Influence of Insulin Receptor Single Nucleotide Polymorphisms on Glycaemic Control and Formation of Anti-Insulin Antibodies in Diabetes Mellitus

Single nucleotide polymorphisms (SNPs) in insulin and insulin receptor genes may influence the interaction between the two molecules, as may anti-insulin antibodies (IAs), commonly found in patients with type 1 diabetes mellitus (T1D) or type 2 diabetes mellitus (T2D) treated with exogenous insulin. We examined the impact of two SNPs in the human insulin gene (INS), rs3842752 and rs689, and two in the insulin receptor gene (INSR) rs2245649 and rs2229429, on disease susceptibility, glycaemic control, and IAs formation in 100 T1D patients and 101 T2D patients treated with insulin. 79 individuals without diabetes were typed as healthy controls. The minor alleles of rs3842752 and rs689 in INS protected against T1D (OR: 0.50, p = 0.01 and OR: 0.44; p = 0.002, respectively). The minor alleles of both rs2245649 and rs2229429 in INSR were risk factors for poor glycaemic control (HbA1c ≥ 80 mmol/mol) in T1D (OR: 5.35, p = 0.009 and OR: 3.10, p = 0.01, respectively). Surprisingly, the minor alleles of rs2245649 and rs2229429 in INSR associated strongly with the absence of IAs in T1D (OR = 0.28, p = 0.008 and OR = 0.30, p = 0.002, respectively). In conclusion, the minor alleles of the investigated INS SNPs protect against T1D, and the minor alleles of the investigated INSR SNPs are associated with poor glycaemic control and the absence of IAs in T1D

Characterization of circulating extracellular traps and immune responses to citrullinated LL37 in psoriasis

Background: The DNA-binding peptide LL37 is a suspected autoantigen in psoriasis. It can be found in neutrophil extracellular traps (NETs) which have been suggested to play a role in the pathogenesis of the disease. Citrullination, the conversion of peptidyl-arginine into peptidyl-citrulline, can be implicated in the formation of NETs. We hypothesized that citrullination increases LL37 immunogenicity and that NETs are a source of LL37.Objectives: We aimed to characterize cytokine responses of B cells and T cells to native and citrullinated LL37 (citLL37) and determine the prevalence and composition of circulating NETs in patients with psoriasis and healthy blood donors (HDs).Methods: Mononuclear cells (MNCs) and serum were isolated from 20 HDs and 20 patients with psoriasis. The MNCs were stimulated with native LL37 and citLL37 and the proportion of cytokine-positive B cells and T cells was determined by flow cytometry. Circulating antibodies against native LL37 and citLL37 as well as circulating NETs were measured by ELISA, as was the content of LL37, citLL37, and IgG in the NETs.Results: CitLL37, but not native LL37, induced IFN-γ-production by T cells and B cells from psoriasis patients, as well as IL-10-production by the patients' CD4+ T cells. Serum from 40% of patients and 55% of HDs contained circulating NETs, of which 63% and 27%, respectively, contained LL37. Only two patients had NETs containing citLL37 and IgG antibodies were found in NETs from three patients and one HD. Post-hoc analysis of the cytokines produced by B cells and T cells after stimulation with citLL37 revealed two clusters of patients consisting of 10 high-responders and 9 low-responders. The high-responders were those that had circulating NETs in combination with an earlier age of onset of the disease.Conclusion: Citrullinated but not native LL37 elicits IFN-γ-responses by T cells and B cells from psoriasis patients, particularly those with circulating NETs and early disease onset, suggesting a role of citLL37 as an autoantigen in this subgroup of patients

Applicability of Small-Molecule Inhibitors in the Study of Peptidyl Arginine Deiminase 2 (PAD2) and PAD4

