Functional characterization of complete and immunodominant epitopes of a novel pollen allergen from Parthenium hysterophorus.

Summary: Background. Parthenium hysterophorus pollen induces chronic clinical conditions such as allergic rhinitis and bronchial asthma. Among the plethora of proteins in the pollens, only few were reported to induce allergy. Currently sensitization to P. hysterophorus pollen allergen is diagnosed by skin prick test (SPT) using the entire pollen extract instead of using the specific allergen. Methods. In P. hysterophorus sensitized patients, SPT was done using the crude pollen extract, 40 kDa allergenic pollen protein and two commercially synthesized allergen epitopes (17 and 24) of P. hysterophorus. Dot-blot of allergen epitopes was done using P. hysterophorus sensitized sera. Crude pollen extract (1, 1.25, 2.5, 5 and 10 µg/mL), 40 kDa allergenic protein (3 µg/mL), and allergen epitopes (3µg/mL) were used to perform Basophil Activation Test (BAT). Results. Crude pollen extract at 2.5, 5, 10 µg/mL and 40 kDa allergenic protein at 3µg/mL concentrations induced wheal and flare reaction by around 15 minutes, whereas commercially synthesized allergen epitopes at 3µg/mL induced wheal and flare reactions in less than 10 minutes. Allergen epitopes (3 µg/mL) revealed strong reactivity with sensitized patient's IgE in dot-blot analysis. Basophil activation Test using crude pollen extract (2.5, 5, 10 µg/mL), 40 kDa allergenic protein (3 µg/mL), and allergenic epitopes (3µg/mL) indicated significant basophil activation (as measured by CD63 expression) in sensitized patients. Conclusions. The 40 kDa allergenic protein and its allergenic epitopes (17 and 24) induced phenotypic and cellular immune responses in P. hysterophorus sensitized individuals. The tested allergenic epitopes (17 and 24) induced faster wheal and flare reactions in comparison with the crude extract and the 40 kDa allergenic protein. The novel 40kDa allergenic protein and its allergen epitopes identified here may be useful for the development of component-resolved diagnosis (CRD) while also serving as a potential therapeutic lead for desensitization treatment for P. hysterophorus pollen induced allergy.

Immunological biomarkers in neuropsychiatric systemic lupus erythematosus: a comparative cross-sectional study from a tertiary care center in South India.

INTRODUCTION: The prevalence of various immunological biomarkers in neuropsychiatric systemic lupus erythematosus (NPSLE) differs among various patients with varied neuropsychiatric manifestations and different populations. We studied the prevalence of these biomarkers; especially the neuron specific autoantibodies in patients with systemic lupus erythematosus (SLE) and compared them among patients with and without neuropsychiatric involvement. METHODOLOGY: This is a comparative cross-sectional study conducted in a tertiary care hospital in South India. The prevalence of immunological biomarkers including complement levels, systemic and brain specific autoantibodies (anti-myelin antibody, anti-myelin oligodendrocyte glycoprotein and anti-myelin-associated glycoprotein antibody) were assessed and compared among those with and without NPSLE and with different NPSLE manifestations. RESULTS: A total of 522 SLE patients were enrolled in the study. The mean age of the study participants was 28.5 ± 8.8 years and 93.5% were women. Neuropsychiatric manifestations were seen in 167 (32%) patients. Seizure was the most common neuropsychiatric manifestation seen in 41.3%, followed by psychosis (18.6%), mood disorder (16.8%), stroke (10.8%), mononeuropathy (10.2%), headache (9.6%), acute confusional state (6.6%) and aseptic meningitis (5.4%). Patients with NPSLE had a higher SLE disease activity index score. Most of the autoantibodies, that is anticardiolipin antibody (aCL), anti-ß2 glycoprotein 1 antibody (ß2GP1), lupus anticoagulant (LA), anti-nucleosome, anti-ribosomal P, anti-Ro52, anti-Ro60 and anti-La, were seen in higher proportion in the NPSLE group, although the difference failed to reach statistical significance. On subgroup analysis, psychosis was significantly higher in patients with anti-ribosomal P positivity than without (11.8% versus 4.1%, p.0.007; odds ratio (OR) 3.1, confidence interval (CI) 1.4-6.8), while stroke had a higher proportion among those with positive b2GP1 IgG (6.3% versus 1.8%, p.0.03; OR 3.6, CI 1.2-11.0). A higher proportion of demyelination was seen among the LA positive than the negative (10.3% versus 0.2%, p.0.03; OR 5.39, CI 1.15-24.17) and anti-myelin oligodendrocyte glycoprotein in mood disorder (14.3% versus 3.4%, p = 0.03; OR 4.66, CI 1.13-19.13). CONCLUSION: No single biomarker correlated with NPSLE. Among different NPSLE manifestations, the prevalence of IgG ß2GP1 in stroke, LA in demyelination, anti-ribosomal P in psychosis and anti-myelin oligodendrocyte glycoprotein in mood disorder were higher. Further studies on the pathogenic mechanisms underlying NPSLE and its different manifestations may help us to identify better biomarkers.

