Anjali Rajadhyaksha

Effect of Proinflammatory Cytokines (IL-6, TNF-α, and IL-1β) on Clinical Manifestations in Indian SLE Patients

Systemic lupus erythematosus (SLE) is an inflammatory rheumatic disease characterized by production of autoantibodies and organ damage. Elevated levels of cytokines have been reported in SLE patients. In this study we have investigated the effect of proinflammatory cytokines (IL-6, TNF-α, and IL-1β) on clinical manifestations in 145 Indian SLE patients. One hundred and forty-five healthy controls of the same ethnicity served as a control group. Clinical disease activity was scored according to SLEDAI score. Accordingly, 110 patients had active disease and 35 patients had inactive disease. Mean levels of IL-6, TNF-α, and IL-1β were found to be significantly higher in SLE patients than healthy controls (P < 0.001). Mean level of IL-6 for patients with active disease (70.45±68.32 pg/mL) was significantly higher (P = 0.0430) than those of inactive disease patients (43.85±63.36 pg/mL). Mean level of TNF-α was 44.76±68.32 pg/mL for patients with active disease while it was 25.97±22.03 pg/mL for those with inactive disease and this difference was statistically significant (P = 0.0161). Similar results were obtained for IL-1β (P = 0.0002). Correlation between IL-6, TNF-α, and IL-1β serum levels and SLEDAI score was observed (r = 0.20, r = 0.27, and r = 0.38, resp.). This study supports the role of these proinflammatory cytokines as inflammatory mediators in active stage of disease.

Anti-C1q antibodies and their association with complement components in Indian systemic lupus erythematosus patients

Systemic lupus erythematosus (SLE) is a prototype autoimmune disease, characterized by immune complex formation and systemic inflammation. Complement components such as C1q and mannose-binding lectin (MBL) play an important role in the clearance of immune complexes. Anti-C1q antibodies are associated with lupus nephritis and reduced levels of the complement components. The objective of this study was to detect anti-C1q antibodies in SLE patients and to evaluate their association with the complement components. Sixty SLE patients were included, of whom 75% had lupus nephritis (LN) and 25% were without renal manifestations (non-LN). The disease activity was assessed at the time of evaluation by the systemic lupus erythematosus disease activity index (SLEDAI). Anti-C1q antibodies, circulating immune complexes, and serum MBL levels were detected by enzyme-linked immunosorbent assay. The anti-C1q antibody prevalence was 58.3%. The LN patients showed 60% anti-C1q positivity with a higher percentage in membranoproliferative glomerulonephritis patients (51.9%). Anti-dsDNA positivity was slightly higher among the anti-C1q positives than in the anti-C1q negatives (65.7% vs. 60%). A higher percentage of reduced C3 and C4 levels was noted among the anti-C1q positives. The LN patients showed a higher percentage of low MBL levels among anti-C1q negatives than in the anti-C1q positives (61.1% vs. 55.6%). Non-LN patients showed a higher percentage of low MBL levels among anti-C1q positives than among anti-C1q negatives (87.5% vs. 57.1%). Anti-C1q antibodies were found in both LN and non-LN patients, but there was no correlation with the clinical severity of the disease.

Mannose binding lectin (MBL) 2 gene polymorphism & its association with clinical manifestations in systemic lupus erythematosus (SLE) patients from western India

Background & objectives: Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease characterized by production of autoantibodies. Mannose binding lectin (MBL) is an important element of the innate defense system. The present study was undertaken to determine whether variant alleles in MBL2 gene were associated with disease severity in SLE patients. Methods: The MBL alleles [-550, -221, +4, Codon 52, Codon 54 and Codon 57] were studied by PCR- RFLP (restriction fragment length polymorphism) method in 100 SLE patients fulfilling ACR (American College of Rheumatology) criteria along with 100 healthy controls. SLE disease activity was evaluated using SLE Disease Activity Index (SLEDAI) score. Results: Homozygosity for MBL variant allele (O/O) was observed in 24 per cent of the SLE patients compared to 16 per cent of the normal controls, while no difference was found for heterozygosity (A/O) (37 vs 35%). A significant difference was reported in incidence of double heterozygosity for mutant allele B and D (B/D) among SLE patients as against control group (P = 0.015). MBL genotypes did not show any association with renal involvement. Interpretation & conclusions: In this study from western India, MBL gene polymorphism showed an influence as a possible risk factor for susceptibility to SLE, but had no direct effect on disease characteristics. Further studies need to be done on a larger number of SLE patients in different regions of the country.

