Rheumatology

OB0001: Rethinking safety profile of drugs for rheumatoid arthritis

Gurvisha Sandhu, B K Thelma; Dewpartment of Genetics, University of Delhi South Campus, New Delhi, India

Background: Current treatment for Rheumatoid Arthritis (RA) comprises synthetic disease-modifying anti-rheumatic drugs and/or biologics, guided by disease activity assessment. Both drug categories exhibit drawbacks, namely limited efficacy, contraindications and side effects. Discovery of small molecules is presently a major thrust area but adverse drug reactions due to unintended activity at off-targets are a leading cause for attrition in clinical trials of such candidates or withdrawal of marketed drugs. Therefore, there is an unmet need for early safety checks in novel drug discovery.

Objectives: Using genetic and cellular network data, Mitogen-activated protein kinase kinase kinase 8 (MAP3K8) was previously prioritized in the laboratory as a potential drug target for RA. This study aims to develop a rational prediction tool for uncovering off-target liabilities of novel inhibitors designed in this study against MAP3K8, to identify 'safer' molecules for pre-clinical development.

Methodology: Firstly, we built a chemo-centric probabilistic model based on high-throughput screening of 2-D chemical similarities among 1.7 million ligand pairs from a Background dataset of >50,000 unique preclinical and clinical small molecule hits, covering 400 kinases. We then calculated Z-scores and expectation values (E-values) for ligand sets of any size, such that the Background fits an extreme value distribution [Figure 1]. E-value <10-4 for query drug against a particular protein was interpreted as statistically significant signal/noise discriminator.

Figure 1: Cheminformatics schema for target profiling Click here to view

Results: On assessing off-targets of FDA-approved drugs to validate the model, E-value predictions matched rationally with their in vitro molecular activity profiles. Of the 13 novel MAP3K8-based small molecule inhibitors, six were 'predicted as specific' with no significant off-targets. In the seven remaining, central scaffold overlapped with a drug candidate for a clinically validated target in cancer and psoriasis, warranting additional investigations.

Conclusions: The cheminformatics tool developed seems robust to identify off-target liabilities for individual chemical agents and is notably applicable universally across human kinome.

OB0002: Evaluation of the effect of human platelet lysate versus fetal bovine serum on human osteoarthritic cartilage derived chondroprogenitors

Upasana Kachroo, Shikha Zachariah, Abel Livingston¹, Boopalan Ramasamy², Elizabeth Vinod1³; Departments of Physiology and¹Orthopaedics,³Center for Stem Cell Research, CMC, Vellore, Tamil Nadu, India,²Department of Orthopaedics, Royal Darwin Hospital, Tiwi, Australia

Background and Objectives: Chondroprogenitors, considered as a mesenchymal stem cell, maybe a contender for cell-based therapy for cartilage repair due to inherent chondrogenesis and good proliferative capacity. Fetal bovine serum (FBS) is a widely used culture additive for expansion and differentiation of chondroprogenitor cells (CPs). However due to concerns related to potential transmission of zoonoses and possibility of transplant rejections following cell implantation, there is a need for a human derived alternative. Human Platelet Lysate (HPL) has been extensively for expanding various cell lines but data related to its use with chondroprogenitors has not been reported. We therefore aimed to culture human chondroprogenitors in HPL and compare them to those grown in FBS.

Methods: CPs were isolated from osteoarthritic knee joints (n=3) following differential fibronectin adhesion assay. Passage 0 cells were considered for assessment of trilineage differentiation, cell cycle analysis, galactosidase senescence assay, CD marker expression and RT-PCR. Cumulative population doubling (CPD) was assessed up to passage 3.

Results: and Conclusion: Results: obtained showed that CD marker expression, number of senescent cells were comparable between the two groups whereas cellular proliferation was significantly higher with HPL (p<0.05). Observations from trilineage differentiation showed that cells in both groups demonstrated capacity for adipogenic and chondrogenic potential, whereas ability for osteogenic differentiation was notably higher with HPL group. Similar Results: were noted in RT-PCR where cells grown in FBS showed lower expression of markers of hypertrophy.

This was the first in vitro study to establish xeno free conditions for expansion and characterization of human CPs. Although Results: presented in the study identify HPL as an effective medium supplement for growth of human articular cartilage derived chondroprogenitors, further evaluation is required for consideration of HPL as an additive in cellular differentiation towards osteogenic lineage for bone regeneration.

Keywords: Chondroprogenitors, FBS, human platelet lysate

OB0003: P-gp and HDAC2 may regulates steroid responsiveness in childhood nephrotic syndrome

Harshit Singh, Narayan Prasad¹, Akhilesh Jaiswal¹, Durga P. Misra, Ravi Mishra, Vikas Agarwal; Departments of Clinical Immunology and¹Nephrology, SGPGIMS, Lucknow, Uttar Pradesh, India

Background: P-glycoprotein (P-gp) over expression in peripheral blood mononuclear cells (PBMCs) has been reported in patients with steroid resistant nephrotic syndrome (NS). Glucocorticoids suppress NFκB-associated co-activator activity by deacetylation of histone by enzyme histone deacetylase (HDAC)-2.Interaction between HDAC2 activity and P-gp expression in childhood NS patients is not clear.

Aim: To evaluate the role of HDAC2 and P-gp expression on PBMCs and steroid responsiveness in patients with childhood NS.

Materials and Methods: 31 patients were recruited at baseline (n = 31) (before initiating steroid therapy);after six weeks of steroid therapy, 24 patients achieved remission (SSNS n=24 mean age, 7.96±3.90), whereas seven patients were resistant to steroids (SRNS, n=7, mean age 10.00±3.55).mRNA expression of HDAC2 and P-gp/MRP-1 and functional analysis of P-gp as well as enzymatic activity of HDAC2 were analyzed at baseline, at 6 weeks of steroid treatment and at the time of relapse, respectively.

Results: The expression of P-gp mRNA was significantly lower in subjects (n=24) who achieved remission at 6-weeks of steroid therapy as compared to baseline and those who were resistant (n=7) to steroids (P<0.005). Similarly, expression of HDAC2 mRNA was significantly higher at baseline and at remission following 6-weeks of steroid therapy as compared to their expression in those who were resistant to steroids (P<0.005).The function of P-gp was significantly lower in NS patients who achieved remission after 6-weeks of steroid therapy as compared to baseline (p<0.005) and those who were resistant to steroid therapy (p<0.005).The enzymatic activity of HDAC2 was significantly higher in SSNS patients as compared SRNS patients at 6-weeks of steroid therapy (p<0.005).

Conclusion: The expression and function of P-gp and HDAC2 may affect steroid response in NS patients. Combined therapy of steroids with P-gp inhibitor and/or HDAC2 inducers may have rationale in the management of steroid resistant NS patients.

OB0004: A peptide vaccination approach to ablate auto reactive B-cells as a novel strategy to treat autoantibody-mediated diseases

Ram Raj Singh; University of California, Los Angeles, CA, USA

We previously demonstrated that the heavy chain variable regions of anti-DNA antibodies contain epitopes that can bind MHC class I molecules. Vaccination of lupus-prone mice with plasmid DNA vectors carrying minigenes that encode such epitopes induced CD8+ cytotoxic T lymphocytes (CTL) that killed anti-DNA antibody-producing B cells, reduced serum anti-DNA antibody levels, retarded the development of nephritis, and improved survival (Fan G and Singh RR, J Exp Med 2002). Treatment with peptides alone did not induce such CD8+ T-cells, as we and others have reported impaired CD8+ regulatory and CTL responses in mice and humans (Singh RR, et al, J Immunol 2002; Stohl W, et al, Lupus 1999). Here, we asked if we could overcome such impairment using synthetic oligodeoxynuleotides containing unmethylated cytidine-phosphate-guanosine dinucletides (CpG-ODN) that can enhance innate and adaptive immunity.

We immunized lupus-prone (NZB/NZW F1) mice that develop lupus that mimics human lupus with CpG-ODN or a control ODN, and assessed CTL responses. We then treated these mice with CpG-ODN conjugated to MHC class I-binding epitopes that we identified using bioinformatic and cellular binding approaches. We monitored lupus mice for proteinuria, serum anti-dsDNA antibodies, and survival.

Immunization with CpG-ODN corrected the impairment in peptide-specific CTL responses in lupus-prone mice. The CpG-ODN conjugated with an MHC class I-binding, anti-DNA Ab VH-derived epitope induced strong peptide-specific CTL responses against B cells from diseased lupus-prone mice, reduced anti-DNA Ab production, inhibited disease development, and improved survival.

Such peptide vaccine-mediated induction of CTLs that ablate autoreactive B cells represents a novel approach to treat autoantibody-mediated diseases.

OB0005: Methanolic extract of choerospondias axillaris fruit decreases inflammatory response in rheumatoid arthritis fibroblast like synoviocytes and collagen-induced arthritis model

Sonia Mann, Ashish Sarkar, Sagarika Biswas*; CSIR-Institute of Genomics and Integrative Biology, New Delhi, India

Background: Rheumatoid arthritis is an autoimmune, systemic disease which mainly affects joints. Recently the focus has been diverted on traditional medicinal plants for treatment of arthritis. Choerospondias axillaris, Lupsi/Lapsi, is an underutilized and edible fruit of family Anacardiaceae possessing many health benefits.