HLA genetics in bipolar disorder.

OBJECTIVE: Bipolar Disorder (BD) is characterized by deregulated adaptive immune processes. Recent genome-wide association studies (GWAS) implicate the major histocompatibility complex (MHC) region in BD. The present study investigates the potential influence of variations in human leukocyte antigen (HLA) on BD risk and/or clinical presentations. This may have relevance to the dysregulated inflammatory processes commonly found in BD. METHOD: DNAs from 475 BD patients and 195 healthy controls (HC) were genotyped for classical HLA class I and II loci. RESULTS: We found that: (i) the HLA-A*02~B*44~DRB1*07 sub-haplotype is less prevalent in BD, vs. HC (pc = 2.4 × 10-2 ); (ii) the 57.1 and the 8.1-derived ancestral haplotypes i.e. HLA-A*02~B*57~Cw*06~DRB1*07~DQB1*09 and HLA-A*02~B*08~Cw*07 are associated with rapid cycling (pc = 1.9 × 10-3 and 1.05 × 10-2 , respectively); (iii) the 8.1AH-derived HLA class II-DRB*03~HLA-DQB1*02 sub-haplotype is more frequent in BD patients with a history of suicidal behaviors (pc = 2.1 × 10-2 ); and (iv) disease onset by an hypomanic episode or by psychotic symptoms are, respectively, more frequent in BD patients bearing the 7.1 AH-derived A*03~B*07~DRB1*15 sub-haplotype (pc = 8.5 × 10-3 ) and the HLA-A*02~B*07~DRB1*15 sub-haplotype (pc = 4.0 × 10-2 ). CONCLUSION: Corroborating the established link between these HLA haplotypes/sub haplotypes and common immune disorders, our findings suggest possible HLA-mediated proinflammatory processes operating in BD.

Association of NKG2D gene variants with susceptibility and severity of rheumatoid arthritis.

NKG2D (KLRK1) is a C-type lectin receptor present on natural killer (NK) cells, γδ, CD8+ and CD4+ T cells. Upon ligand binding, NKG2D mediates activatory and co-stimulatory signals to NK cells and activated CD4+ T cells, respectively. Polymorphisms in NKG2D predispose to infectious diseases, cancer, transplantation and autoimmune disorders. We studied the influence of this NK receptor polymorphism on predisposition to and modification of the disease phenotype in patients with rheumatoid arthritis (RA). Eight different single nucleotide polymorphisms (SNP) in the NKG2 gene were genotyped in 236 patients with RA and 187 controls using Taqman 5' nuclease assays. NKG2D genotype/allele frequency did not differ between patients and controls. Subgroup analysis showed that the frequency of A allele of NKG2D9 and T allele of NKG2D10 was significantly higher in patients with deformities (a marker of severe disease) [11 versus 5%, Pc = 0·03, odds ratio (OR) = 2·44, 95% confidence interval (CI) = 1·09-5·98 and 10 versus 4%, Pc = 0·04, OR = 2·45, 95% CI = 1·05-6·39, respectively], while the frequency of alleles G of NKG2D9 and A of NKG2D10 was greater in patients without deformities (Pc = 0·03, OR = 0·41, 95% CI = 0·17-0·91 and Pc = 0·04, OR = 0·41, 95% CI = 0·16-0·96). Similar trends of association were observed with deforming phenotype of RA in female patients and deforming young onset RA subgroups. Haplotype analysis revealed that the frequency of haplotype G-C-A-G-A-T-C-C was higher in patients than in controls (12 versus 8%, P = 0·04, OR = 1·61, 95% CI = 1·01-2·55), suggesting that it may predispose to RA. Our study suggests that the NKG2D gene polymorphisms may modify the risk of development and severity of RA.

