific concern score. General overuse score high in 248 (99.2%). General harm score was high in 246(98.4%) patients. Thus, 242 (96.8%) patients were classified as indifferent, 4 (1.6%) accepting, 4 (1.6%) sceptical and none as ambivalent!

There were weak correlations (Spearman; r-square<0.1) between the subset scores, and there was no statistically significant correlations of these scores with age, income, educational status or occupation.

Conclusion: Though general harm and overuse scores were high, patients had low scores on the scales specific for RA medication. Thus most of them were classifiable as “indifferent” unlike previous studies in established RA. Further studies are required to assess if these beliefs change with increasing disease duration.

PC0010: Ultrasound imaging in evaluation of small joints of hand in rheumatoid arthritis

P P Amritha, manisha Ashwin Daware, B N Kishore Kumar; Narayana Hrudayalaya, Bengaluru, Karnataka, India

Background: Rheumatoid arthritis (RA) is a common disease in our population which significantly affects the functioning capacity of patients. With early and adequate treatment it is possible to halt disease progression and prevent irreversible joint damage.

Objectives

• To highlight the role of high frequency gray-scale and power Doppler ultrasound in small joints of hand in patients with RA and correlate with clinical and radiographic findings
• To determine whether sonography can be used as a reliable and cost effective investigation in grading the severity of RA.

Methods: In this study, 47 early RA patients with disease duration less than 2 years were included. Rheumatoid factor (RF) and ESR of the patients were measured. Bilateral hand joints and wrists were examined by ultrasonography (US) and radiography. Sonographic examination included evaluation of wrists, the second, third, and fifth metacarpophalangeal (MCP) joints; second, third and fifth proximal interphalangeal (PIP) joints. Relationship between laboratory parameters, radiographic and US findings were analyzed.

Results: US was significantly more sensitive in detecting bone erosions compared to radiography. PDUS was positively correlated with ESR(P<0.01), but not correlated with RF and disease duration (P > 0.05). GSUS was positively correlated with RF (P<0.05) but not correlated with ESR and disease duration (P > 0.05). Bone erosion was positively correlated with disease duration, and RF (P < 0.05) and was not correlated with ESR (P>0.05).

Figure 1: Mild synovial thickening in the dorsal aspect of left wrist joint with mild articular margin erosions Click here to view

Conclusion: US is very valuable in the diagnosis of early RA in synovitis synovitis, joint effusion, tenosynovitis, and bone erosion. Ultrasonography and clinical and laboratory parameters had a great correlativity. In view of the advantages of low cost and convenience, ultrasound may be a better choice during early RA diagnosis.

PC0012: Sleep quality in rheumatoid arthritis

Anil Kumar, Rohini Handa, Sundeep Kumar Upadhyaya, Sirinder Jit Gupta, Bindhyachal Kumar Gupta; Department of Rheumatology, Indraprastha Apollo Hospital, New Delhi, India

Objective: Poor sleep quality is one of the most common problem in rheumatoid arthritis patients and it significantly negatively affect the HRQoL. No study related to sleep quality in RA has been conducted in India. The aim of this study was to assess the sleep quality and factors influencing sleep quality in Indian RA patients.

Methods: This prospective questionnaire based cross sectional study enrolled, 80 patients of RA with age ≥18 years. A detailed history of basic demographic data was taken. Sleep quality was assessed by Hindi version of Pittsburgh sleep quality index (PSQI). Other patient related outcomes measured were VAS 100 for pain, DAS28 and CDAI for disease activity, HAQ-DI Indian version for function disability and modified Kuppuswamy's Socio-Economic scale 2018 for socioeconomic status. Patients were divided into good sleeper (PSQI was < 5.0) and poor sleeper (PSQI was ≥5.0) and these two groups were compared.

Results: The mean age of study participants was 49.05 ± 12.37 years and mean disease duration was 7.8 ± 6.32 years. 81% of study participants were female. The prevalence of poor sleep quality in present study was 67.50% and the mean global PSQI score was 6.52 ± 3.02. The mean VAS Pain, HAQ-DI and DAS28 were 49.49, 1.275 and 4.02 respectively. Poor sleepers had significantly higher DAS28 (4.48± 1.26 vs 2.79 ± 1.09; p=0.0001) and CDAI (20.41 ± 11.33; p=0.0001). HAQ-DI score and VAS pain score were also significantly higher in poor sleeper. Poor sleep quality was not associated with age, sex, disease duration, drugs used and socioeconomic status.

Conclusion: The majority of Indian RA patients have poor sleep quality. Poor control of disease activity and functional disability are associated with poor sleep.