Objective: Initially Phytochemical analysis of methanolic extract of Choerospondias axillaris has indicated the presence of phytochemicals such as phenols, flavonoids, flavonols, alkaloids etc. As these compounds are of pharmacological interest, coupled with the use of this plant in traditional medicine, encouraged us to check in vitro and in vivo anti-inflammatory activity of Choerospondias axillaris fruit extract.

Methods: The antioxidant in vitro activity was measured by means of the free radicals scavenging assays of the extract. Bioactive compounds of Choerospondias axillaris were identified via LC-MS/MS analysis. The anti-inflammatory effect of methanolic extract was investigated in Rheumatoid arthritis (RA) and Osteo arthritis (OA) primary cells. The study was further extended in collagen-induced arthritis (CIA) rat paw edema model. Further, the medicinal influence of anti-inflammatory bioactive compound of Choerospondias axillaris methanolic extract, were supported by docking studies of ascorbic acid, salicylic acid, quercetin and diclofenac with TNF-α and IL-6.

Results: Both in-vitro and in-vivo studies showed significant decrease in the inflammation. Also, to check the medicinal efficacy of anti-inflammatory bioactive compound of Choerospondias axillaris methanolic extract, docking studies of ascorbic acid, salicylic acid, quercetin and diclofenac with TNF-α and IL-6 was carried out. Docking analysis indicated that salicylic acid inhibits TNF-α with -7.58 kcal/mol binding energy and 2.78 μM inhibitory constant whereas quercetin inhibits IL-6 with -6.36 kcal/mol and 21.9 μM inhibitory constant.

Conclusion: Observed Results: suggest the possible use of Choerospondias axillaris fruit for reducing the inflammatory indicators and symptoms in case of inflammatory diseases such as RA.

OB0006: Synovial cell-derived micro particles as source of auto antigens and their in vitro effects on synoviocytes in rheumatoid arthritis

Benita N R Michael, K G Chengappa, V S Negi; Department of Clinical Immunology, Jawaharlal Institute of Postgraduate Medical Education and Research, Pondicherry, India

Background: The role of Cell-derived micro particles (MPs) in the pathogenesis of Rheumatoid arthritis is not clearly delineated. The present study investigated the hypothesis that MPs may be a source of auto antigens and drive disease progression in the synovium.

Methods: Five ml of synovial fluid (SF) was collected from knee joints of 41 DMARD naïve patients with RA and 30 patients with OA. Cell-free synovial samples were stained with Annexin-V-APC with either anti-vimentin Alexa Fluor-488 or anti-Glucose regulated protein-78 (GRP78) Dylight-488 for flow cytometry analysis. RA and OA fibroblast-like synoviocytes (FLS) (Cell applications, USA) were co-cultured with respective synovial fluid-derived MPs in vitro for 24 hours. The supernatant was assayed for inflammatory analytes by multiplex assays.

Results: Elevated proportion of AnnexinV+Vimentin+ MPs were present in RA [0.8(1.30)] [median (IQR)] compared to OA [0.3(0.33); p<0.001]. Elevated proportion of AnnexinV+GRP78+ MPs were also found in RA [0.3(0.28)] compared to OA [0.1(0.2); p<0.01]. Among 16 cytokines studied, RA synovial fluid-derived MPs stimulated RA FLS to produce CXCL1, CXCL2, CXCL5, CXCL6, CCL2, CCL8, CCL15, IL8, TNFa, RANTES, and BAFF at high concentrations compared to unstimulated FLS. CXCL6, CCL2, CCL8, CCL15, RANTES, IL6, VEGF, and BAFF were released at higher levels when RA FLS were stimulated with RA synovial fluid-derived MPs compared to OA FLS stimulated with OA synovial fluid-derived MPs. CXCL6, CCL8, and RANTES were higher when RA FLS were stimulated with RA Synovial fluid-derived MP as opposed to MP free SF. However, the stimulatory effects of MPs were lesser than that observed with positive controls (Il-1beta and LPS) [Figure 1].

Figure 1: Levels of CXCL6, CCL2, CCL8, CCL15, RANTES, IL6, VEGF, and OA FLS cell stimulation as-says (a-h). Cells were either left unstimulated (negative control) or stimulated with synovial fluid MP, MP free synovial fluid (2.5 μl), IL-1β (125 pg/well) (thermoflished scientific, USA) (positive control), and lipopolysaccharides from  Escherichia More Details coli) O26;B6 [LPS (1 μg/well) (sigma asdrice, USA) positive control] for 24 h. The data are expressed as mean ± standard deviation of three independent experiments (n = 0). The variation between two independent groups was statistically assessed by unpaired t-test with Wekh's correction. Horizontal lined indicates standard deviation, CXCL, (C-X-C) motif ligand; CCL, (C-C) motif ligand; IL6: Interleukin-6, RANTES: Regulated on activation, normal T-cell expressed and secreted, VEGF: Vascular endothelial grouth factor, BAFF: B-cell activating factor, NV: Negative control (Unstimulated cells), MP: Microparticle, SF: Synovial fluid, LPS: Lipopolysaccharides, RA FLS: Rheumatoid arthritis fibroblast-like synoviocytes, OA FLS: Osteoarthritis fibroblast-like synoviocytes, *P < 0.05, **P < 0.01, ***P < 0.001 Click here to view

Conclusions: MPs are a source of auto antigens in RA synovium. MPs may stimulate synoviocytes and contribute to local production of autoantibodies, neovascular angiogenesis, and activation of autoreactive cells in RA.

OB0007: Accumulation of deleterious mitochondrial DNA in peripheral blood mononuclear cells of rheumatoid arthritis patients

Kumar Sagar Jaiswal, Shweta Khanna, Arup Ghosh, Prasanta Padhan, Sunil K Raghav, Bhawna Gupta; School of Biotechnology, Kalinga Institute of Industrial Technology, Bhubaneswar, Odisha, India

Background: Due to absence of a defined etiology Rheumatoid Arthritis has become a systemic, debilitating and chronic inflammatory disorder affecting 1% of the total world population. Studies report blend of mitochondrial, environmental, genetic and epigenetic factors responsible for disease progression. Mitochondria are involved in cellular functions and are a deciding factor for fate of immune cells. Mutations in mitochondrial DNA (mtDNA) may alter cellular functions, thus resulting in inflammation. Finding “one for all” cure is difficult due to genetic variations among populations; thus deciphering genetic, epigenetic information and difference in regulatory network between populations can lead us to design a cure based on the principles of personalized medicine.

Objectives: This study aims to identify single nucleotide polymorphisms (SNPs), copy number variation due to deletion or duplication and heteroplasmy in mtDNA hampering overall function of mitochondria.

Methods: mtDNA were sequenced using NGS from 23 RA patients and 17 healthy controls (HC). Further, bioinformatics analyses were used to identify SNPs, changes in heteroplasmy, copy number variations in RA patients and HC along with haplogroup analysis to find any association between mutated mtDNA and RA.

Results: A total of 382 single nucleotide variants were observed in mtDNA; among which 91 (23.82%) of mutations were present in hypervariable region and 291 (76.18%) in coding region of patients and controls. A non-synonymous heteroplasmy in ND1 gene was found at a single position 3533 in increased number of RA patients as compared to HC. Upon analysing copy number variations we could observe a significant increase of duplications in RA mtDNA samples.

Conclusion: The findings of this study indicate altered mtDNA of immune cells and possible role of mitochondria of immune cells in development of RA. The variants identified can be used as prognostic marker for individuals at high risk of developing RA.

OB0008: Role of altered CD14 expression triggering the fibrotic pathophysiology in the patients with systemic sclerosis

Dipanjan Bhattacharjee¹, Aniruddha Bagchi^1,2, Deepak Rath¹, Ayindrila Saha¹, Sanchaita Misra¹, Sudipta Chatterjee¹, Sulagna Chatterjee¹, Pradyot Sinhamahapatra¹, Alakendu Ghosh³; ¹Institute of Post Graduate Medical Education and Research/SSKM Hospital, Departments of³Rheumatology and²Pharmacology, Institute of Post Graduate Medical Education and Research/SSKM Hospital, Kolkata, West Bengal, India

Background: Fibrocytes, the cells of leukocyte lineage, after activation, express markers of fibroblasts [CD90, α-smooth muscle actin (αSMA), Collagen-1(Col1)] by reducing leukocytes markers (CD34, CD45, CD14). Fibrocyte activation and transdifferentiation may trigger the fibrotic pathophysiology.

Objectives: Isolation and characterization of blood-borne fibrocytes and its involvement in scleroderma pathophysiology.

Methods: Bloods were collected from 10 diffused cutaneous SSc (dSSC) patients [ACR, 2013] and 10 age-sex matched healthy controls. Characterization of fibrocytes was identified with specific antibodies by FACS.

Results: Two subpopulation of: CD14high and CD14Low have been found within the CD45+ cells. The percentage of these two subpopulations are inversely represented in scleroderma patients (CD14high: 12 % and CD14Low: 84%) with respect to healthy control (CD14high: 80% and CD14Low: 20%). The expression of Col-I, αSMA and CD90 are significantly higher (p= 0.0004, p=0.001, p=0.02 respectively) in the CD14high population of patients than controls. Patients have an increased expression of Col-I, αSMA and CD90 (p< 0.0001, p=0.006, p=0.001 respectively) in the CD14high population compared to the CD14Low population. There is a clear indication that the patients have higher amount of CD90 expressing cells among the total CD14 population than healthy controls. Percent of CD14high cell population is not correlated (r2 =0.09, p=0.05) with the disease duration, rather showing a positive significant correlation with mRDSS (r2 =0.2, p=0.02).

