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.

Expanding the clinical and immunological phenotype of prolidase deficiency: A case report.

Prolidase deficiency (PD) is a rare autosomal recessive disorder associated with recurrent infections, immune dysregulation, and autoimmunity. PD is characterized by persistent dermatitis, skin fragility, and non-healing ulcerations on the lower limbs as its main dermatologic characteristics. Herein, we report a boy with PD due to a novel variant in PEPD who had abnormal facies, cognitive impairment, corneal opacity, recurrent infections, and persistent non-healing leg ulcers. Th17 lymphocyte counts and phosphorylated-STAT5 expression following IL-2 stimulation were reduced in our patient as compared to healthy control.

High expression of pro-inflammatory cytokine genes IL-1ß and IL-1R2 upon TLR4 activation in Takayasu arteritis.

Toll-like receptors (TLR) 4 and its endogenous ligands are highly expressed in aorta. In the present study, we have explored the effect of TLR-4 activation by pro-inflammatory and angiogenic factors in PBMCs of patients with Takayasu Arteritis (TA). In the screening cohort, PBMCs of TA (n = 6) and healthy controls (n = 6) were stimulated with LPS and cultured. mRNA expression of 84 genes were quantitated by RT2 Profiler™ PCR Array kit in PBMCs. Validation set of additional PBMCs from TA (n = 7) and healthy controls [HC) (n = 7) were then stimulated with LPS to study expression of selected genes with delta Ct > 0.1 in the screening cohort. Significant gene expressions were correlated with Indian Takayasu arteritis activity scores (ITAS 2010). Increased expression of CCL2 was observed only in unstimulated PBMCs of patients with TA [median relative difference (RD) of 2.37] as compared to HC (RD 1.37, p < 0.03) in validation cohort, while stimulation with TLR4 ligand led to increased mRNA expression of IL-1ß (RD 7.9, p < 0.028) and IL-1R2 (RD 0.08 p < 0.013) genes as compared to that of HC [RD of 5.32 for IL-1ß and 0.01 for IL-1R2, respectively] in validation cohort. TLR4 activation also led to significantly higher expression of HPSE, TIMP1 and low expression of VEGFB, S1PR1, SERPINF1, ANGPLT4, ANGPT2, TIE1 and NOS3 genes in the screening cohort. But expression of VEGFB was not significant in validation cohort. The significant gene expressions, however, did not correlate with ITAS [ITAS2010 and ITAS-A (CRP)]. TLR4 activation leads to increased expression of IL-1ß and IL-1R2 genes in PBMCs of patients with TA.

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.

Severe COVID-19, multisystem inflammatory syndrome in children, and Kawasaki disease: immunological mechanisms, clinical manifestations and management.

Multisystem inflammatory syndrome (MIS-C) is a pediatric hyperinflammation disorder caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). It has now been reported from several countries the world over. Some of the clinical manifestations of MIS-C mimic Kawasaki disease (KD) shock syndrome. MIS-C develops 4-6 weeks following SARS-CoV-2 infection, and is presumably initiated by adaptive immune response. Though it has multisystem involvement, it is the cardiovascular manifestations that are most prominent. High titres of anti-SARS-CoV-2 antibodies are seen in these patients. As this is a new disease entity, its immunopathogenesis is not fully elucidated. Whether it has some overlap with KD is still unclear. Current treatment guidelines recommend use of intravenous immunoglobulin and high-dose corticosteroids as first-line treatment. Mortality rates of MIS-C are lower compared to adult forms of severe COVID-19 disease.

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.

CXCL13 levels in serum but not in saliva are elevated in Asian Indian patients with primary Sjögren's syndrome.

