DNA methylation profiling of labial salivary gland tissues revealed hypomethylation of B-cell-related genes in primary Sjögren's syndrome.

The objective of this epigenetic study was to investigate the cellular proportions based on DNA methylation signatures and pathways of differentially methylated genes in labial salivary gland (LSG) tissues of individuals with Sjögren's syndrome (SS). Two methylation array datasets from the Gene Expression Omnibus repository (GSE166373 and GSE110007) were utilized, consisting of 159 LSG tissues from 77 SS cases and 82 non-SS controls. The raw data underwent analysis using the Chip Analysis Methylation Pipeline (ChAMP) in R statistical tool, which identified differential methylation probes and regions. The EpiDISH and minfi packages in R were employed to identify proportions of epithelial cells, fibroblasts, and immune cells, as well as immune cell subsets. The results showed that proportions of immune cells were increased, while proportions of epithelial cells and fibroblasts were significantly decreased in the LSG of individuals with SS compared to non-SS controls. Specifically, proportions of B-cells and CD8 T-cells were increased, while CD4 T-cells, Treg, monocytes, and neutrophils were decreased in the LSG of individuals with SS. Pathway analysis indicated that genes involved in immune responses to Epstein-Barr virus infection were significantly hypomethylated in SS, and gene set enrichment analysis highlighted the hypomethylation of genes involved in the somatic recombination of immune receptors in SS. Additionally, Disease Ontology analysis showed enriched pathways related to multiple myeloma, arthritis, and the human immunodeficiency virus. The study also revealed significant hypomethylation of the WAS gene on chromosome X in LSG tissues of individuals with SS. Overall, the findings suggest an increased proportion of B-cells and genes related to B-cell function, as well as hypomethylation of genes involved in immune responses and immune receptor recombination, in LSG tissues of individuals with SS compared to non-SS controls.

Angiogenesis related genes in Takayasu Arteritis (TAK): robust association with Tag SNPs of IL-18 and FGF-2 in a South Asian Cohort.

We performed genetic association study for genes encoding angiogenic and angiostatic proteins in patients with Takayasu arteritis (TAK). A total of 96 SNPs involving 60 genes were studied. Genotyping was performed in Fluidigm 96.96 Dynamic Array chip. All statistical analysis for SNP evaluation was performed using PLINK software. Initial analyses revealed five SNPs from three genes [IL-18 (encodes Interleukin-18), FGF2 (encodes Fibroblast Growth Factor-2), and ANGPT1 (encodes Angiopoietin-1)] as significantly different between controls and cases (uncorrected p < 0.05). After permutation-based analysis, two tag SNPs on the promoter region of IL-18 (rs187238 and rs1946518) and one 3'UTR tag SNP (rs1476217) of FGF2 were significantly associated with susceptibility to TAK, with p and OR (95% CI) of 0.0006 and 1.64 (1.25-2.17), 0.03 and 1.28 (1.02-1.64) & 0.016 and 1.33 (1.05-1.67), respectively; while, the two tag SNPs of ANGPT1 gene (rs6469101 and rs16875900) showed a trend (p = 0.055 & p = 0.051, respectively after permutation based correction). There is robust linkage disequilibrium between the two tag SNPs of IL-18 gene as validated by 1000 genome data of South Asian population; the eQTL effects of these tag SNPs of IL-18 and FGF2 genes on adjacent genes further suggest that these tag SNPs act as genetic risks for development of TAK in South Asians, with possible functional implications towards future biomarker development. Genotype phenotype study by genetic model-based analysis also revealed associations between genotype subsets and clinical features like fever, visual loss, left subclavian and coronary artery involvement in our TAK patients.

In vitro effects of curcumin on proinflammatory cytokines and expression of their genes in minor salivary gland tissue of patients with Sjogren's syndrome.