Discussion: patients' having altered expression of CD14+ cells with increased number of CD45+ / CD14high population which significantly express high amount of Col1.The increased number of CD90+/αSMA+ population of cells in patients showing the involvement of activated circulating fibroblasts that might be a potential inducer of uncontrolled fibrosis in Scleroderma. The higher expression of Col-1 and activation markers of fibroblasts by CD45+ / CD14high subpopulation indicating that this predominant subpopulation in patients might acts as a potent contributor of fibrosis.

OB0009: Local, organ-specific mechanisms may explain the heterogeneity of SLE

Ram Raj Singh^{1, 2, 3, 4}, Anna U Eriksson¹, Peter J Kim¹, Darshan Randhawa¹, Rachael L Philips¹, Jennifer King¹, Miguel-Angel Gutierrez^1,2; ¹Department of Medicine, Division of Rheumatology, Autoimmunity and Tolerance Laboratory,²Molecular, Cellular, and Integrative Physiology Graduate Program,³Molecular Toxicology Interdepartmental Program,⁴Department of Pathology and Laboratory Medicine, David Geffen School of Medicine, University of California at Los Angeles, Los Angeles, California, USA

Background: Current treatments for lupus involve suppressing systemic immunity, thus increasing the risk of adverse effects. Identifying organ-specific mechanisms and targets may lead to organ-specific treatments. Skin harbors specialized subsets of dendritic cells (DC), including Langerhans cells (LC) in the epidermis, and LangdDC in the dermis. These DCs take antigens from skin and deliver them to skin-draining lymph nodes in order to induce immunity (in case of skin infection) or tolerance (to avoid autoimmunity against skin). We previously reported that the migration of LC is impaired in lupus (Eriksson and Singh, J Immunol 2008), and that this defective migration played a role in the loss of tolerance to skin antigens in lupus (King J…Singh RR, J Immunol 2015). Skin also harbors specialized T-cells called dendritic epidermal γδ T-cells (DETC). Here, we investigated the interplay between LCs and DETCs in the pathogenesis of lupus dermatitis using the MRL model.

Methods: To be able to track and selectively deplete skin-DC in vivo, we generated langerin-driven eGFP gene knock-in and diphtheria toxin receptor knock-in mice, respectively, in genetically lupus-prone MRL model.

Results: DETCs directly modulated LC migration via direct cell-to-cell interaction. Lupus-prone mice had reduced DETCs and reduced LC-migration; both these defects were corrected by glycolipid-treatment that ameliorates lupus dermatitis. Finally, selective depletion of LC accelerated cutaneous lupus. LC-depletion or glycolipid-treatment did not affect anti-DNA antibodies or lupus nephritis.

Conclusion: Skin-DETCs regulate the migration of skin-DCs, which is impaired in lupus leading to autoimmune dermatitis. Glycolipid treatment that corrects this defect improves lupus dermatitis but does not affect disease in other organs.

Implications: We identified a novel mechanism whereby interplay between tissue-resident specialized immune cells regulates local immunity in an organ. Such specialized local regulation of autoimmunity at the tissue level can be targeted to develop tissue-specific treatment without affecting systemic immunity.

OB0010: Effect of curcumin on pro-inflammatory cytokines in primary sjögren's syndrome

Jayakanthan Kabeerdoss, Pulukkol Sandhya, M Hindhumathi, Debashish Danda; Department of Clinical Immunology and Rheumatology, Christian Medical College, Vellore, Tamil Nadu, India

Introduction: Curcumin reduces disease severity and ameliorates lupus-like/ Sjögren's Syndrome-like disease in mice model. The immunological basis of these effects is unknown. This study examined the effect of curcumin on pro-inflammatory cytokines secreted by salivary gland in patients with primary Sjögren's syndrome (pSS).

Methods: Minor salivary gland (MSG) tissue was collected after obtaining written consent from patients undergoing biopsy as a part of evaluation for suspected pSS . The tissue was treated with phytohemagglutinin (PHA) alone as well as PHA with curcumin (30μM) and cultured in RPMI 1640 medium for 48 hours at 37°C in CO2 incubator. After the incubation period, culture supernatant and tissue were stored in the ultra-deep freezer(-80 °C). IL-6 levels were measured in supernatant by commercially available ELISA kits. RNA was extracted from tissue using Tri reagent method. Expression of pro-inflammatory cytokines, IL-6, IL-8, TNF-α, IL-1β, IL-4, IL-21, IL10 and IFN-γ was done by qPCR. Differences between groups were tested by Student's t-test.

Results: Forty-seven patients were recruited. Eight patients satisfied ACR/EULAR criteria for pSS. Seven patients with absent glandular inflammation and negative serologies constituted controls. In pSS group, but not in controls, median IL-6 levels in supernatant was less in curcumin treated as compared to PHA alone (5.5 (0.7 -1.3) vs 18.3 (12-32) ng/ml;p=0.04). mRNA expression levels of IL-6 and IL-1β were lower in curcumin treated groups as compared to PHA alone in both cases and controls (p= 0.0009 & p=0.04 respectively). There was no difference in other cytokine levels between the treatment groups. mRNA expression levels of IL-4, IL-21 and IL10 were below detectable range.

Conclusion: Curcumin reduces secretion of IL-6 levels in salivary gland tissue of patients with pSS. Curcumin suppressed PHA induced mRNA expression levels of IL-6 and IL-1β in MSG tissue of pSS and sicca controls.

OB0011: NMR-based serum, urine and muscle metabolomics in inflammatory myositis for diagnosis and activity assessment: Serum metabolomics can differentiate active from inactive myositis

Latika Gupta, Dinesh Kumar¹, Umesh Kumar¹, Anupam Guleria¹, Ritu Raj¹, Ramnath Misra*; Department of Clinical Immunology,¹Centre of Biomedical Research, SGPGIMS, Lucknow, Uttar Pradesh, India

Objective: To identify changes in metabolomics profiles in serum, urine and muscle of patients with myositis with active and inactive disease.

Methods: Sera (n=116), urine (n=114) and muscle (n=11) from patients classifiable as myositis by the ACR-EULAR criteria [34 years (23.5 - 50.5 IQR), M/F 28:88] were compared with healthy controls [n=18 and 12 respectively, age= 44 (35-50) years, M/F-8:10]. For the muscle biopsies, two disease controls were used for comparison. To study effect of fasting state on urine metabolomics, 50 paired urine samples obtained in fasting and non-fasting states were analyzed.

Metabolic profiles were obtained at 800 MHZ NMR spectrometer and compared using multivariate partial least-squares discriminant analysis (PLS-DA). The discriminatory metabolites were identified based on variable importance in projection (VIP) statistics (p-value <0.05). Paired T tests were done for metabolites in urine and muscle after normalizing for creatinine. MDAAT>=1 was used to define active myositis.

Results: and discussion: Metabolomics profiles in IIM were distinct from healthy controls [Figure 1]a.Patients with active myositis exhibited differential clustering from those with inactive myositis [Figure 1]b with lower amino-acid metabolites [Figure 1]c elevated phenylalanine-to-tyrosine ratio and glutamate to glutamine ratio [Figure 1]d and low creatinine [Figure 1]e with significant discriminatory potential [Figure 1]f.

Figure 1: Serum metabolomics (a) three dimensional score plot obtained from PCA analysis of 1D 1H CPMG NMR based serum metabolic profiles showing clustering and separation between healthy control and inflammatory myositis patients (b) two dimensional score plot after PCA analysis of sera from myositis patients with active and inactive disease (c) screening of metabolites responsible form the group separation in the score plot identified based on variable importance in projection score > 2.0 (d) variable importance in projection projection suggesting elevated phenylalanine-to-tyrosine ratio (PTR) and glutamate to glutamine ration (EQR) (e) variable importance in projection projection suggesting elevated significantly decreased in the sera of active myositis patients (f) representative ROC curves of discriminatory metabolites Click here to view

Urine in IIM exhibited higher creatine and lower urea and creatine than healthy controls [Figure 2]a and [Figure 2]b. However, urine metabolomics exhibited similar clustering in active and inactive disease [Figure 2]c. In urine, paired samples in fasting and non-fasting state exhibited overlapping clustering [Figure 2]d apart from higher creatine and creatinine [Figure 2]e.

Figure 2: Urine metabolomics (a) three dimensional score plot obtained from PCA analysis of 1D 1H CPMG NMR based urine metabolic profiles showing clustering and separation between healthy control and inflammatory myositis patients (b) variable importance in projection projection suggesting elevated creatinine, and low urea and creatine in myositis versus healthy controls. (c) PCA suggests no differential clustering between active and inactive myositis. (d) In urine, paired samples obtained in fasting versus nonfasting state were fairly similar, (e) apart from differences in creatine and creatinine Click here to view

Muscle in IIM exhibited almost absent levels of sucrose, mannose, uridine, histamine, inosine and carnitine compared with infection associated myositis.