Clinical and immunological profile of systemic lupus erythematosus

Pediatric onset systemic lupus erythematosus (SLE) is not uncommon and female to male ratio varies. Pediatric SLE patients have more severe disease at onset, higher rates of organ involvement and more aggressive clinical course than adults. We compared the clinical and immunological parameters among pediatric SLE and adult SLE from Western India. Twenty five children and 60 adult patients fulfilling American College of Rheumatology SLE criteria were included. Anti-nuclear antibodies, anti-dsDNA and complement (C3, C4) levels were tested. Of 25 pediatric SLE patients studied, 24% showed CNS involvement vs. 8.3% in adults SLE (P=0.0499). Lupus nephritis was seen in 75% adult patients vs. 52% among children. Hepatosplenomegaly was noted more among adult SLE 26.8% vs 12% among children. Alopecia was an exclusive features among adult SLE.

Profile of neurological manifestations in systemic lupus erythematosus

Abstract Introduction To study the pattern of neurological involvement in systemic lupus erythematosus (SLE) and its correlation with investigation, disease activity and response to treatment. Methods This observational study was carried out from June 2007 to May 2008. Diagnosed cases of SLE (based upon ARA criteria) who present with neurological manifestations at the time of diagnosis or develop neurological manifestations anytime during the course of the disease were followed up for six months. Both prospective and retrospective cases were included. Results Of the 35 patients with neurological manifestations of SLE, presenting to KEM hospital, Mumbai from the period of June 2007 to May 2008, 94% were females. The commonest age group was 20–29 years. Manifestations observed were seizures (66%), altered sensorium (20%), psychosis (9%), hemiparesis (9%), headache (6%), peripheral neuropathy (6%), depression (3%), cognitive decline (3%) and myelopathy (3%). With appropriate and adequate treatment, at the end of the study, 86% patients improved neurologically and in terms of SLE disease activity, while 11% died and 4% remained the same state neurologically. Conclusion Neurological involvement in SLE is seen relatively early during the course of the disease and correlates with disease activity. It is commonly seen in patients with SLE who receive inappropriate and/or inadequate treatment.

Clinical and autoimmune profile of scleroderma patients from Western India.

Background. Systemic sclerosis (SSc, scleroderma) is a disorder characterized by fibrosis of skin and visceral organs. Pathogenesis of scleroderma is complex and is incompletely understood as yet. Autoantibodies in SSc represent a serologic hallmark which have clinical relevance, with diagnostic and prognostic potential. Objectives. To study distribution of clinical manifestations and to identify frequency of autoantibodies among subtypes of scleroderma patients from Western India. Methodology. One hundred and ten scleroderma patients were clinically classified according to the American College of Rheumatology/European League Against Rheumatism (ACR/EULAR) criteria. All these patients were in active stage of disease. Clinical manifestations were recorded at the time of presentation. Autoantibodies were tested in them by indirect immunofluorescence test and ELISA. Immunoglobulin levels were estimated by nephelometer. These parameters were further correlated with clinical presentation of the disease. Results. Scleroderma patients had M : F ratio of 1 : 10 where mean age at evaluation was 34.7 ± 10.7 years and a mean disease duration was 43.7 ± 35 months. Clinical subtypes showed that 45 patients (40.9%) had diffused cutaneous (dcSSc) lesions, 32 patients (29.1%) had limited cutaneous (lcSSc) lesions, and 33 patients (30%) had other autoimmune overlaps. The overall frequency of ANA in SSc patients studied was 85.5%. The frequency of anti-Scl70, anti-centromere, anti-endothelial cell antibodies (AECA), and anti-keratinocyte antibodies (AKA) was 62.7%, 22.7%, 30%, and 40.9%, respectively. Anti-Scl70 antibodies were significantly high (75.6% versus 46.9%) among dcSSc patients (P < 0.0115) whereas anti-centromere antibodies were significantly high (9% versus 38%) among lcSSc patients when these two subtypes were compared (P < 0.0044). Conclusion. This study supports that there are geoepidemiological variations among scleroderma patients for their clinical presentation, autoantibody profile, and immune parameters across the country.