Curcumin reduces disease severity and ameliorates lupus-like/Sjögren's Syndrome-like disease in mice model. The immunological basis of these effects is largely unknown. This study examined the effects of curcumin on pro-inflammatory cytokines secreted by minor salivary glands in patients with primary Sjögren's syndrome (pSS). Minor salivary gland (MSG) tissue samples were collected from patients undergoing biopsy for suspected pSS. The tissues were treated with phytohemagglutinin (PHA) alone as well as PHA with curcumin (30 µM) and cultured in RPMI 1640 medium for 48 h at 37 °C in CO2 incubator. After the incubation period, culture supernatant and tissues were stored in the freezer (-80 °C). IL-6 levels were measured in supernatant by ELISA kit. Gene expressions of pro-inflammatory cytokines, namely IL-6, IL-8, TNF-α, IL-1ß, IL-4, IL-10, IL-17, IL-21, and IFN-γ, were measured by qPCR. IL-6 secretion levels and gene expressions were compared statistically between groups by Student's t test. Forty-seven patients were screened. Eight patients satisfied ACR/EULAR criteria for pSS. Seven patients with absent glandular inflammation and negative serology constituted sicca controls. These 15 subjects were included in final analysis. In pSS group, but not in controls, median IL-6 levels in supernatant were less in curcumin-treated as compared to PHA-alone subset [5.5 (0.7-13.34) vs 18.3 (12-32) ng/ml; p = 0.0156]. mRNA expression levels of IL-6 were also lower in curcumin-treated samples as compared to PHA alone, when cases and controls were analyzed together as well as in cases alone (p = 0.0009 and p = 0.0078, respectively); however, mRNA expression of IL-1ß was lower in curcumin-treated samples as compared to PHA alone, only when cases and controls were analyzed together (p = 0.0215). There was no difference in other cytokine gene expression levels between the subsets under the in-vitro experimental conditions. In conclusion, curcumin reduced mRNA expression as well as secretion of IL-6 levels by salivary gland tissue of patients with pSS. Curcumin also suppressed PHA-induced mRNA expression levels of IL-6 and IL-1ß in MSG tissue of patients with pSS and controls when analyzed together as a combined group.

High expression of S100 calgranulin genes in peripheral blood mononuclear cells from patients with Takayasu arteritis.

BACKGROUND: Toll-like receptors (TLR) 1 to 4 are highly expressed in aorta. Activation of TLR4 causes transmural arteritis in Human temporal artery-SCID chimera model. Neither TLR-4 nor its ligands have been studied in TA patients as yet. Aim of this study was to examine the expression of TLR4 and its endogenous ligands in peripheral blood mononuclear cells (PBMCs) of patients with Takayasu arteritis (TA). METHODS: mRNA expression of TLR4, RAGE and various endogenous TLR4 ligands were quantified in PBMCs of 24 TA patients and 19 sex and age matched healthy controls by real time PCR using specific primers and SYBR Green qPCR master mix. S100A8/A9 and S100A12 were measured in cell culture supernatant of PBMCs from TA patients and healthy controls, both in un-stimulated state as well as, after lipopolysaccharides (LPS) stimulated cultures for 4 h. Expression of S100A8/A9 in aortic tissues was assessed by immunohistochemistry. RESULTS: The mRNA expression of S100A8, S100A9, S100A12 and TLR4 were higher, while expression of RAGE and HSP70 were lower in TA as compared to healthy controls. Induction with LPS led to increase in secretion of both S100A8/A9 and S100A12 levels in TA as well as healthy controls. The fold of induction, measured by LPS stimulated/unstimulated control was higher in healthy controls [2.88 (1.7-3.53) fold] as compared to TA [1.345 (1-1.82) fold]; p < 0.05. Numerically, S100A8/A9 was also higher in healthy controls [2.04 (1.7-5.6) fold] as compared to TA [1.38 (1.09-3.6) fold], but it didn't reach statistical significance; p = 0.129. Mild to moderate intensity expression of S100A8/A9 protein was noted in aortic tissues from patients with TA. CONCLUSION: mRNA expression of TLR4 and its ligand S100A8, S100A9, and S100A12 in PBMCs of TA patients was higher as compared to healthy controls. LPS stimulation led to higher induction of S100A12 secretion in healthy controls as compared to TA. Expression of S100A8/A9 was detected in inflamed aortic tissues from patients with TA.