Conclusion: Serum metabolomic profiling using NMR has the potential to discriminate active from inactive myositis patients. Muscle metabolites holds potential to distinguish inflammatory myositis from infectious polymyositis.

OB0012: Peripheral T helper subset profiling is different in various subsets of idiopathic inflammatory myositis

Anamika Kumari Anuja, Harshit Singh, Durga P Misra, Vikas Agarwal, Latika Gupta; Departemnt of Clinical Immunology, SGPGI, Lucknow, Uttar Pradesh, India

Introduction: There is dearth of biomarkers in Idiopathic Inflammatory Myositis(IIM) to identify ongoing inflammation in the muscle and distinguish it from inactivity or damage.

Objective: Since myositis is autoantibody mediated and tertiary lymphoid organogenesis(TLO) reported in the diseased muscles, we looked for peripheral blood T helper subset profiling as a reflection of ongoing muscle inflammation.

Methods: Twenty-six patients of IIM(ACR EULAR criteria) were compared with 15 healthy controls(HC) and 21 patients with sarcoidosis. Peripheral blood mononuclear cells were stained with combinations of antibodies to identify Th1, Th17, Th17.1 and Treg cells after stimulation assays (BD Biosciences). Myositis Specific and Associated autoantibodies were tested by the line immunoassay(Euroimmune, Germany).

Results: All T helper subsets were higher in myositis as compared with healthy controls [Figure 1]a, [Figure 1]B, [Figure 1]C, [Figure 1]d. Between various IIM subsets, polymyositis had higher Th1 and Treg cells [Figure 2]b and [Figure 2]c while Th17 and Th17.1 cells(c) were higher in Overlap Myositis [Figure 2]a and [Figure 2]d as compared with healthy controls. Patients with sarcoidosis had similar subset profiling as myositis [Figure 5]a, [Figure 5]b, [Figure 5]c, [Figure 5]d, [Figure 5]e, [Figure 5]f.

Figure 1 Click here to view

Figure 2 Click here to view

Figure 3 Click here to view

Figure 4 Click here to view

Figure 5 Click here to view

Patients who were had either arthritis or were positive for myositis specific autoantibodies had higher Th17.1 cells [Figure 3]a(iii) and [Figure 3]b(iii) than those negative for MSA. There was no difference in T cell profile between autoantibody subsets [Figure 6]a, [Figure 6]b, [Figure 6]c, [Figure 6]d.

Figure 6 Click here to view

There was no difference in subsets between active and inactive disease although active disease had lower Th1/Treg, Th17/Treg and Th17.1/Treg ratios.

Conclusion: T Helper cell subsets are distinct from HC but similar to sarcoidosis patients. However, they differ in various types of myositis, suggesting that possibly T cell populations(Th1, Th17) might be migrating to muscle tissue, hence, lesser in peripheral blood. Autoantibody positivity is associated with elevated Th17.1 population suggesting plasticity in TLO which needs to be explored further. However,T cell profiling cannot distinguish active from inactive disease limited predictive potential as a biomarker.

OC0001: Comparison of methotrexate and glucosamine in primary knee OA

Biswadip Ghosh, Subhankar Hadar, Meghna Saha; Department of Rheumatology, IPGME and R and SSKM Hospital, Kolkata, West Bengal, India

Background: Osteoarthritis (OA) is most common rheumatologic disease but it attracts little attention in comparison from rheumatologists. Many patients with osteoarthritis show some features of inflammation, though the traditional thinking is that, OA is a degenerative disease.

Objective: To compare effect of methotrexate and glucosamine in primary Inflammatory knee OA.

Methods: 344 patients (40-70 years of age) of primary knee OA were examined. 249 patients with local clinical inflammation were recruited, subdivided in systemic Non-Inflammatory group (77) and Inflammatory group (172) based on serum levels of ESR and CRP. Those 172 patients of inflammatory group were randomly allocated to Methotrexate (15-20 mg/wk) or Glucosamine (1500mg/day) group and observed for three months. WOMAC was calculated before and after treatment. 137 patients completed the study of which 59 patients received glucosamine and 78 patients received methotrexate. MRI of knee (3 tesla) was done in every patient.

Results and Analysis: No significant difference in WOMAC, ESR or CRP was found in glucosamine group after three months. Methotrexate produced significant changes in WOMAC, ESR and CRP after three months in primary knee OA patients. There is decrease in swelling of knees in nearly all patients and improvement in pain in most of them after three months of taking methotrexate.

Conclusion: Large number of patients of primary knee OA show signs of local inflammation. Many of them have raised acute phase reactants like ESR and CRP, signifying systemic inflammation. These patients may be benefitted with methotrexate administration.

Funding: Department of Science and technology, Government of West Bengal, India.

OC0002: Utility of testing conventional and non-conventional anti-phospholipid antibodies in suspected obstetric anti-phospholipid syndrome

G Arvind, K Jayakanthan, Aswin Nair, Ashsih J Mathew, Ruchika Goel, John Mathew, John Anthony Jude Prakash¹, Sukesh Nair², Debashish Danda; Departments of Clinical Immunology and Rheumatology,¹Microbiology and²Immuno-haematology and Transfusion Medicine, CMC, Vellore, Tamil Nadu, India

Background: Anti-phospholipid syndrome (APS) is an important cause for recurrent pregnancy losses (RPL). Conventional APS antibodies (aPLs) like lupus anti-coagulant (LA), anti-cardiolipin( ACL) and anti-beta 2 glycoprotein I ( anti-β2 GP I) are not present in significant number of obstetric APS(OAPS) patients, leading to a state described as “ sero-negative” OAPS ( SNOAPS). Recent literature shows non-conventional aPLs like Anti phosphatidylserine-prothrombin complex (Anti-PSPT) and Anti-Annexin V (Anti-Ann V) can be positive in up to 50% of SNOAPS patients.

Objectives: Testing performance of conventional and non-conventional aPLs in suspected OAPS patients (obstetric events as defined in the Sydney classification criteria for APS)

Methods: We performed a retrospective chart review of 84 patients who underwent combined testing for non-conventional aPLs for suspected OAPS from May 2015 to August 2019 at our department. Patients were categorized into OAPS cases (n=42, median age 31 years) and controls (n=42, median age 31.5 years) based on their fulfilment of clinical definition of OAPS events defined by Sydney criteria. Conventional aPLs were tested by Methods: adapted in Sydney criteria and Anti PSPT /Anti Ann V were tested by commercial ELISA.

Results: 31 cases (73.8%) were 'sero-positive' & 11 cases (26.2%) were truly 'sero-negative' for conventional aPLs. Four (36.4%) of the SNOAPS patients were positive for Ant-PSPT and/or Anti AnnV antibodies. Performance of the various aPLs in suspected OAPS is displayed in [Table 1].

Table 1: The performance of the various conventional and nonconventional anti-phospholipid syndrome antibodies in suspected obstetric anti-phospholipid syndrome cases Click here to view

Conclusion: In a delicate situation like RPL, performance of non-conventional aPLs on their own, though not as sensitive as conventional aPLs, still demonstrate better specificity. The real value of testing Anti PSPT & Anti Ann V in RPL, is combined testing with conventional aPLs wherein they improve the sensitivity and accuracy of diagnosis of OAPS by 10% & 3.5% with only 2.4% drop in specificity. Non-conventional aPLs should be tested in SNOAPS.

OC0003: Demographic, clinical, laboratory and genetic studies of patients with Palindromic rheumatism: a single center experience

Padmanabha Shenoy, Sanjana Joseph, Sreelakshmi Sreenath, Ansu Abu Alex, Sageer Babu, Manjesh V Pai; Care, Centre for Arthritis and Rheumatism Excellence, Cochin, Kerala, India

Introduction: Palindromic rheumatism (PR) is characterized by multiple, episodic and recurrent attacks of arthritis, without residual joint damage occurring at irregular intervals.

Aim: To do a comprehensive demographic, clinical laboratory and genetic studies on patients diagnosed with PR.

Patients and Methods: 350 patients diagnosed with PR based on PR classification criteria from 2014-2018 were included in the study. Whole exome sequencing was done in PR and RA cohorts with the help of Medgenome.

Results: Most of the patients were under the age of 49yrs (n=183) and had female predominance of 72% (n=255). 83 patients showed disease symptoms for greater than 10 years. The major symptoms were redness (54% of patients) and swelling (40% cases). 47% of patients showed Rheumatoid factor positivity (n=334), 60% were ACPA positive (n=328) and 137 cases were both positive. Majority of the patients were on hydroxychloroquine (n=322) and colchicine (n=302) . Prednisolone was given in 83 patients who was tapered from average dose of 3.5mg to 1.84mg and stopped in 18 patients. Methotrexate, Sulfasalazine and leflunomide were also used as second line agents.

Whole exome sequencing was done to find variants that are associated with prevention of persistent inflammation in PR patients (protective variants) in contrast to RA patients (n=10 each).The frequency of IL10RA, IL10RB and HLADRB1 were more in PR. And there were 18 alleles unique to PR and 20 alleles unique to RA and 7 in common. Detailed analysis has to be done further to elucidate the mechanisms unique to PR.