Human and animal model studies suggest CXCL13 is a potential biomarker in primary Sjögren's syndrome (pSS). CXCL13 has not been studied in Indian patients with pSS. pSS cases classified by American European Consensus Group (AECG) or American college of Rheumatology(ACR) 2012 criteria, attending rheumatology clinic between July 2014 and July 2015 were included. Hospital staff and healthy, non-blood related family members of patients constituted the control group. pSS cases underwent clinical evaluation, laboratory investigations, ESSDAI and ESSPRI scoring. Unstimulated saliva was collected by the spitting method. Salivary and serum CXCL13 were quantified by indirect ELISA. CXCL13 positivity was determined using Receiver Operator Characteristic (ROC) curve. STATA13.1 (StataCorpLP,Texas,USA) software was used for statistical analysis. In this study, 45 pSS cases and 42 healthy controls were recruited. In pSS, median levels of serum CXCL13, but not salivary CXCL13 was significantly higher as compared to the corresponding levels in healthy controls (p < 0.001). Using cutoff of 43.03 pg/ml obtained by ROC, serum CXCL13 positivity was seen in 31/43(72.1%) cases and 10/34 (29.4%) controls, respectively. Serum CXCL13 levels among pSS patients on treatment, treatment naïve patients and healthy controls were statistically different. Serum CXCL13 positivity was associated with oral symptoms (p = 0.02), ocular signs (p = 0.03) and hyperglobulinemia (p = 0.01). There was no association of salivary CXCL13 level with any of the clinical variables. While serum CXCL13 was elevated in pSS, salivary CXCL13 was not. In conclusion, serum CXCL13 positivity was found to be associated with oral symptoms, ocular signs and hyperglobulinemia in pSS.

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.

Addition of second-line steroid sparing immunosuppressants like mycophenolate mofetil improves outcome of Immunoglobulin G4-related disease (IgG4-RD): a series from a tertiary care teaching hospital in South India.

IgG4-related disease (IgG4-RD) is a systemic fibro-inflammatory disease. This disease may be associated with elevated serum and tissue IgG4 levels. Early treatment prevents fibrosis and organ damage. We retrospectively studied the clinicopathologic correlation and outcome of treatment in IgG4-RD. This single-center retrospective study was done using electronic records of patients subjected to assay of serum IgG4 levels in our laboratory by nephelometry. There were 473 patients with suspected IgG4-RD. Of them, 41 patients fulfilled comprehensive diagnostic criteria for IgG4-RD and 432 had diseases other than IgG4-RD. Clinical and histopathological data including tissue IgG4/IgG ratio, other relevant laboratory findings as well as management data of 41 patients with IgG4-RD were analyzed. There were 29 males and 12 females with mean age of 44.1 ± 2.19 years. Thirteen patients had definite, 19 had probable and 9 had possible IgG4-RD. Male predominance, multiple organ involvement and IgG4 responder Index were significantly higher in definite IgG4-RD as compared to probable and possible IgG4-RD. Serum IgG4 level was elevated in 37 patients (90.2%). Glucocorticoids were used in 35 patients (85.4%) and second-line immunosuppressive agent in 23 patients (65.7%). Of the 21 patients on follow-up, 19 (90.7%) had clinical improvement at the first follow-up visit. Nine (90%) out of the ten patients who were assessed by IgG4 responder index, also had shown improved score with treatment. Patients with IgG4-RD in our series showed favorable responses to treatment with glucocorticoids and addition of steroid sparing immunosuppressive agents (mainly mycophenolate mofetil) helped successful tapering of steroids, while maintaining the improvement.

Impaired toll like receptor 9 response in pulmonary tuberculosis.

BACKGROUND & AIM: Innate immune responses are important in susceptibility to pulmonary tuberculosis (TB). In order to test the hypothesis that Toll-like receptor (TLR) 2 function would be abnormal in patients with active pulmonary TB we compared the cytokine responses of peripheral blood mononuclear cells (PBMC) to innate immune ligands in a case-control study. METHODS: PBMC from 19 untreated pulmonary TB patients, 17 healthy controls, and 11 treated pulmonary TB patients, were cultured for 24h with TLR 2 ligand (PAM-CSK) and other TLR ligands (muramyl dipeptide, flagellin, lipopolysaccharide (LPS), CpG oligodeoxynucleotide (CpG-ODN)). Interleukin-8 (IL-8) was estimated in the supernatant by ELISA. Messenger RNA expression for inflammatory cytokines was quantitated using real time PCR. RESULTS: The important findings were (1) reduced PBMC secretion of IL-8 in response to all ligands in active TB; (2) normal to increased PBMC secretion of IL-8 in response to all ligands except CpG ODN (TLR 9 ligand) in TB patients who had recovered; (3) absence of difference in mRNA expression for a consortium of inflammatory pathway genes between healthy controls, active pulmonary tuberculosis and treated pulmonary tuberculosis patients. CONCLUSION: There was a generalized post-translational suppression of the IL-8 response to innate immune ligands in active TB. There appears to be a defect of TLR 9 signaling in patients with tuberculosis, the nature of which needs to be further explored.

