Manisha Patwardhan

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-nucleosome antibodies as a disease marker in systemic lupus erythematosus and its correlation with disease activity and other autoantibodies

BACKGROUND Detection of anti-nucleosome antibodies (anti-nuc) in patients with systemic lupus erythematosus (SLE) has been well established and it is claimed that their presence is associated with disease activity. AIMS The aim of this study is to evaluate the incidence of anti-nuc antibodies and to correlate them with disease activity and its association with other autoantibodies like anti-nuclear antibodies (ANA), anti-double stranded DNA (anti-dsDNA), anti-histone antibodies (AHA), as well as autoantibodies to histone subfractions like H1, (H2A-H4) complex, H2B, and H3. METHODS This cross-sectional study included 100 SLE patients referred from the Rheumatology, Dermatology, and Nephrology Departments. SLE disease activity was evaluated by using SLE-Disease Activity Index (SLEDAI) score. A patient was defined as having active SLE when the SLEDAI score was more than 5.0. Fifty normal controls were also tested as a healthy control group. Anti-nuc antibodies, anti-dsDNA, and AHA were tested by Enzyme-Linked Immunosorbent Assay (ELISA) and ANA was detected by an indirect immunofluorescence test. RESULTS All patients studied were in an active stage of disease and were untreated, of which 44 patients had renal biopsy-proven kidney involvement, which was categorized as lupus nephritis (LN) and 56 patients did not show any renal manifestations (SLE without LN). Anti-nuc antibodies were positive in 88%, anti-dsDNA in 80%, and AHA in 38% of the cases. ANA was positive in all SLE patients studied. None of the normal controls was found to be positive for these antibodies. Although a slightly higher incidence of autoantibodies were noted in LN, there was no statistical difference noted between LN and SLE without LN groups for anti-nuc and anti-dsDNA antibodies (p > 0.05). A higher incidence of autoantibodies to ANA specificities were noted in anti-nuc positive cases, but there was no statistical difference between anti-nuc positive and anti-nuc negative cases for ANA specificities among LN and SLE without nephritis groups (p > 0.05). CONCLUSIONS Anti-nuc antibody detection could be a better tool for the diagnosis of SLE. Although there was no significant difference in LN and SLE without LN groups, this study suggests that anti-nuc detection can be useful as an additional disease activity marker to other laboratory tests.

Autoantibody profile and other immunological parameters in recurrent spontaneous abortion patients

Background: An autoimmune cause and related immunological alterations resulting in recurrent spontaneous abortion (RSA) have been suggested in patients with unknown etiology. Materials and Methods: This study evaluated the autoantibody profile and other immunological parameters among RSA patients and normal pregnant women from Mumbai western India. Fifty RSA patients with unknown cause and greater than three consecutive abortions along with 50 normal pregnant women were studied for various auto antibodies such as ANA, anti-dsDNA, ANCA, AECA, 2 micro globulin, anti-HLA antibodies and ACLA using immunofluorescence microlymphocytotoxicity and ELISA. Immunological parameters such as HLA class I monoclonal antibody expression, CD3 (T cell), CD19 (B cell), and CD56 (NK cell) were estimated by flow cytometry. Results: The results revealed 34% positivity of all auto antibodies tested among patients. ANA(12%), ANCA (20%), AECA (24%), ACLA (8%), anti-dsDNA(0%), β2 microglobulin (14%), and anti-HLA antibodies(10%) among RSA patients were identified. An increased expression of HLA class I specific monoclonal antibody (10%) with HLA A3 (16%) specificity were found to correlate with shared HLA alleles among the RSA couples. Among normal pregnant (control) group ANA (2%), ANCA (2%), AECA (3%), ACLA (4%) and increased expression of CD56 with reduced HLA class I monoclonal were observed. Conclusion: Our findings suggest a possible role of various autoantibodies along with the related immunological parameters underlying RSA.

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.