Conclusion: This study is a large case series of patients with PR from a single center looking at comprehensive demographic, clinical and laboratory parameters. More studies on genes involved in inflammation and immune response has to be studied further to identify the molecular and genetic mechanisms in PR.

OC0004: Assessing the risk of maculopathy in Indian patients using hydroxychloroquine for rheumatic complaints: A cross sectional observational study

Arindam Nandy Roy, Yarram Ashok Kumar, Syeda Sana Fatima; Department of Rheumatology, Yashoda Hospital, Secunderabad, Telangana, India

Background: Hydroxychloroquine (HCQ) has shown benefits in treating rheumatic diseases such as SLE , RA and scleroderma. The risk of developing irreversible maculopathy and consequent vision loss is a possible serious complication with HCQ use. Modern day screening Methods: can detect retinopathy early in such patients.

Objective: Primary aim was to assess the prevalence of HCQ maculopathy in Indian patients with rheumatic diseases by modern day screening Methods: Secondary aim was to look for the risk factors of HCQ maculopathy.

Methods: This cross-sectional study was carried out in the Dept of Rheumatology,Yashoda Hospital, Secunderabad between July 2017 to March 2019. 984 subjects having different rheumatic diseases, who had used HCQ for 1 year and beyond and evaluated with at least Humphrey Visual Fields were included. Retinopathies other than HCQ were excluded. Informed consent was taken.

All data regarding Age, Gender, BMI, Diagnosis, Comorbidities, Daily dosage and Duration of HCQ use and Screening Methods: (Humphrey Visual Fields, Spectral Domain Ocular Coherence Tomography, Fundus Auto Fluorescence, Multifocal Electroretinogram, Fundus Fluorescein Angiography) were noted.

Chi Square test was used for statistical analysis and p<0.05 was considered significant.

Results: Maculopathy was found in 13.5% patients on HCQ beyond 1 year No statistical association was seen between HCQ maculopathy and Gender (p= 0.189), BMI (p=0.289), diagnosis (p=0.865), Comorbidities and daily dosage of HCQ (p=0.171).

However, a significant correlation was found between HCQ maculopathy and Age {8.4 %< 30 years VS 20.3%>60 years (p=0.033)}, Weight {8.7 %<50kg VS 15.1%>60kg(p=0.045)}, HCQ duration {11.7%<5 years VS 21.1%>5 years (p=0.002)} and Cumulative dose {283.79 g VS 231.33 g (p=0.006)}.

The detection rates of maculopathy by different Methods: were HVF (13.5%), SDOCT (13.3%), mfERG (12.8%), FAF (12.1%), FFA (11.8%), HVF+SDOCT (13.3%), HVF+mfERG (12.8%) and HVF+FAF (12.1%)

Conclusion: Hydroxychloroquine maculopathy is not infrequently seen in rheumatic disease patients in India.

OC0005: Infliximab usage in kawasaki disease: Sharing an experience over 3.5 years

Priyankar Pal, Mandira Roy, Jigna Bathia, Alolika Mondal, Mallar Mukherjee, Anil Kr Singhi; Pediatric Rheumatology and Cardiology Unit, Institute of Child Health, Kolkata, West Bengal, India

Introduction: Tumor Necrosis Factor (TNF-α) blocker Infliximab (IFX) is emerging as an important drug in management of Kawasaki Disease (KD). This study was undertaken to evaluate the effectiveness of Infliximab in severe KD on 33 children over a period of 3.5 years.

Objectives:

1. Determine effectiveness in IVIg resistant KD.

2. Evaluate it's response if any on developing/ increasing coronary artery aneurysms (CAAs).

Methodology: Study was carried out in IPD of Rheumatology Unit of Institute of Child Health, Kolkata. 33 children aged between 6 weeks to 7 years with KD were included who received IFX after 1-2 doses of IVIG due to persistent fever/ increasing CAA or developing new CAA. Patients were analyzed for response in terms of achievement of defervescence in days, normalization of CRP and improvement in echocardiographical findings specially CAAs.

Results: IFX was used in 1) IVIG resistant fever(18/33) 2) increasing CAAs post IVIg) (10/33). Overlapping indications ie. IVIG resistance with increasing CAAs was also present (7/33). 3 patients received IVIG twice before receiving Infliximab. IFX was given at 5mg/kg and defervescence was achieved within 24 hours of administration in 23 of the 25 resistant cases along with normalization/ drastic fall in CRP. 2 children whose fever persisted were later diagnosed as SOJIA.

17 children with severe CAAs were followed up by echocardiography weekly till 6 weeks, monthly till 3 months, and then at 6 months and 1year.15 of them had significant reduction in size, giant aneurysms being converted to medium or small sized aneurysms over 6 to 18 months.

Conclusion: Infliximab showed 100% success in IVIG resistant patients. Its beneficial effect on the CAAs seems promising, but further follow-up is required for validation.

OC0006: Short and long-term outcome of immunosuppressive therapy among Indian juvenile lupus nephritis patients

Rudra Prosad Goswami, Parasar Ghosh; Department of Rheumatology, IPGME and R, Kolkata, West Bengal, India

Background: Juvenile-onset systemic lupus erythematosus (JSLE) is more severe than adult-onset disease, with higher proportion of lupus nephritis (LN). There is a paucity of long-term outcome data of LN in JSLE from India.

Objective: To describe the short and long-term renal response and flare rates in JSLE LN patients.

Methods: Single centre longitudinal observational study including JSLE LN patients (fulfilling ≥4 American College of Rheumatology criteria, age of onset ≤16 years, biopsy proven LN (classes III, IV or V)) receiving induction with intravenous cyclophosphamide (CyC, n=25, median dose 5400mg, inter-quartile range (IQR): 5000-5400) or mycophenolate mofetil (MMF, n=19, median dose 2gm/day, IQR: 2-2.63) and maintenance azathioprine (n=19, 75mg/day, IQR: 75-100) or MMF (n=21, 1.5gm/day, IQR 1.5-2).

Results: We analysed the Results: of 44 JSLE LN patients [short-term and long-term renal responses in [Figure 1]. Induction failure-rate was 9.1% (4/44, 95% confidence interval (CI): 3.1-21.7). They were treated with either cyclophosphamide (n=1, failed with MMF) or rituximab (n=3, failed with cyclophosphamide). Maintenance failure-rate was 25% (10/40, 95%-CI: 14.2-40.2). Among maintenance failure, 7/10 were early (occurred ≤2 years of FU). Time to maintenance failure was 24 months (IQR: 24-42). In univariate analysis early maintenance failure was associated with lower prednisolone dose in the second year (3.6±1.8 mg/day vs 5.9 ± 3.9 mg/day, p=0.049); prednisolone ≤ 5mg/day from 1st year FU (66.7%, 2/3 vs 13.5%, 5/37, p=0.02) and prednisolone ≤ 5mg/day from 2nd year FU (37.5%, 3/8 vs 10%, 3/30, p=0.05). Patients receiving MMF maintenance experienced numerically higher early maintenance failure (23.8%, 5/21 vs 10.5%, 2/19, p=0.27). In multivariate logistic regression receipt of prednisolone ≤5mg from first year of FU predicted early maintenance failure (Odd's ratio 15.5, 95%-CI:1.13-212, p=0.04).

Figure 1: Renal responses and flare rates of juvenile lupus nephritis patients over 5 years Click here to view

Conclusion: Both short-term and long-term outcomes of JSLE LN were good. Majority of renal flares were early and linked to premature prednisolone dose reduction.

OC0007: Low dose prednisolone plus tacrolimus therapy is more effective in Pla2r-Ve membranous glomerulonephritis patients

Akhilesh Jaiswal, Narayan Prasad, Vikas Agarwal, Vinita Agrawal, Harshit Singh; Department of¹Nephrology,²Clinical Immunology and³Pathology, SGPGIMS, Lucknow, Uttar Pradesh, India

Background: Idiopathic Membranous Nephropathy (IMN); an autoimmune concomitant nephrotic syndrome of the adult is mainly associated with PLA2R antibody expressed on podocytes. The lack of exact mechanisms involved in the pathogenesis of IMN is transmitted to its therapeutic management. We propose the efficacy of low dose Tacrolimus (Tac) plus prednisolone and associated changes in anti-PLA2R in adult IMN.

Methods: Total 101 membranous nephropathy patients were treated with combination of prednisolone 1mg/kg alt-day) and Tac 0.1mg/kg/day (trough 6-10 ng/ml first 6M and 4-6 ng/ml for next 3M) then both taper by 1/3 every month up to 12M. Out of 101 patients; 15 diabetic; 7 lupus; 1HBV and 1 ankylosing spondylitis patients were excluded. Finally; 77 Patients were followed and evaluated for the anti-PLA2R level at baseline; 3M; 6M; 12M and end of follow-up (17-61; median 38 months). CR; PR; relapse; and side-effects were recorded. Of the 77 patients; at 3M 60(77.92%; CR-37; PR-23); at 6M 61(79.22%; CR-53; PR-8); at 12M 53(68.86%; CR-47; PR-6) achieved remission. At end of follow-up; out of 54 responsive patients 37(68.51%; CR-36; PR-1) remained in remission and 17(31.48%) patients relapsed.