Study of serial serum myeloid-related protein 8/14 as a sensitive biomarker in Takayasu arteritis: a single centre study.

The aim of the study was to explore utility of serial serum myeloid-related protein 8/14 (MRP8/14) as a biomarker of clinical disease activity and angiographic progression in Takayasu arteritis (TA). Serum MRP8/14 levels were assayed by commercial ELISA for 85 TA patients and 24 healthy controls at baseline, and for 56 and 21 TA patients during follow-up visits R1 and R2, respectively. Disease was categorised as active, indeterminate and stable according to Indian Takayasu Arteritis score (ITAS 2010), ITAS-A(CRP) and angiography. Patients were divided into responders and non-responders/relapsers based on treatment response. Non-parametric tests were used for inter-group comparisons at baseline and during follow-up time points. Generalised Estimating Equation was used to study association between changes in serial MRP8/14 levels and disease activity. At baseline, median MRP8/14 levels were higher in patients with TA than healthy controls [7353 (4524 to11283) vs 4896 (3194 to 8474.5) ng/ml, p = 0.011]. Patients with active disease had higher levels [8552 (5463-12488)] than stable disease [5292.5 (3140.5-7310)], p = 0.002, and healthy controls [4896 (3194-8474.5)], p = 0.001. Changes in serial MRP8/14 level were associated with changes in disease activity, independent of steroid dose, p = 0.000. At R1, MRP 8/14 levels were lower than baseline in responders (n = 38) [9146.0 (6296.8-13693.8) vs 6501 (4314.8-8304.5), p = 0.004], but did not change in non-responders/relapsers (n = 14) [6693.5(4210.8-10516.3) vs 7755.0(5342-10741.0), p = 0.42]. Similar trend was observed at R2. MRP8/14 levels increased during follow-up in 66% and 26.3% of angiographic progressors and non-progressors, respectively. MRP8/14 in TA may act as a novel biomarker with prognostic implications.

Anti-C1q Antibody is Associated with Renal and Cutaneous Manifestations in Asian Indian Patients with Systemic Lupus Erythematosus.

INTRODUCTION: C1q play an important role in clearance of immune complexes and apoptotic cell debris. Impaired clearance leads to exposure of C1 native antigen and development of anti-C1q antibody formation. Anti-C1q antibody is well studied in Systemic Lupus Erythematosus (SLE). Significance of anti-C1q Ab in Indian SLE patients and their clinical manifestations is not clear. AIM: The aim of this study was to investigate associations between anti-C1q antibody and clinical as well as serological markers of SLE. MATERIALS AND METHODS: Retrospective study of SLE patients fulfilling either American College of Rheumatology (ACR) 1990 or Systemic Lupus International Collaborating Clinics (SLICC) 2012 classification criteria were recruited from inpatients and outpatients services of the Clinical immunology and Rheumatology Department, Christian Medical College at Vellore, India between March 2013 and January 2015. Anti-C1q antibody was assayed by ELISA (Demeditec Diagnostics GmbH, Germany). Logistic regression analysis was performed to find the association of anti-C1q antibodies with serological and clinical parameters in SLE including Lupus Nephritis (LN). RESULTS: Sixty nine patients (54.76%) out of 126 SLE patients had LN. Anti-C1q levels were higher in patients with LN as compared to those without (p<0.05). Anti-C1q antibody was also significantly associated with positive C1q immunofluorescence staining in renal biopsy specimens (p<0.05). Overall, renal Systemic Lupus Erythematosus Disease Activity Index (SLEDAI) {OR 1.35 (1.08-1.69)}, low C4 {OR 3.11 (1.04-9.26)} and mucocutaneous manifestation {OR 4.72 (1.38-16.05)} were independently associated with anti-C1q levels in serum. CONCLUSION: Renal SLEDAI, low C4 and mucocutaneous manifestations were independently associated with raised anti C1q antibody in SLE patients.

Interleukin-17F and interleukin-6 gene polymorphisms in Asian Indian patients with Takayasu arteritis.