Fc γ R IIIB polymorphisms: their association with clinical manifestations and autoantibodies in SLE patients from Western India

Background:  Receptors for the Fc fragment of immunoglobulin G (Fc γ Rs) represent the link between the humoral and cellular immune responses. Polymorphisms of Fc γ R, mainly IIA, IIB, IIIA, IIIB have been identified as genetic factors influencing susceptibility to disease or disease course of a prototype autoimmune disease like systemic lupus erythematosus (SLE). Fc γ alleles may be associated with inefficient removal of apoptotic cells or antigens and hence may be associated with higher risk of SLE.

APO-1/Fas gene: Structural and functional characteristics in systemic lupus erythematosus and other autoimmune diseases

Systemic lupus erythematosus (SLE) is an autoimmune disorder affecting multiple organ systems. It is characterized by the presence of autoantibodies reactive against various self-antigens. Susceptibility to SLE is found to be associated with many major histocompatibility complex (MHC) and non-MHC genes, one of which is APO-1/Fas gene, which is present on chromosome 10 in humans. The APO-1/Fas promoter contains consensus sequences for binding of several transcription factors that affect the intensity of Fas expression in cells. The mutations in the APO-1/Fas promoter are associated with risk and severity in various autoimmune diseases and other malignancies. The APO-1/Fas receptor is expressed by many cell types. Two forms of APO-1/Fas protein that are involved in regulation of apoptosis have been identified. Fas receptor-mediated apoptosis plays a physiological and pathological role in killing of infected cell targets. In this review, we have focused on APO-1/Fas gene structure, promoter variants and its association with SLE and other autoimmune diseases. Functional aspects of Fas receptor in apoptosis are also discussed.

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.

Fc gamma receptor polymorphisms in systemic lupus erythematosus and their correlation with the clinical severity of the disease

Receptors for the Fc domains of IgG (Fc γ R) play a critical role in linking humoral and cellular immune responses. The various Fc γ R genes may contribute to differences in infectious and immune related diseases in various ethnic populations. Polymorphisms of Fc γ R mainly Fc γ R IIA, IIB, IIIA, IIIB have been identified as genetic factors influencing susceptibility to disease or disease course of a prototype autoimmune disease like Systemic Lupus Erythematosus (SLE). Activated and inhibitory Fc γ Rs seem to play an important role in the pathogenesis of SLE, in initiation of autoimmunity, the subsequent development of inflammatory lesions and finally immune clearance mechanisms. This review focuses on the role of Fc γ R polymorphism and their association with clinical manifestations and initiation of autoantibody production, inflammatory handling of immune complexes and disease development in SLE patients.

Fc γ RIIA Genotypes and Its Association with Anti-C1q Autoantibodies in Lupus Nephritis (LN) Patients from Western India

To identify Fc γ RIIA genotypes in Systemic Lupus Erythematosus (SLE) patients and their association with anti-C1q antibodies. Methods. Fc γ RIIA genotyping was done in eighty Indian SLE patients and eighty healthy controls using allele-specific PCR. Anti-C1q antibodies were measured by ELISA. Results. LN patients showed higher SLEDAI (6–32) as compared to SLE patients without renal manifestations and had SLEDAI between 6–23. Fc γ RIIA polymorphic frequency in SLE patients was R131/H131 (67.5%), R131/R131 (20%) and H131/ H131 (12.5%) as against that of normal population (62.5%, 10%, and 27.5%), respectively. Sixty two patients (77.5%) showed positivity for anti-C1q antibodies. LN patients showed elevated levels of anti-C1q antibodies (258.2 u/ml ± 38.5 U/mL) as compared to SLE patients without nephritis (134.6 ± 24.6 U/mL). Among anti-C1q positive patients, 71% had R131/H131 genotype, 22.6% had R131/R131 and remaining 6.4%, patients had H131/H131 genotype. All anti-C1q positive patients with R131/R131 genotype had elevated levels of anti-C1q antibodies (>100 U/ml), whereas among anti-C1q negative patients, none had R131/R131 genotype. Conclusion. This first report on Indian SLE patients supports the hypothesis that Fc γ RIIA R131 variant over expression may constitute a susceptibility factor for development of severe SLE manifestations in LN patients.