Results: Out of 77 patients; 51 (66.3%) were anti-PLA2R positive. Remission rate was significantly low in PLA2R+ve than PLA2R-ve (36/51 vs 24/26; p=0.03) at 3M; (36/51 vs 25/26; p=0.009) at 6M and (31/51 vs 22/26; p=0.03) at 12M. There were significant correlations between PLA2R level and 24h proteinuria at baseline; 3M and at 6M. During therapy 4 patients develop cutaneous tenia; 1 osteonecrosis of the femur head; 1 corpus tunnel syndrome; 4 onset diabetes; 3 tremor; and 14 patients experienced GI symptoms. To note; 4 females had pregnancy and successful delivery in our cohort of patients.

Conclusion: S PLA2R+ve patients showed poor response compare to PLA2R-ve patients. Remission with Tacrolimus and prednisolone therapy is comparable to historical Ponticelli regimen

OC0008: Prevalence of musculoskeletal pain and its risk factors among adolescent school children: A cross sectional survey

Maumita Kanjilal, Uma Kumar, Ravi Kant Arya¹, Gajendra Kumar Gupta¹, Deepika Agrawal¹; Department of Rheumatology, AIIMS, New Delhi,¹Department of Community Medicine, Santosh University, Ghaziabad, Uttar Pradesh, India

Background: The lack of physical activity, sedentary behaviour and increase in the use of visual display unit is an alarming concern among adolescent school children which is leading to increased musculoskeletal pain.

Objectives: The aim of the study is to find prevalence of musculoskeletal pain and its association with duration of physical activity and the screen time spent on smart phone and TV in adolescent school children.

Methods: The cross-sectional study was conducted at Government schools in Delhi. Children in the age group 10-19 years without any known musculoskeletal disorders or any disease participated in the study. The demographic details, Nordic Muscular Questionnaire, the level of sedentary lifestyle and physical activity were assessed through the questionnaire consisting of the time spent on smart phone/TV and International physical activity questionnaire.

Results: 1600 children participated in the study with 855 (53 %) boys and 745 (47 %) girls in the age group 10-19 years participated in the study. The prevalence of musculoskeletal pain was found to be 63% (95% CI :55-70) in past 12 months and recent 7 days period as per Nordic musculoskeletal Questionnaire. The data analysis (χ2) suggested that musculoskeletal pain was significantly associated with female gender (p=<0.0001), late adolescence age (p=<0.0001), duration of physical activity less than 60 minutes per day (p=<0.0001), duration of watching TV (p=0.033) and smart phone usage(p=<0.0001) more than 2 hours per day.

Conclusion: 63% adolescents had musculoskeletal pain. Use of mobile phone/watching TV more than the recommended screen time duration of more than 2 hours per day, moderate physical activity of less than 60 mins per day among adolescent is a major risk factor for the development of musculoskeletal pain. Significant association of pain with late adolescent and girls further endorses this observation.

OC0009: Assessment of liver fibrosis by transient elastography in patients with rheumatoid arthritis on long-term methotrexate therapy

Prashant Bafna, Anupam Wakhlu, Sumit Rungta¹, Puneet Kumar, Urmila Dhakad; Departments of Clinical Immunology and Rheumatology and¹Medical Gastroenterology, King George's Medical University, Lucknow, Uttar Pradesh, India

Background: Although methotrexate (MTX) is widely available effective DMARD in rheumatoid arthritis (RA), long term therapy may be associated with increased risk of liver fibrosis. A noninvasive method like transient elastography (TE) is used for assessing hepatic fibrosis, as monitoring with liver enzymes and albumin alone are unreliable for assessing liver dysfunction in liver fibrosis.

Objectives: Primary: To study the prevalence of liver fibrosis in patients with long term methotrexate therapy for RA

Secondary: To correlate duration and cumulative dose of methotrexate use with the risk of developing liver fibrosis

Methods: In this cross-sectional study, 40 patients fulfilling the 2010 ACR/EULAR criteria of RA on methotrexate therapy for > 3years were assessed. Ethical approval was obtained. The median duration and cumulative dose of MTX use were calculated from their previous records. Hepatic stiffness by TE method (by FibroScan) was determined for the included patients. The duration and cumulative dose of MTX with liver fibrosis was correlated using Spearman correlation formula.

Results: The mean age of patients was 49±9.4years. There were 37 females and 3 males. The median duration of MTX use was 336weeks (IQR 240-432 weeks). Nine(22.5%) patients were on MTX for >10years. The median cumulative dose of MTX was 5.6gm (IQR 4.6gm-7.3gm). Thirty(75%) patients had a cumulative dose between 5 to 10 gm and 4(10%) had >10gm. Mean hepatic stiffness was 5.26kPa±2.16kPa. Six(15%) patients had liver stiffness between 7.5 to 9.5kPa. Two(5%) patients had liver stiffness of 11.6kPa and 12.2kPa with their corresponding cumulative dose of 7.6gm and 13.8gm respectively. Their stiffness improved to < 7.5kPa by 6 months after discontinuation of methotrexate. No correlation was found between the dose and duration of methotrexate use with the liver fibrosis (p=0.08, r=0.06).

Conclusion: Methotrexate, when conventionally prescribed in low-dose weekly schedule is possibly safe for long-term use in rheumatoid arthritis

OC0010: Cardiovascular risk assessment in Indian rheumatoid arthritis patients: Comparison of risk algorithms and carotid intima media thickness

Hafis Muhammed, Durga P Misra, Sujata Ganguly, Sarit Sekhar Pattanaik, Saurabh Chaturvedi, Harshit Singh, Mohit K Rai, Anamika Anuja, Namita Mohindra1, Neeraj Jain¹, Sudeep Kumar², Vikas Agarwal; Departments of Clinical Immunology and Rheumatology,¹Radiodiagnosis and²Cardiology, Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India

Background: Rheumatoid arthritis(RA) patients have increased cardiovascular(CV) risk with no data on CV risk scores in Indian patients. We aimed at studying CV risk in RA patients.

Methods: Patients fulfilling 2010 ACR/EULAR criteria for RA were included. Presence of CV risk factors were recorded. 10-year-CV risk was predicted using Framingham Risk scoring using lipids(FRS-Lipids), Framingham Risk scoring using body mass index(FRS- BMI), QRISK-2, SCORE and the algorithm recommended by ACC/AHA (ASCVD) in patient who were 40 years or older. Carotid Intima Media Thickness(CIMT) was measured on far-wall of the common carotid artery. Subclinical atherosclerosis was defined as CIMT >0.90 mm or presence of plaque.

Results: 334 patients (M: F = 49:285, Mean age =47.16±12.57 years) were enrolled. 6% were smokers, 12% had diabetes mellitus(DM) and 21% had hypertension. Mean CIMT was 0.70±0.15 mm. In univariate analysis mean CIMT significantly differed according to gender and presence or absence of erosions, extra articular manifestation (EAM), DM and hypertension. CIMT correlated significantly with age, disease duration, systolic blood pressure and total cholesterol. Multiple regression analysis showed age, EAM and male gender as independent predictors of CIMT (r2=0.431 for final regression model). All risk scores had moderate correlation with CIMT with maximum for QRISK (r=0.570). Percentage of patients with predicted >10% risk varied from17.6% to 41.9% between scores. Agreement between scores in predicting risk was moderate in general with maximum agreement between QRISK2 and FRS-Lipids (weighted kappa:0.790). ASCVD and QRISK-2 showed maximum sensitivity when subclinical atherosclerosis assessed by CIMT was taken as standard (AUC: 0.822 and 0.806 respectively) [Figure 1].

Figure 1: ROC curve Click here to view

Conclusions: Prevalence of risk factors and subclinical atherosclerosis were calculated. Age, EAM and male gender independently predicted CIMT. While further validation is required in terms of clinical end points, our findings suggest that risk algorithms cannot be used indiscriminately in RA.

OC0011: Subclinical atherosclerosis in Indian patients with spondyloarthropathy: Clinical and serological associations

Mohit Kumar Rai, Mantabya Singh, Harshit Singh, Saurabh Chaturvedi, Anamika Anuja, Kritika Singh, Namita Mohindra, Neeraj Jain, Sudeep Kumar, Vikas Agarwal, Durga Prasanna Misra; Departments of Clinical Immunology and Rheumatology, Nephrology, Radiodiagnosis and Cardiology, SGPGIMS, Lucknow, Uttar Pradesh, India

Background:

We evaluated traditional and novel risk factors associated with subclinical atherosclerosis (SCA) in spondyloarthritis, in a cross-sectional study.

Objectives:

• To compare mean carotid intima-medial thickness (CIMT) between patients with spondyloarthropathy (n=105) fulfilling ASAS 2010 criteria, and healthy controls (n=55).
• To compare traditional and novel biomarkers (microparticles, endothelial microparticles, inflammatory cytokines associated with increased cardiovascular risk, serum osteoprotegerin, serum apolipoprotein A1) between patients with spondyloarthritis and healthy controls.
• To identify factors associated with SCA in spondyloarthropathy.

Methods: SCA was defined by carotid artery plaques, or increased CIMT >2 standard deviations compared with Indian reference standards for age and sex. Total microparticles (TMP) were measured in plasma after ultracentrifugation using microbeads of 7 mm size (TMP were of size 0.1-1 mm); of these, microparticles positive for CD31 and CD146 were taken as endothelial microparticles (EMP). Serum cytokines (IL-1b, IL-6, TNF-a, IL-17, IL-27, IL-33), osteoprotegerin and apolipoprotein A1 were measured by ELISA using manufacturer instructions. Univariate analysis compared patients with spondyloarthropathy with healthy controls. Linear regression was used to identify the determinants of CIMT in spondyloarthropathy. Binomial logistic regression was used to identify factors associated with SCA in spondyloarthopathy.