Gut inflammation and microbiome in spondyloarthritis.

Spondyloarthritis (SpA) is chronic inflammatory disease involving joints and the spine. Bowel inflammation is common in SpA, which may be classified as acute or chronic. Chronic gut inflammation is most common in SpA patients with axial involvement as compared to those presenting with peripheral involvement alone. The pathogenesis of gut inflammation in SpA could be explained by two factors-over-activation of immunological cells and altered gut microbiome. This is exemplified by SpA animal models, namely HLA-B27-expressing transgenic animals and SKG mice models. Immunological mechanisms include homing of activated T cells from gut into synovium, excess pro-inflammatory cytokines secretion by immune cells such as IL-23 and genetic variations in immunological genes. The evidence for role of gut microbiome in SpA is gradually emerging. Recently, metagenomic study of gut microbiome by sequencing of microbial nucleic acids has enabled identification of new microbial taxa and their functions in gut of patients with SpA. In SpA, the gut microbiome could emerge as diagnostic and prognostic marker of disease. Modulation of gut microbiome is slated to have therapeutic potential as well.

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.

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.

Gut microbiota of preterm infants in the neonatal intensive care unit: a study from a tertiary care center in northern India.

Introduction: Disruptions of the gut microbiota of preterm infants admitted to the neonatal intensive care unit (NICU) during the first 2 weeks of life are of critical importance. These infants are prone to various complications, including necrotizing enterocolitis (NEC) and sepsis. Studying the gut microbiota will improve outcomes in preterm infants. In the present study, we examined the gut microbiota of preterm infants admitted to the NICU in the first month of life. Methods: Neonates admitted to the NICU were recruited, and stool samples were collected weekly from the seventh day of the infant's life until the 30th day of life. DNA was extracted using a DNeasy Powersoil DNA isolation kit. 16S rRNA gene sequencing targeting the V3-V4 region was performed using the MiSeq platform. Sequenced reads were processed on DADA2 pipeline to obtain an amplicon sequence variant (ASV) table. All bioinformatic and statistical analyses were performed using different packages in the R statistical framework. Results: Fourteen preterm infants were recruited, and 48 samples were collected. Alpha diversity metrics, observed ASV count, and Shannon index were found to have no differences in any clinical variables. Permutational multivariate analysis of variance (PERMANOVA) showed discrimination of neonates by gestational age and administration of probiotics. Differential abundance analysis showed a decreased abundance of Bifidobacterium Breve in extremely preterm infants (gestational age <28 weeks) compared to moderate preterm infants (gestational age 29-32 weeks). Supplementation with probiotics decreased Acinetobacter and increased Bifidobacterium in the gut of preterm neonates regardless of gestational age. Conclusion: Gestational age and probiotic supplementation alter the gut microbiota of preterm infants admitted to the NICU.

A pilot study of childhood-onset Takayasu arteritis using whole exome sequencing suggests oligogenic inheritance involving classical complement, collagen, and autoinflammatory pathways.