Citrullination, the conversion of peptidyl-arginine into peptidyl-citrulline, is involved in the breakage of self-tolerance in anti-CCP-positive rheumatoid arthritis. This reaction is catalyzed by peptidyl arginine deiminases (PADs), of which PAD2 and PAD4 are thought to play key pathogenic roles. Small-molecule PAD inhibitors such as the pan-PAD inhibitor BB-Cl-amidine, the PAD2-specific inhibitor AFM-30a, and the PAD4-specific inhibitor GSK199 hold therapeutic potential and are useful tools in studies of citrullination. Using an ELISA based on the citrullination of fibrinogen, we found that AFM-30a inhibited the catalytic activity of PADs derived from live PMNs or lysed PBMCs and PMNs and of PADs in cell-free synovial fluid samples from RA patients, while GSK199 had minor effects. In combination, AFM-30a and GSK199 inhibited total intracellular citrullination and citrullination of histone H3 in PBMCs, as determined by Western blotting. They were essentially nontoxic to CD4(+) T cells, CD8(+) T cells, B cells, NK cells, and monocytes at concentrations ranging from 1 to 20 muM, while BB-Cl-amidine was cytotoxic at concentrations above 1 muM, as assessed by flow cytometric viability staining and by measurement of lactate dehydrogenase released from dying cells. In conclusion, AFM-30a is an efficient inhibitor of PAD2 derived from PBMCs, PMNs, or synovial fluid. AFM-30a and GSK199 can be used in combination for inhibition of PAD activity associated with PBMCs but without the cytotoxic effect of BB-Cl-amidine. This suggests that AFM-30a and GSK199 may have fewer off-target effects than BB-Cl-amidine and therefore hold greater therapeutic potential

Peptidylarginine Deiminase 2 Gene Polymorphisms in Subjects with Periodontitis Predispose to Rheumatoid Arthritis

Epidemiologic studies have shown associations between periodontitis and rheumatoid arthritis (RA), but a causal relationship has not been established. Citrullination of gingival proteins by human peptidylarginine deiminases (PADs) or PAD from Porphyromonas gingivalis has been proposed to generate autoantigens in anti-CCP-positive RA. This study investigated whether the association between periodontitis and RA is influenced by single nucleotide polymorphisms (SNPs) in the genes encoding PAD2 and PAD4 that catalyze aberrant citrullination in RA and often are overexpressed in inflamed gingival connective tissue in subjects with periodontitis. The study included 137 RA patients and 161 controls with self-reported periodontitis. Periodontitis onset preceded RA onset by 13 years on average and was not associated with any of the SNPs investigated. In subjects with periodontitis, carriage of the minor alleles of rs2057094 and rs2235912 in PADI2 significantly increased the risk of RA (odds ratios 1.42 [p = 0.03] and 1.48 [p = 0.02], respectively), and this effect was driven by the anti-CCP-negative RA patients. The minor alleles of these SNPs only increased risk of anti-CCP-positive RA in individuals with periodontitis and a history of smoking. These data suggest that individuals with periodontitis carrying the minor alleles of SNPs rs2057094, rs2076616 and rs2235912 in PADI2 may be at increased risk of RA

Probiotics Partly Suppress the Impact of Sugar Stress on the Oral Microbiota—A Randomized, Double-Blinded, Placebo-Controlled Trial

The aim was to test if probiotics counteract oral dysbiosis during 14 days of sugar stress and subsequently help restore oral homeostasis. Eighty healthy individuals received either probiotics (n = 40) or placebo lozenges (n = 40) for 28 days and rinsed with a 10% sucrose solution 6–8 times during the initial 14 days of the trial. Saliva and supragingival samples were collected at baseline, day 14, and day 28. Saliva samples were analyzed for levels of pro-inflammatory cytokines, albumin, and salivary enzyme activity. The supragingival microbiota was characterized according to the Human Oral Microbiome Database. After 14 days of sugar stress, the relative abundance of Porphyromonas species was significantly higher (p = 0.03) and remained significantly elevated at day 28 in the probiotic group compared to the placebo group (p = 0.004). At day 28, the relative abundance of Kingella species was significantly higher in the probiotic group (p = 0.03). Streptococcus gordinii and Neisseria elongata were associated with the probiotic group on day 28, while Streptococcus sobrinus was associated with the placebo group on day 14 and day 28. On day 28, the salivary albumin level was significantly lower in the probiotic group. The present study demonstrates a potential stabilizing effect on the supragingival microbiota mediated by consumption of probiotics during short-term sugar stress