Application of a Simple In-House PCR-SSP Technique for HLA-B* 27 Typing in Spondyloarthritis Patients.

Background. Microlymphocytotoxicity (MLCT) and flowcytometry (FC) are the conventional serological methods to detect HLA-B* 27. Due to some disadvantages in these methods, most of the HLA laboratories have now switched over to molecular methods. Molecular techniques based on commercial kits are expensive; as such many laboratories with limited funds in developing countries cannot afford these techniques. Aims. Our main aim was to standardize a simple inexpensive in-house PCR-SSP technique for HLA-B* 27 typing. Materials and Methods. Sequence Specific primers were designed to amplify all the subtypes of B* 27 using IMGT-HLA sequence database. Accuracy was checked by retyping of 90 PCR-SSOP typed controls. Results. The presence of 149 bp specific band with control band on 2% agarose gel showed B* 27 positivity. No discrepancies were found when compared with PCR-SSOP results. The frequency of HLA-B* 27 was found to be significantly increased (68.75% versus 4.40%, O.R 46.909: P value 6.62E − 32) among 700 SpA patients as compared to controls. Clinically, 54% of patients had polyarticular arthritis with SI joints involvement (68%) and restricted spine flexion (60%). Conclusion. In-house PCR-SSP technique is very simple and inexpensive technique to detect B* 27 allele, which was strongly associated with SpA patients from Western India.

Association between the angiotensin-converting enzyme gene insertion/deletion polymorphism and susceptibility to systemic lupus erythematosus in an Indian population

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by intense inflammation andmultiple organ damage (1). There is much evidence for genetic susceptibility to SLE and several candidate genes including that encoding the angiotensin-converting enzyme (ACE) have been identified (2). Biologically, ACE elevates the levels of angiotensin II, a vasoactive peptide, growth factor and potent pro-inflammatory modulator that contribute to tissue remodelling (3, 4). The ACE gene contains a 287-base-pair repeat insertion/deletion (I/D) polymorphism within intron 16 (5), and studies have shown that D/D homozygotes have an approximate twofold higher level of tissue and plasma ACE compared with I/I homozygotes (6). Although the D/D genotype and the concomitant increase in serum ACE and angiotensin II levels could contribute to the pathogenesis of SLE (3, 4), there are conflicting data with regard to the association of the ACE D/D genotype and susceptibility to the disease (7). The aim of the current study was to investigate ACE I/D gene polymorphisms and serum ACE levels in Indian SLE patients, and to determine any correlations to the clinical features of SLE. One hundred and nine SLE patients (six male, 103 female; mean age 27.8 9.7 years, range 12–60 years) were recruited to this single-centre prospective observational study at the King Edward Memorial Hospital, Mumbai, Maharashtra, India, between January 2010 and January 2012. The clinical details of the SLE patient group are given in Supplementary Table S1. SLE was diagnosed according to the revised and updated criteria of the American Rheumatism Association for the classification of SLE (8, 9). Of the SLE patients, 39 were untreated at the point of inclusion in the study, and 70 were receiving corticosteroid treatment. A total of 100 unrelated healthy individuals without SLE or other autoimmune disease, or a history thereof, and matched for ethnicity, sex, and age, were also included in the study as controls. Approval for the study was given by the institutional ethics committee, and written informed consent was obtained from all participants. The study was conducted in accordance with the principles of the Declaration of Helsinki. In a case–control study, the ACE I/D genotypes and serum ACE levels were determined for 109 SLE patients and 100 ageand sex-matched controls using polymerase chain reaction (PCR) amplification and an enzyme-linked immunosorbent assay (ELISA), respectively (Supplementary Material). The observed genotype frequencies of the controls (Table 1) did not differ significantly from the frequencies predicted by the Hardy– Weinberg equilibrium (p 1⁄4 0.11). There was a strong statistical trend for an increase in the D/D and I/D genotypes in SLE, while there was a statistically significant increase in the D allele (Table 1). Serum ACE levels were significantly higher in the SLE patient group compared with controls (Table 1). In both SLE patients and controls, the D/D genotype correlated with the highest levels of serum ACE (Figure 1). SerumACE levels in SLE patients were also increased significantly when compared with controls of the same ACE I/D genotype (Figure 1). There was no significant difference in the prevalence of ACE I/D genotypes or alleles or in the levels of serum ACE in relation to SLE activity, as measured by the SLE Disease Activity Index (SLEDAI), or to the presence of vasculopathy or lupus nephritis (Table 1). SLEDAI scores did not differ significantly in relation to the D/D, D/I, and I/I ACE genotypes: the median, 25th and 75th percentiles, and range were, respectively, 14, 11, 22, 6–51; 13, 10, 18, 3–34; and 10, 15, 21, 8–27 (p 1⁄4 0.20, Kruskal–Wallis test), and no significant correlation was found between SLEDAI scores and serum ACE levels [r 1⁄4 –0.09 with 95% confidence intervals (CI) –0.28 to 0.11; p 1⁄4 0.37, Spearman’s rank correlation test]. The current study provides evidence of an association between SLE and the D allele of the ACE I/D genotype, and between SLE and elevated levels of serum ACE in an Scand J Rheumatol 2015;44:425–430 425