OBJECTIVES: To assess genetic association between single nucleotide polymorphisms (SNPs) in genes encoding T-helper cytokines and Takayasu Arteritis (TA) susceptibility in Asian Indian population. METHODS: In Phase-1, the genomic DNA of 120 TA patients and 119 healthy controls were genotyped for SNPs rs1800795 (interleukin (IL)-6), rs763780 (IL-17F), rs1800871, rs1800872, rs1800896 (IL-10) and rs1800468, rs1800469, rs1800470 (transforming growth factor-ß). Allele frequencies between cases and controls were compared using chi-squared test and also reassessed empirically (pe) by 10,000 permutations. In Phase-2, additional 98 TA patients and 101 controls were genotyped for replicating the significant associations noted in Phase-1 of the study. RESULTS: All 8 SNPs in Phase 1 were in Hardy-Weinberg proportions. The G allele at rs763780 (IL-17F) was significantly associated with TA (p=0.014). We also found that rs1800795 (IL-6) was associated with tuberculosis (p=0.001) under a dominant model. In Phase-2 replication part of the study, the rs763780 showed a trend towards association with TA (p=0.08), and the magnitude and direction of the odds ratio (OR) also were consistent with results of Phase-1. In the combined analysis, protective association of the G allele of rs763780 with TA was again significant [OR (95% CI)=0.44 (0.25-0.77); p=0.0029]. The G allele was also significantly associated (p<0.05) with underlying tuberculosis (TB) and occurrence of syncope in TA. CONCLUSION: G allele of rs763780 in IL-17F gene was protectively associated against susceptibility to TA. GG genotypes of rs1800795 in IL-6 was also associated with occurrence of tuberculosis in our patients with TA.

High salivary soluble L-selectin and interleukin-7 levels in Asian Indian patients with primary Sjögren's syndrome.

In present study, we aimed to study salivary soluble L-selectin (sL-selectin), interleukin-7(IjL-7), and lymphotoxin-α levels in primary Sjögren's syndrome (pSS) and their clinical as well as serological correlations. pSS patients fulfilling either the American European Consensus Group (AECG) and/or the American college of Rheumatology (ACR) criteria were recruited. Age- and sex-matched hospital staff were recruited as healthy controls. Unstimulated saliva was collected by the spitting method; sL-selectin, IL-7, and lymphotoxin-α were measured in the saliva using commercial ELISA kits. Forty-three patients with pSS and 31 healthy controls were included in the study. Increased levels of sL-selectin and IL-7 were found in the saliva of patients as compared to controls. Lymphotoxin-α was undetectable in the saliva of pSS patients and controls. Salivary sL-selectin positively correlated with rheumatoid factor (r = 0.47; p < 0.003). No other variable including ESSDAI was significantly associated with salivary sL-selectin and IL-7 levels. Indian patients with primary Sjögren's syndrome have higher salivary sL-selectin and IL-7 levels than healthy controls.

Alterations of mucosal microbiota in the colon of patients with inflammatory bowel disease revealed by real time polymerase chain reaction amplification of 16S ribosomal ribonucleic acid.

BACKGROUND & OBJECTIVES: Alterations in microbial communities closely associated with the intestinal mucosa are likely to be important in the pathogenesis of inflammatory bowel disease (IBD). We examined the abundance of specific microbial populations in colonic mucosa of patients with ulcerative colitis (UC), Crohn's disease (CD) and controls using reverse transcription quantitative polymerase chain reaction (RT-qPCR) amplification of 16S ribosomal ribonucleic acid (16S rRNA). METHODS: RNA was extracted from colonic mucosal biopsies of patients with UC (32), CD (28) and patients undergoing screening colonoscopy (controls), and subjected to RT-qPCR using primers targeted at 16S rRNA sequences specific to selected microbial populations. RESULTS: Bacteroides-Prevotella-Porphyromonas group and Enterobacteriaceae were the most abundant mucosal microbiota. Bacteroides and Lactobacillus abundance was greater in UC patients compared with controls or CD. Escherichia coli abundance was increased in UC compared with controls. Clostridium coccoides group and C. leptum group abundances were reduced in CD compared with controls. Microbial population did not differ between diseased and adjacent normal mucosa, or between untreated patients and those already on medical treatment. The Firmicutes to Bacteroidetes ratio was significantly decreased in both UC and CD compared with controls, indicative of a dysbiosis in both conditions. INTERPRETATION & CONCLUSIONS: Dysbiosis appears to be a primary feature in both CD and UC. Microbiome-directed interventions are likely to be appropriate in therapy of IBD.