Polymorphisms in the promoter region of iNOS predispose to rheumatoid arthritis in south Indian Tamils.

Nitric oxide synthase (NOS) catalyses the production of nitric oxide (NO) from L-Arginine, which participates in diverse biological processes including inflammation and apoptosis. Macrophages, chondrocytes, osteoblasts and osteoclasts express inducible NOS (iNOS) at the site of synovial inflammation. NO produced at the inflamed joint may contribute to peri-articular bone loss, mediate apoptosis and regulate Th1/Th2 balance in rheumatoid arthritis (RA). Variations in the promoter region of NOS gene regulate the nitric oxide synthase expression and iNOS (NOS2) polymorphisms have been associated with susceptibility to autoimmune disorders. Hence, this study was conducted to identify the possible contributions of NOS2 -1659G/A, -1026C/A, -277A/G promoter polymorphisms towards development of RA in South Indian Tamils. A total of 242 (219 females, 23 males) patients with RA (mean age 41.2 ± 10.9 years, disease duration 8.5 ± 4.3 years) and 279 age- and sex-matched healthy individuals of South Indian Tamil ethnicity were genotyped for NOS2 -1659C/T, -1026G/T and -277A/G promoter polymorphisms by TaqMan chemistry. Nature of disease (erosive or nonerosive), the presence of extra-articular manifestations, seropositivity for rheumatoid factor and anticyclic citrullinated peptide, serum C-reactive protein (CRP) level and response to therapy were assessed for all patients. The three single nucleotide polymorphisms (SNPs) were in Hardy-Weinberg equilibrium. The frequency of GG genotype and G allele of NOS2-277 was higher in patients (pc = 5.7 × 10-9 , OR = 6.09, 95% CI = 3.09-12.8 and pc = 4 × 10-13 , OR = 2.37, 95% CI = 2.06-3.62, respectively) compared to controls. Similarly, the frequency of NOS2-1026 (rs2779249) GT genotype and the T allele was higher in patients with RA (pc = .01, OR = 1.61, 95% CI = 1.09-2.36, and pc = .04, OR = 1.40, 95% CI = 1.02-1.91, respectively). However, no significant difference in frequency of NOS2-1659C/T polymorphism was observed between patients and controls. None of the studied SNPs were associated with erosive disease, seropositivity or extra-articular manifestations. The -277A/G and -1026 G/T promoter polymorphisms in iNOS may confer susceptibility to RA in South Indian Tamils.

Human leukocyte antigen-G polymorphism influences the age of onset and autoantibody status in rheumatoid arthritis.

The study was conducted to investigate the frequency of three gene polymorphisms in the 3'-untranslated region (3'-UTR) of human leucocyte antigen-G (HLA-G) gene in south Indian patients with rheumatoid arthritis (RA) and analyze their influence on disease susceptibility, phenotype and treatment response. HLA-G 14 bp insertion (Ins)/deletion (del) (rs66554220), HLA-G +3142G>C (rs1063320) and +3187A>G (rs9380142) polymorphism was analyzed in 221 RA patients and 200 healthy controls. Frequency of HLA-G genotypes or alleles did not differ between patients and controls. Analysis based on rheumatoid factor (RF) status revealed that the frequency of allele 'A' (rs9380142) was significantly higher in RF-positive than in RF-negative patients [84% vs 74%, Yates-corrected P value (Pc) = 0.04, odds ratio (OR) = 1.8, 95% confidence interval (CI) = 1.0-3.2]. A similar difference was maintained in RF-positive female patients than their RF-negative counterparts (83% vs 71%, Pc = 0.02, OR = 1.9, 95% CI = 1.0 to 3.4) and between RF-positive and RF-negative young onset RA (YORA) patients (84% vs 73%, Pc = 0.03, OR = 1.9, 95% CI = 1.0-3.2), suggesting that rs9380142 polymorphism influenced RF status. The 14 bp Ins allele of rs66554220 was significantly more prevalent in RF-positive YORA than in RF-positive late onset RA (LORA) patients (51% vs 25%, P = 0.03, OR = 3.1, 95% CI = 1.1-9.8). Frequency of the four major haplotypes [InsGA (48%), DelGA (22%), DelCG (18%), DelCA (9.7%)] observed did not differ between cases and controls. HLA-G does not appear to be a risk factor for development of RA in south Indian Tamils but may act as a genetic modifier of clinical phenotype in terms of autoantibody production, gender preference and age at disease onset.