Anti-C reactive protein antibodies in Indian patients with systemic lupus erythematosus

Systemic lupus erythematosus (SLE) is characterized by over production of autoantibodies. C-reactive protein (CRP) is a phylogenetically highly conserved plasma protein that participates in the systemic response to inflammation. Anti-CRP antibodies might have biological functions of pathogenetic interest in SLE. We evaluated anti-CRP antibodies in Indian SLE patients and their association with anti-dsDNA antibodies and complement levels (C3 and C4). One hundred SLE patients diagnosed according to the American College of Rheumatology criteria were included. Disease activity was assessed using SLE disease activity index (SLEDAI). Anti-CRP autoantibodies were detected by enzyme linked immunosorbent assay. Anti-dsDNA antibodies were detected by indirect immunofluroscence test (Euroimmun Lubeck, Germany). High sensitivity CRP and complement levels (C3, C4) were detected using a Nephelometer. (BN ProSpec, Dade Behring, Germany). Anti-CRP antibodies were detected in 26% of SLE patients. Mean age of disease onset among anti-CRP positives was 22.4 ± 7.5, and 26.6 ± 9.3 years among anti-CRP negatives (P > 0.05). Anti-dsDNA positivity was significantly higher among anti-CRP positives (32.7%) as compared to anti-CRP negatives (16%) (P = 0.00519). No statistically significant difference was observed in SLEDAI scores of anti-CRP positive group and anti-CRP negative group (P > 0.05). We observed a positive correlation between anti-CRP antibodies and anti-dsDNA antibodies.

A functional SNP MCP-1 (−2518A/G) predispose to renal disorder in Indian Systemic Lupus Erythematosus patients

Abstract Systemic Lupus Erythematosus (SLE) is a clinically heterogeneous chronic, inflammatory autoimmune disorder that affects multiple organs where exact etiology of the disease is not yet clearly understood. Various evidences suggest that genetic polymorphisms in inflammatory mediators like cytokines and chemokines may influence development of the disease. Here, we investigated whether functional polymorphism at the Monocyte Chemoattractant Protein‐1 (MCP‐1) regulatory region associates with disease phenotype in Indian SLE patients. This case control study included 200 SLE patients and 201 ethnically matched healthy controls. Genotyping of MCP‐1 (−2518 A/G) polymorphism was performed using PCR‐RFLP method. Serum MCP‐1 levels were detected by bead‐based multiplex immunoassay. Serum MCP‐1 levels were found to be higher in patients compared with healthy individuals (p < 0.0001). A significant difference for MCP‐1G allele frequency (OR = 1.9, 95%CI = 1.4–2.6, p < 0.0001) was observed among SLE patients against healthy individuals. A significant difference in the distribution of MCP‐1 −2518GG (OR = 3.0, 95%CI = 1.4–6.7, p = 0.0041) and AG + GG genotypes (OR = 2.0, 95%CI = 1.4–3.0, p = 0.0005) was also noted among SLE patients when compared with healthy individuals. A significant association was observed between A/G and G/G versus A/A genotypes with renal manifestations (p < 0.0001, Pc < 0.001). Serum MCP‐1 levels in active LN patients were found to be significantly higher than inactive LN (p = 0.0059), mild LN (p = 0.0061) as well as non‐LN patients (p = 0.0001). These findings suggest that −2518G allele of MCP‐1 −2518 A/G polymorphism is associated with renal disorders and may influence MCP‐1 gene expression among Indian SLE patients.