Association between heat shock protein 70 gene polymorphisms and clinical outcomes in intensive care unit patients with sepsis.

OBJECTIVE: The objective of the following study is to evaluate the associations between single nucleotide polymorphisms (SNPs) in the Heat Shock Protein 70 (HSP70) gene, gene expression of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α) and medical intensive care unit (MICU) stay and organ failure in sepsis. MATERIALS AND METHODS: MICU patients with sepsis were genotyped for rs1061581, rs2227956, rs1008438 and rs1043618 polymorphisms in HSP70 gene using polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis or allele-specific PCR. Messenger ribonucleic acid (mRNA) expression of IL-6 and TNF-α were quantitated in peripheral blood lymphocytes. Outcomes were recorded. RESULTS: 108 patients (48 male) aged 40.7 ± 16.0 (mean ± standard deviation) years included H1N1 infection (36), scrub typhus (29) and urosepsis (12). Seventy-one (65.7%) had dysfunction of three or more organ systems, 66 patients (61.1%) were treated by mechanical ventilation, 21 (19.4%) needed dialysis. ICU stay was 9.3 ± 7.3 days. Mortality was 38.9%. One or more SNPs were noted in 101/108 (93.5%) and organ failure was noted in only 1/7 patients without a single SNP. The A allelotypes of rs1061581 and rs1008438 were associated with hematological dysfunction (P = 0.03 and 0.07) and longer ICU stay (P = 0.05 and 0.04), whereas IL-6 and TNF-α mRNA levels were associated with central nervous system dysfunction. CONCLUSIONS: HSP70 genotypes may determine some adverse outcomes in patients with sepsis.

Clostridium leptum group bacteria abundance and diversity in the fecal microbiota of patients with inflammatory bowel disease: a case-control study in India.

BACKGROUND: Alterations in the fecal bacterial flora occur in inflammatory bowel disease (IBD). We examined the abundance and diversity of Clostridium leptum group, an important group of carbohydrate-fermenting bacteria, in the feces of patients with IBD and compared them with healthy controls. METHODS: Seventeen healthy controls (HC), 20 patients with Crohn's disease (CD) and 22 patients with ulcerative colitis (UC) participated in the study. DNA extracted from fecal samples was amplified by PCR targeting 16S rRNA gene sequences specific to C. leptum group. The PCR product was subjected to temporal temperature gradient electrophoresis (TTGE) and the number and position of individual bands were noted and diversity was estimated. The identity of bands at different positions was confirmed by cloning and sequencing. Real time quantitative PCR with Mesa Green, targeted at specific 16S rRNA gene sequences, was used to quantitate C. leptum group and its most prominent constituent, Faecalibacterium prausnitzii. RESULTS: Twenty five different operational taxonomic units (OTUs, equivalent to species) were identified constituting the C. leptum group in these participants. Their sequences were deposited in GenBank [accession numbers GQ465348 to GQ465370]. OTU number was significantly reduced in CD (7.7 ± 3.7, mean ± SD) and UC (9.0 ± 3.0) compared to HC (11.9 ± 2.2) (P=0.0005). The Simpson D index of alpha diversity was not significantly different between the three groups. Total numbers of C. leptum group bacteria and F. prausnitzii were reduced in both CD and UC compared to HC (P=0.0036 and P<0.0001 respectively). Disease activity did not influence numbers of C. leptum or F. prausnitzii in patients with CD or UC. CONCLUSION: C. leptum numbers and diversity were significantly reduced in both CD and UC suggesting that alterations noted were not specific to one disease. This could contribute to reduced short chain fatty acid production in IBD.