IRF5rs2004640 single nucleotide polymorphism is associated with susceptibility to rheumatoid arthritis in South Indian Tamils.

Polymorphism of interferon regulatory factor 5 (IRF5), a latent transcription factor gene has been associated with various auto-immune diseases. Our aim was to study the IRF5rs2004640 gene polymorphism and its association with disease susceptibility, disease phenotype and treatment response in South Indian Tamil patients with rheumatoid arthritis (RA).The study was conducted on 217 RA patients fulfilling the American College of Rheumatology (ACR) 2010 criteria and 482 healthy controls (HCs) without family history of autoimmune disease. The IRF5rs2004640 genotyping was performed using a TaqMan 5' allelic discrimination assay. We found that the IRF5rs2004640T allele [P < 0.0001, odds ratio (OR) 3.25, 95% confidence interval (CI) 2.55-4.12] and TT genotype (P < 0.0001, OR 4.60, 95% CI 3.23-6.57) were significantly more frequent in RA patients as compared with HCs. No association was found between IRF5rs2004640 polymorphism, clinical manifestations, autoantibody profile and treatment response. IRF5rs2004640 T (mutant) allele may be a susceptibility factor conferring risk for RA in South Indian Tamils, whereas G allele (wild type) may be protective.

HLA class II alleles influence rheumatoid arthritis susceptibility and autoantibody status in South Indian Tamil population.

Rheumatoid arthritis (RA) is a complex multifactorial autoimmune disease characterized by inflammatory arthritis. The precise etiology and pathogenesis of RA remains elusive but evidence points towards stochastic interactions between genetic and environmental factors. This study investigated the distribution of human leucocyte antigen (HLA)-DRB1/DQB1 alleles in South Indian patients with rheumatoid arthritis (RA) and their influence on RA susceptibility and clinical phenotype. Low resolution HLA-DRB1 and -DQB1 typing was performed in 271 RA patients and 233 healthy controls by polymerase chain reaction (PCR) using sequence-specific primers (SSP). HLA-DRB1*10 was found to be more frequent in patients (Pc = 0.004, OR = 2.23, 95% CI = 1.5-3.34) than controls. This difference persisted in RF positive (Pc = 9 × 10-6 , OR = 2.45, 95% CI = 1.62-3.74), ACPA positive (Pc = 0.007, OR = 2.10, 95% CI = 1.35-3.29), ACPA negative (Pc = 0.001, OR = 2.45, 95% CI = 1.50-3.97) and both RF and ACPA positive subgroup of patients (Pc = 0.003, OR = 2.22, 95% CI = 1.41-3.51). On the contrary, the HLA-DRB1*13 (Pc = 0.01, OR = 0.43, 95% CI = 0.25-0.73) and HLA-DRB1*14 (Pc = 0.003, OR = 0.43, 95% CI = 0.26-0.69) alleles were over-represented in controls than patients. Further, distribution of the prominent Caucasian RA risk allele DRB1*04 did not differ between patients and controls in our study population. We did not find any association between DQB1 alleles and RA susceptibility or autoantibody status. The haplotypes DQB1*05-DRB1*10 (P = 6.8 × 10-6 , OR = 2.46, 95% CI = 1.63-3.79) and DQB1*06-DRB1*15 (P = 0.03, OR = 1.41, 95% CI = 1.02-1.96) were more frequent in patients while DQB1*05-DRB1*14 (P = 8.4 × 10-4 , OR = 0.44, 95% CI = 0.26-0.74) and DQB1*06-DRB1*13 (P = 9.5 × 10-4 , OR = 0.40, 95% CI = 0.21-0.72) were higher in controls. To conclude, HLA-DRB1*10 is associated with RA while HLA-DRB1*13 and HLA-DRB1*14 alleles confer protection in south Indian Tamils.