Takayasu arteritis (TA) is a chronic granulomatous inflammatory disease affecting the aorta and its branches. Paediatric TA (pTA) may present from 6 months after birth till the adolescent age group. Genetics and pathogenesis of pTA are not fully understood. Earlier studies reported monogenic mutation in NOD2, XIAP, and STAT1 genes in patients with pTA. TA, a relatively rare disease, is more common in geographical pockets, including India. We hypothesized that South Asian patients with pTA, namely, those of Indian subcontinent origin, may have clinically relevant and unique pathogenic variants involving one or more genes, especially those linked to genetically driven vasculitic illnesses, including autoinflammatory pathologies. Children with pTA fulfilling EULAR/PRINTO/PReS classification criteria and presenting with clinical symptoms to the Paediatric Rheumatology clinic of Christian Medical College, Vellore, were included. Blood samples were collected after getting informed consent from parents or guardians and assent forms from children. DNA was extracted from whole blood using the Qiagen DNA extraction kit. Initially, the common variant in Indian population, namely, ADA2 c.139G > A; p.Gly47Arg, was screened, followed by whole exome sequencing. Fourteen children were recruited for the study. Median age of patients was 11 years (4 months-14 years) with a male-to-female ratio of 4:10. Distribution of angiographic subsets by Numano's classification of included children were as follows: type 5 (n = 7), type 4 (n = 5), and type 3 (n = 2). We identified novel variants in ten different genes. This include variants in genes of classical complement pathway, namely, C2, C3, C6, C7, and C9, and other genes, namely, CYBA, SH3BP2, GUCY2C, CTC1, COL5A1, and NLPR3. Two of 14 patients have heterozygous pathogenic variants; this implies that combination of heterozygous variants in C3 and COL5A1 might lead to disease development, suggesting digenic inheritance. One patient has a homozygous variant in CYBA. None of the patients were identified to have ADA2 variants. Whole exome sequencing reveals combination of rare variants in genes C3, COL5A1, and CYBA associated with disease development in children with Takayasu Arteritis. Key Points • We identified novel variants in genes of classical complement pathway, namely, C2, C3, C6, C7, and C9, and other genes, namely, CYBA, SH3BP2, GUCY2C, CTC1, COL5A1, and NLPR3. • Two of 14 patients have heterozygous pathogenic variants in C3 and COL5A1; this may have implications in disease development, suggesting digenic inheritance. • One patient has homozygous variant in CYBA. • None of the patients were identified to have ADA2 variants.

Study of pathogenic T-helper cell subsets in Asian Indian patients with Takayasu arteritis.

The relapses and refractory disease are a challenge in the management of patients with Takayasu arteritis (TAK). We quantified pathogenic CD4 + memory T helper cells bearing surface markers CD161 and/or p-glycoprotein (MDR1) in patients with TAK. Peripheral blood mononuclear cells of 21 patients with TAK and 16 age-matched controls were stained with anti-CD3, anti-CD4, anti-CD45RA, anti-CD161 and anti-p-glycoprotein antibodies and subjected to flow cytometry by FACS ARIAIII. Eighteen patients underwent follow-up immunophenotyping. Intracellular staining for interleukin-17 and interferon-γ was performed for 18 patients and 11 controls. Surgical arterial biopsies of 6 TAK and 5 non-inflammatory controls were subjected to immunohistochemistry with anti-CD161 and anti-p-glycoprotein. At baseline the frequency of MDR1 + CD4 + and CD161 + MDR1 + CD4 + memory T cells was higher in TAK than controls (p = 0.002 and 0.01, respectively). After stimulation, the frequency of IFN-y + CD161 + cells was higher in TAK than controls (p = 0.028). Modal fluorescence intensity of CD161 + MDR1 + CD45RA - CD4 + cells was higher in active as compared with stable disease (p = 0.041). At 6 months, MDR1 + and CD161 + MDR1 + memory CD4 + T cells decreased significantly only in patients who had complete/partial response to treatment (p = 0.047 and 0.02, respectively). To conclude, MDR1 + and MDR1 + CD161 + CD4 + memory T-helper cells are increased in patients with TAK. These cells decreased only in patients with response to treatment during subsequent follow-up.

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.

Intravenous Immunoglobulin in Kawasaki Disease-Evolution and Pathogenic Mechanisms.

Kawasaki disease (KD) is an acute vasculitis of childhood that affects the medium vessels with a special predilection to the involvement of coronary arteries. The major morbidity of this disease is due to coronary artery aneurysm, which occurs in about 25-30% of untreated cases. For decades now, intravenous immunoglobulin (IVIg) has consistently been shown to reduce the risk of CAAs to less than 5%. However, the mechanism of immunomodulation remains unclear. Several studies on the role of IVIg in the modulation of toll-like receptor pathways, autophagy, and apoptosis of the mononuclear phagocytic system, neutrophil extracellular trap, and dendritic cell modulation suggest a modulatory effect on the innate immune system. Similarly, certain studies have shown its effect on T-cell differentiation, cytokine release, and regulatory T-cell function. In this review, we discuss the potential mechanisms underlying the immunomodulatory actions of IVIg in patients with Kawasaki disease. Furthermore, we provide a summary of the evidence regarding various infusion protocols and dosages utilized in the treatment of KD patients.