Milind Nadkar

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.

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.

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

Do high sensitivity C-reactive protein and serum interleukin-6 levels correlate with disease activity in systemic lupus erythematosuspatients?

Introduction: Systemic Lupus Erythematosus (SLE) is an inflammatory autoimmune disease where an interplay between acute phase proteins and cytokines are involved in disease activation. Aim and Objectives: This case control study was performed to investigate interrelationship between high sensitivity C-reactive proteins (hs-CRP), Interleukin-6 (IL-6) levels and disease activity among SLE patients. Materials and Methods: One hundred forty one clinically diagnosed SLE cases were included and disease activity was noted by SLE Disease Activity Index (SLEDAI). Serum IL-6 levels were measure by cytokine multiplex assay. Serum hs-CRP, C3 and C4 levels were measure by nephelometer. The Pearson correlation test was used for correlation between hs-CRP, Il-6 and SLEDAI. Results: Based on SLEDAI, 126 patients (89.4 %) had active disease and 15 patients (10.6%) had inactive disease. Mean hs-CRP levels in SLE patients were significantly higher (12.1+ 11.5 mg/L) than controls (2.41+ 1.37 mg/L) (P < 0.0001). Hs-CRP levels among active SLE were significantly higher (13.5+ 11.4 mg/L) as compared with inactive SLE (4.4 + 2.9 mg/L) (P=0.0002). Similarly, IL-6 levels in SLE patients were significantly higher among active SLE (26.9 + 15.5 pg/ml) as compared with inactive SLE (13.9+ 10.2 pg/ml) (P=0.0001). An inverse correlation between Il-6 and hemoglobin levels between active and inactive SLE was noted (r=-0.46, P <0.0001). Conclusion: This study suggests a good correlation between hs-CRP, IL-6 and SLE disease activity indicating their direct involvement in inflammatory conditions associated with disease.

Association of clinical and serological parameters of Systemic Lupus Erythematosus patients with Epstein-Barr virus antibody profile†

Epstein‐Barr viral infection is one of the known environmental factors involved in development of Systemic Lupus Erythematous (SLE). Though not much is known about the exact role of Epstein‐Barr virus (EBV) in SLE pathogenesis, the theory of switching of lytic and lysogenic cycles of EBV in memory B cells fits well with the periods of waning disease activity and intermittent flares in SLE patients. In this study, we investigate the association of EBV antibody profile with clinical and serological parameters in SLE. Eighty‐seven clinically diagnosed SLE patients fulfilling the American College of Rheumatology (ACR) classification criteria and fifty healthy individuals were enrolled in this case control study. Anti‐VCA IgM, anti‐VCA IgG, and anti‐EBNA IgG were detected by ELISA technique. Antibodies concentrations between two groups were compared using Mann‐Whitney whereas the difference in categorical data was compared using Chi‐square considering statistical significance at P < 0.05. This study demonstrated a significant increase in EBV VCA‐IgG, VCA‐IgM, and EBNA‐IgG antibodies levels of SLE patients when compared to healthy controls (P < 0.05). High seroprevalence was seen in both the study groups for EBV VCA‐IgG and EBNA‐IgG antibodies when compared to VCA‐IgM antibodies. A significant increase was noted in the anti‐VCA‐IgG levels with dsDNA autoantibody positivity (P < 0.05). Though there was no significant association between EBV antibody profile and clinical manifestations, 100% seropositivity for anti‐VCA‐IgG was seen in SLE patients with renal manifestations. Association of anti‐VCA IgG levels with presence of anti‐dsDNA antibodies suggests a possible role of EBV as an environmental trigger in pathogenesis of SLE.

Catalytic antibodies in patients with systemic lupus erythematosus

OBJECTIVE Antibodies with catalytic (hydrolytic) properties to DNA or RNA have been reported in systemic lupus erythematosus (SLE). However, it is well known that ethnicity plays an important role in the presentation of SLE and severity of the disease; hence, these data may not truly represent a general feature of all SLE patients. Therefore, we have analyzed the hydrolyzing activity of immunoglobulin G (IgG) of SLE patients from the Indian population with an aim to decode whether the catalytic antibody response represents part of an active disease process. METHODS IgGs were isolated from the sera of 72 consecutive patients diagnosed with SLE. As a control, IgGs from healthy donors were used. The catalytic activity of IgG was measured by PFR-MCA and affinity-linked oligonucleotide nuclease assay. RESULTS IgGs from patients with SLE from the Indian subcontinent displayed significantly higher hydrolysis rates of both the surrogate substrate, PFR-MCA, and the DNA than IgG from healthy individuals. Intergroup comparisons of the IgG-PFR-MCA interactions with clinical manifestations of the disease demonstrated a significantly increased level of hydrolysis among the patients with renal involvement who tested positive for anti-dsDNA antibodies. The PFR-MCA hydrolysis also appears to be associated with the active disease (p=0.0988, vs. inactive group). CONCLUSION The prevalence of catalytic antibodies represents a general feature of SLE patients, irrespective of their origin.

Adipokine interactions promote the pathogenesis of systemic lupus erythematosus

Background Adipokines are chemical mediators released from adipose tissue involved in regulation of appetite, insulin sensitivity, immune system and inflammatory responses. Adipokines contributes to low grade inflammatory response in autoimmune disease like Systemic Lupus Erythematosus (SLE) but the pathophysiology is yet not clear. The aim of this study is to understand role of adipokine interactions in SLE disease pathogenesis. Methods Sixty newly diagnosed treatment naïve SLE patients fulfilling the ACR criteria and forty age‐sex matched healthy subjects were enrolled in thiscase‐control study. Disease activity in SLE patients was evaluated using SELENA‐SLEDAI. Array of adipokines, C1q circulating immune complexes (C1q‐CIC), anti‐C1q, anti‐ribososmal P0 (anti‐RibP0) and anti‐mitochondrial antibodies (AMA) levels were detected by ELISA. Antinuclear antibodies (ANA) and anti‐dsDNA autoantibodieswere detected by Indirect Immunofluorescence (IIF), while antigen specificities were detected by Immunoassay blot. Serum levels of C3 and C4 complement factors were assessed by nephlometer. Results Statistically significant elevation in progranulin, adipsin and resistin levels was seen among SLE patients when compared to healthy controls (p < 0.0001). Leptin and omentin levels were significantly reduced in SLE patients (p < 0.0001). There was no statistically significant difference in serum adiponectin, chemerin and visfatin levels when these two groups were compared (p > 0.05). Adiponectin, adipsin and resistin levels were elevated in SLE patients with renal manifestations (p < 0.05). Reduced leptin levels were significantly associated with presence of renal manifestations (p < 0.05). Adiponectin levels positively correlated with disease activity (r = 0.294, p = 0.027) whereas negatively correlated with C3 levels (r = −0.439, p = 0.0007). A positive correlation was observed between hypocomplementemia and leptin levels (p < 0.05). Leptin levels were negatively correlated with disease activity, anti‐dsDNA, C1q‐CIC and anti‐C1q levels (p < 0.05). A significant positive correlation was observed between progranulin levels and anti‐ribosomal P0 antibodies (r = 0.499, p < 0.0001). Conclusion Adipokines levels and associated clinical manifestations suggest involvement of adipokines in disease pathogenesis of SLE. SLE disease activity and complement components may suggest regulatory effect of adipokines (adiponectin and leptin) on disease pathogenesis. Further studies on adipokines in SLE patients with renal manifestations may propose them as prognostic markers in renal damage. Trial Registration: NA