Results: Despite lower BMI, total cholesterol, triglycerides and higher HDL cholesterol, CIMT was significantly higher in patients with spondyloarthropathy [Table 1]; on linear regression, body mass index (BMI, p=0.026), total cholesterol (p=0.031), endothelial microparticles (0.006), serum IL-1b (p=0.001) and IL-27 (p=0.040) were independent predictors of CIMT in spondyloarthropathy. Fourty- two (40%) patients had SCA; 15 (14.3%) had carotid plaques. Spondyloarthropathy patients with SCA had higher age, disease duration, BMI, waist-hip ratio, LDL cholesterol and serum IL-1b; binomial logistic regression identified independent association of age and LDL cholesterol with SCA.

Table 1: Comparison between patients with spondyloarthritis and healthy controls Click here to view

Conclusions: Both traditional and novel cardiovascular risk factors (endothelial microparticles, IL-1b, IL-27) associate with high prevalence of SCA in Indian spondyloarthropathy patients.

OC0012: Scoring system based on inflammatory parameters to screen for infection and disease flare and bacterial versus viral infections in the background of inflammatory rheumatic disease

S Chandrashekara, P Renuka, K R Anupama, Devaraj, J Prakruti, R Veena; ChanRe Rheumatology and Immunology Centre and Research,¹ChanRe Diagnostic Centre, Bengaluru, Karnataka, India

Background: In patients with autoimmune rheumatic disease, differentiating infections (viral or bacterial) from disease exacerbation is a challenge. The leucocyte response, CRP, NLR and pro-calcitonin behave differently and could be used to differentiate them.

Objective: To develop and validate two clinical risk scores for the screening of infections and disease flare and in infection to distinguish bacterial or viral in AIRD patients.

Materials and Methods: The retrospective cohort study included 115 consecutive AIRD patients (January-December 2018) for derivation and 90 patients (January-July 2019) for internal validation. The clinical risk score was derived by assigning points based on multivariate logistic regression co-efficient. The points were summed to derive the risk score for each patient.

Results: Of the 115 patients, 77 (66.96%) had infections and 38 (33.04%) had disease flare. Corresponding bacterial and viral infections noted was 44 (57.14%) and 33 (42.86%). The final infection and flare risk score included total leucocyte count, NLR, CRP and procalcitonin. Bacterial vs viral infection risk score included CRP, NLR and procalcitonin. ROC curve obtained for both models showed fair discrimination (AUC= 0.7). The categorization of patients for infection vs. flare risk score was as follows: >9 high probability of infection, 5 to 9 moderate possibility of infection present and <5 infection less likely. Patients with bacterial vs. viral risk were scored as: >8 risk of bacterial infection, 5 to 8 borderline risk of bacterial infection and <5 viral infection likely. In validation cohort, proportion of patients noted with risk score ≥5 was 79.71% for infection and 78.43% for bacterial infection.

Conclusion: We derived 2 simple risk scores to aid in screening and appropriate treatment selection for AIRD patients in hospital setting. Clinical features and other markers may improve classification of patients. External validation has to be performed to evaluate generalizability of risk score.

PB0001: Alteration of serum biomarkers in primary knee OA

Meghna Saha, Subhankar Hadar, Biswadip Ghosh; Department of Rheumatology, IPGME and R and SSKM Hospital, Kolkata, West Bengal, India

Background: Osteoarthritis (OA) is a leading cause of pain and disability. Many patients with osteoarthritis show some features of inflammation. Traditional thinking is that, OA is a degenerative disease with main site of pathology at the cartilage.

Objective: To demonstrate evidence and extent of inflammation in primary knee OA.

Methods: 249 patients with knee OA with local clinical inflammation were recruited, subdivided in systemic Non-Inflammatory group (77) and Inflammatory group (172) based on serum levels of ESR and CRP. WOMAC of these patients were measured. Then OPG, RANKL, MPO, Fibrin, Human COLL2-1 NO2 from the serum were measured by ELISA and compared with 48 healthy controls.

Results: The serum level of RANKL is significantly higher in all patients of knee OA, when compared with healthy control group and serum level of OPG is significantly lower in both Non-Inflammatory group and Inflammatory group, when compared with healthy control group. Serum level of MPO was found to be significantly lowered in both the inflammatory and Non-inflammatory patient groups. However, fibrin and Human COLL2-1 NO2 showed no significant change in both the patient groups when compared with healthy control group.

Conclusions: Increased serum level of OPG and decreased level of RANKL indicate abnormality in bone. COLL2-1 NO2, a marker of cartilage damage is not altered in OA patients in comparison to healthy controls. MPO, a marker of inflammation, is significantly lowered in all patients of Knee OA. Serum level of Fibrin, an acute phase reactant, showed no significant change in patients with primary OA. So, it seems that the pathogenesis in knee OA is localised inflammation and role of bone may be no less than cartilage. It is time to shift our attention outside cartilage in knee OA, particularly in patients with systemic inflammation, as evidenced by raised ESR/CRP in many patients.

PB0002: Methotrexate treatment is associated with reduction of neutrophil reactive oxygen species and CD177 in RA patients

Varun Dhir, Urvashi Kaundal, Aastha Khullar, Biman Saikia; PGIMER, Chandigarh, India

Background: Methotrexate (MTX) is the gold-standard DMARD in rheumatoid arthritis, and may work through inhibiting neutrophils. This may involve changes in ROS production and neutrophil activation.

Objectives: To compare ROS production and activation markers CD177, CD11b and CD64 in neutrophils of naïve RA and methotrexate treated RA patients.

Methods: This was a single-center cross-sectional study, which recruited rheumatoid arthritis (RA) patients and healthy controls. Both naïve and MTX treated RA patients (>=15 mg/week x 6 mos) recruited. Neutrophils were separated by using Hetasep and density-gradient centrifugation. Reactive oxygen species (ROS) were detected at baseline and after PMA (1.62 ug/ml) stimulation using dihydrorhodamine assay on FACS. ROS by Luminol detected after adding PMA (0.4 ug/ml) and Luminol (50 uM) by luminescence detector. Activation markers CD177, CD11b and CD64 were assessed on FACS.

Results: This study included total 121 subjects, which included 53 (F:M= 43:10) RA on methotrexate (MTX-RA), 47 (F:M=39:8) naïve (naïve-RA) and 21 healthy controls. Mean DAS28-3 was significantly higher in naïve-RA than MTX-RA (6.2, 4.9, p<0.001). At baseline, neutrophils of naive-RA compared to MTX-RA showed higher MFI on DHR assay (11049, 7301, p=0.009). This remained higher even after PMA stimulation. There was no significant difference between MTX-RA and healthy controls after PMA. Even on luminol assay, after PMA stimulation, there was higher ROS production in naïve-RA than MTX-RA. CD177 had higher expression in naïve-RA than MTX-RA patients (MFI: 46620, 34475, p=0.02) and healthy controls (46620, 26855, p= 0.001). No significant difference in CD11b and CD64 levels between naïve RA and MTX-RA.

Conclusions: MTX treated RA patients had reduced (similar to healthy controls) levels of ROS production in neutrophils. CD177 expression was also significantly reduced in MTX- treated patients. One of the ways MTX acts in RA may be through reducing neutrophil activation and ROS.

PB0003: Female sex hormone mediated protein regulations in rheumatoid arthritis

Debolina Chakraborty, Uma Kumar¹, Sagarika Biswas; CSIR-Institute of Genomics and Integrative Biology,¹AIIMS, New Delhi, India

Background: Female prevalence in certain diseases more than male, rise several questions which is still unclear and need to be revealed. Rheumatoid Arthritis (RA) which is a persistent systemic disease associated with inflammation of joints leading to joint cartilage destruction is one such autoimmune disease with female to male ratio 4-5:1 (age<50) and 2-3:1 in later ages. Controversies whether female sex hormone has positive or negative influence on RA progression emerge an interest to search for the pathways regulated by sex hormones in RA.

Objectives: For diminishing this controversy, this study will deal with the proteins which are differentially expressed with hormonal (Estrogen: Most important female sex hormone regulating RA) regulations in RA patients leading to their in-depth study for understanding the mechanism of estrogen regulated disease associated proteins.

Methods: Expression level of pro-inflammatory cytokines TNFα, IL6 and IL17 were measured before and after estrogen induction in RA and OA primary fibroblast like synoviocytes compared with LPS induced and normal SW982 synovial cells. Differential protein profiling will be studied.

Results: Inflammatory status of RA FLS compared to OA and secondary cell lines by estrogen induction is deduced. Differentially proteins expression which links estrogen with inflammation can reveal a potential reason for the prevalence of women affected by RA.

Conclusions: Expression changes of specific identified proteins can directly link estrogen mediated pathways in regulating disease pathogenesis. This study will provide an idea for analyzing the reason for different RA conditions in complete female life cycle and RA remission in pregnancy period as well as will provide the instances for increase in RA severity during nulliparity. These proteins can also become potential targets for therapy and can provide an instant for different ways of treatment for different sexes in RA.

PB0004: Major histocompatibility antigen HLA-DQB1*0601 is associated with rheumatoid arthritis among Indians in a replication study: Evidence of gene-environment interaction with LPG stove use

Able Lawrence, Anshul Dhar, Swayam Prakash, Suvrat Arya, Amita Aggarwal, Suraksha Agarwal; Sanjay Gandhi Postgraduate Institute of Medical Science, Lucknow, Uttar Pradesh, India

Background: Shared Epitope interaction with smoking does not fully explain MHC association in Rheumatoid Arthritis (RA). We previously showed HLA-DQB1*0601 with restricted Asian distribution as susceptibility allele in India. Pristane Induced arthritis susceptibility is determined by rat DQ ortholog RT1-B.

Objective: To confirm DQB1*0601 association with RA and explore interactions with smoking and household fuel use.

Methods: Patients with RA (ACR 2010 criteria) and healthy controls of similar ethnic Background were included. Data on environmental exposure like tobacco use, active and passive smoking and exposure to household smoke during cooking including biomass fuels (wood, coal, cow-dung cake) and hydrocarbon fuels LPG and kerosene was collected. Patients sex, age of onset, duration of exposure, birth year, RF , anti-CCP were also recorded. HLA-DQB1*0601 was genotyped using Sequence-specific PCR. The association of Chi-squared test was used to test association. Gene environment interaction was studied using Cox regression after stratifying by genotype.

Results: 175 patients (21 males) and 263 healthy controls were included. The median age of onset of RA was 37(range 17-67 years) and median duration of disease was 9 years (range 2mo to 35 years). DQB1*0601 was present in 45/175 (25.7%) of patients and 47/263 of controls (17.9%), OR 1.59 (P<0.005) confirming association with RA. LPG exposure was associated with earlier onset of RA (median 40 years versus 44 years) only in the presence of DQ6.1 with a hazard ratio of 2.5 (CI 1.2-5, P=0.014).

Conclusions: This replication study confirms the association of DQB1*0601 with RA. There is signal of gene-environment interaction between DQ6.1 and exposure to LPG stoves.

Figure 1: Earlier onset of rheumatoid arthritis among LPG stove exposed only in the presence of DQB1*0601: Evidence of gene-environment interaction. Kaplan Maier hazard function amone DQB1*0601 negative (N = 128) and DQB1*0601 positive subjects (N = 45) LPG use is associated with a hazard ratio of 2.85 (1.23–6.63) P = 0.015 only among DQB1*0601 positive subjects Click here to view

Reference

1. Lawrence A, Prakash S, Bharadwaj U, Aggarwal A, Misra R, Agrawal S. Major histocompatibility antigen HLA-DQ6.1 (DQA1*0103/DQB1*0601) increases rheumatoid arthritis risk independent of shared epitope among Indians. Arthritis Rheumatol 2016;68 Suppl 10.

PB0005: Correlation of calcium pyrophosphate dihydrate crystals with varying grades of osteoarthritis

Elizabeth Vinod^1,2, Tephilla Epsibha Jefferson¹, Soosai Manickam Amirtham¹, Neetu Prince¹, Upasana Kachroo¹, Boopalan Ramasamy³; ¹Department of Physiology, CMC,²Centre for Stem Cell Research, CMC, Vellore, Tamil Nadu, India,³Department of Orthopaedics, Royal Darwin Hospital, Tiwi, Australia

Background: Accurate diagnosis of osteoarthritis (OA) is the first important step in ensuring appropriate management of the disease. A multitude of tests involving assessment of biomarkers help in assessment of severity and grading of osteoarthritic damage. However, most of these tests are time consuming and are limited by the paucity in synovial fluid volume. In majority of OA effusions, calcium pyrophosphate dihydrate and basic calcium phosphate crystals are found. Therefore, the aim of our study was to evaluate whether a correlation existed between the amount of calcium containing crystals present in synovial fluid and severity scoring of osteoarthritis to propose a quick and inexpensive technique for disease assessment.

Materials and Methods: 12 Adult male NZW rabbits were used to create low-and high-grade OA (n=6 each) using monosodium-iodoacetate. At 16 weeks, synovial fluid and joints were harvested for histopathological analysis (H&E, Safranin O and Collagen II). OA grading was established based on OARSI scoring and the calcium containing crystals in synovial fluid (5μl) were assessed by light microscopy following staining with Alizarin red on a wet mount preparation (three independent blinded investigators). Presence of calcium containing crystals was confirmed using Fluo-4 AM staining. For statistical analysis, Mann Whitney U test was used to compare low- and high-grade OA groups, whereas Pearson correlation was used to assess the relation between number of calcium crystal clumps vs the grade of OA.

Results: and Conclusion: The clumps counted in low-grade OA were significantly lower than high-grade OA, in addition to showing a positive correlation (coefficient: 0.65; P=0.02) between positively stained clumps vs OA severity. A positive correlation between the amount of calcium containing crystals and severity scoring of OA thus enables us to consider Alizarin red staining of synovial fluid crystals as an effective and rapid, bed-side method for detecting progression of the disease.

PB0006: Immunomodulation of CD8+ T-cells from patients with rheumatoid arthritis by lactobacillus rhamnosus

Archana Tripathy, Prasanta Padhan, Sunil Raghav and Bhawna Gupta; School of Biotechnology, Kalinga Institute of Industrial Technology, Bhubaneswar, Odisha, India

Background: Rheumatoid arthritis (RA) is a progressive autoimmune disease with systemic complications and early deaths. Though the pathogenesis of RA is not yet fully elucidated, it is known to be induced by environmental factors on a genetically susceptible Background. Microbes are also considered and often discussed as potential triggers for initiation and perpetuation of RA. Alterations in gut microbiota create an imbalance between pro-inflammatory and anti-inflammatory immune responses and trigger the development of RA.

Objective: Identify the role of Lactobacillus rhamnosus (L. rhamnosus) in modulating CD8+Tcell response in RA patients and determine the effect of Escherichia coli (E. coli) towards acute inflammation in RA.

Methods: Hundred RA patients and hundred healthy donors participated in this study. Clinical variations like disease duration, number of inflamed joints, type of bones deformities, CRP, RF, Anti-CCP, ESR were recorded for each patient and DAS 28 scores were calculated with the help of the clinician. Different cytokines and transcription factors level was checked after stimulation of CD8+T cells with E. coli and L. rhamnosus by flow cytometry. We analyzed the TLRs profiling and the proliferation rate of CD8+Tcells upon bacterial infection.

Results: Significant increased of different inflammatory cytokines in patients with RA compared to healthy donors after stimulation with E. coli (p<0.005). In CD8+ T cells, L. rhamnosus treatment decreased the expression level of proinflammatory mediators which leads to down regulate Th1 response in CD4+T cell (p<0.05). L. rhamnosus infection significantly increased the mRNA expression of TLR2 which enhances the proliferation capacity of CD8+ T cells (p<0.005) where as E. coli significantly upregulated the transcript of TLR1 and 4.

Conclusion: In summary, our data suggest that co-culture of L. rhamnosus with CD8+T cells can effectively suppress CD8+T cell mediated inflammation by simultaneous down regulation of Th1 response which may leads to suppression of disease symptoms.

PB0007: TLR7 mediated activation of cd8+ t cells leads to downregulation of inflammatory mediators in patients with rheumatoid arthritis

Nitish Swain, Archana Tripathy, Prasanta Padhan, Sunil Raghav, Bhawna Gupta; School of Biotechnology, Kalinga Institute of Industrial Technology, Bhubaneswar, Odisha, India

Background: Rheumatoid arthritis (RA) is a systemic inflammatory, autoimmune disease of unknown origin characterized by aberrant immune responses towards self peptides. The disease is thought to be induced by environmental or genetic factors. Cytokine mediated destruction of the synovial joints is the signature symptom of the progression of this disease. Toll- like Receptors (TLRs) are one of the most prominent innate immune receptors which recognize an array of components ranging from microbial compounds to nucleic acids. Research has shown the presence of TLRs in the CD8+ T cells in case of RA patients and this study focuses on finding the role of TLR7 in the pathogenesis of RA.

Objective: To determine the role of TLR7 in CD8+ T cells of patients with RA.

Methods: 40 RA patients and 35 healthy control (HC) participated in this study. Clinical variations like disease duration, number of actively inflamed joints, bone deformities, CRP, RF, Anti-CCP, ESR were recorded for each patient and DAS 28 scores were calculated. We analyzed the expression of different cytokine transcripts by real-time PCR and protein level expression by flow cytometry as well as immunoblotting after stimulation of TLR7 present on CD8+T cells by its agonist imiquimod.

Results: We found that TLR7 ligand responsiveness significantly increased the mRNA expression of different inflammatory mediators in CD8+T cells of RA patients compared to healthy individuals. Flow cytometry analyses revealed that there was no significant increase in the level of the cytokines that were found at the mRNA level. Significant up regulation of Tristetraprolin (TTP) in treated CD8+ T cells might be the explanation for translation inhibition and mRNA decay of inflammatory mediators (p< 0.05).

Conclusion: Our data suggests that activation of CD8+ T cells via TLR7 agonist decreased expression of effector molecules to a similar level as in HC.