Peptidylarginine deiminase 4 (PAD4) activity in early rheumatoid arthritis.

Objectives: Peptidylarginine deiminases (PADs) are a family of calcium-dependent enzymes catalysing the conversion of arginine residues to citrulline, which may constitute a risk factor in rheumatoid arthritis (RA) pathogenesis. We investigated PAD activation by serum (PADAct) in early RA, and the associations between PAD activation and disease characteristics, treatment response, and progression of radiographic damage.Method: Sera from disease-modifying anti-rheumatic drug (DMARD)-naïve early RA patients (n = 225), classified according to the 2010 American College of Rheumatology/European League Against Rheumatism criteria, and healthy controls (n = 63) were analysed for PAD4 activating capacity at 0, 3, 12, and 24 months using a high-performance liquid chromatography fluorometric method. Associations for PADAct were evaluated by Mann-Whitney U and chi-squared tests. Changes in PADAct levels were compared using the Wilcoxon signed-rank test.Results: PADAct positivity occurred in 42% (n = 95) of the patients and was more prevalent in anti-citrullinated protein antibody (ACPA)-positive vs ACPA-negative patients (47% vs 20%, p = 0.002), but not in rheumatoid factor (RF)-positive vs RF-negative patients (44% vs 38%, p = 0.49). PADAct-positive were younger than PADAct-negative patients [mean ± sd 48.7 ± 13.5 vs 53.2 ± 13.7 years, p = 0.011]. Median [25th, 75th percentile] PADAct levels were higher in patients than in controls (8768 [7491, 11 393] vs 7046 [6347, 7906], p < 0.0001) and decreased after initiation of DMARD treatment, but were not associated with treatment response or progression of radiographic damage after 2 years of follow-up.Conclusion: Serum capacity to activate PAD4 was associated with ACPA and RF positivity and earlier disease onset in early RA patients, and decreased after initiation of DMARD treatment, indicating that anti-PAD treatment could potentially be beneficial in RA.

Salivary, gingival crevicular fluid and serum levels of ghrelin and chemerin in patients with periodontitis and overweight.

BACKGROUND AND OBJECTIVE: Nutrition and body weight are modifying factors for periodontitis. The purpose of this study was to quantify two molecules (ghrelin and chemerin), released in association with food intake and obesity, in periodontally healthy and diseased individuals with respect to different body mass categories. MATERIAL AND METHODS: The two main groups (patients with chronic periodontitis and periodontally healthy/gingivitis volunteers) were subdivided into groups of subjects with normal weight [body mass index (BMI) <25] and groups of overweight/obese subjects (BMI ≥25). Subgingival bacteria were analysed and the levels of acylated and total ghrelin, chemerin and interleukin-1ß (IL-1ß) were assessed in saliva, gingival crevicular fluid and serum. RESULTS: The amount of Treponema denticola present subgingivally was significantly higher in the groups of patients with chronic periodontitis as well as in periodontally healthy/gingivitis individuals with BMI ≥25 than in periodontally healthy/gingivitis individuals with BMI <25. The amount of total ghrelin in gingival crevicular fluid differed significantly between the groups, with the lowest levels found in the group of patients with chronic periodontitis and BMI ≥25. The levels of chemerin in gingival crevicular fluid were significantly higher in each chronic periodontitis group than in periodontally healthy/gingivitis individuals with BMI <25. However, the level of IL-1ß in the gingival crevicular fluid was most differentiating between the groups, with the highest levels found in the group of patients with chronic periodontitis and BMI <25 and the lowest levels in periodontally healthy/gingivitis individuals with BMI <25. No significant differences between any groups were seen for chemerin or for acylated ghrelin in the stimulated whole saliva, or for acylated and total ghrelin in peripheral blood serum. The BMI correlated with the serum level of chemerin. CONCLUSION: Low ghrelin and high chemerin levels in the gingival crevicular fluid might be linked to periodontal disease and overweight/obesity. However, unlike IL-1ß, the levels of chemerin and ghrelin in gingival crevicular fluid are not reliable indicators of periodontal destruction.

Benzamidine derivatives inhibit the virulence of Porphyromonas gingivalis.

We have previously shown that benzamidine-type compounds can inhibit the activity of arginine-specific cysteine proteinases (gingipains HRgpA and RgpB); well-known virulence factors of Porphyromonas gingivalis. They also hinder in vitro growth of this important periodontopathogenic bacterium. Apparently growth arrest is not associated with their ability to inhibit these proteases, because pentamidine, which is a 20-fold less efficient inhibitor of gingipain than 2,6-bis-(4-amidinobenzyl)-cyclohexanone (ACH), blocked P. gingivalis growth far more effectively. To identify targets for benzamidine-derived compounds other than Arg-gingipains, and to explain their bacteriostatic effects, P. gingivalis ATCC 33277 and P. gingivalis M5-1-2 (clinical isolate) cell extracts were subjected to affinity chromatography using a benzamidine-Sepharose column to identify proteins interacting with benzamidine. In addition to HRgpA and RgpB the analysis revealed heat-shock protein GroEL as another ligand for benzamidine. To better understand the effect of benzamidine-derived compounds on P. gingivalis, bacteria were exposed to benzamidine, pentamidine, ACH and heat, and the expression of gingipains and GroEL was determined. Exposure to heat and benzamidine-derived compounds caused significant increases in GroEL, at both the mRNA and protein levels. Interestingly, despite the fact that gingipains were shown to be the main virulence factors in a fertilized egg model of infection, mortality rates were strongly reduced, not only by ACH, but also by pentamidine, a relatively weak gingipain inhibitor. This effect may depend not only on gingipain inhibition but also on interaction of benzamidine derivatives with GroEL. Therefore these compounds may find use in supportive periodontitis treatment.

Periodontal pathogens affect the level of protease inhibitors in gingival crevicular fluid.

In periodontitis, an effective host-response is primarily related to neutrophils loaded with serine proteases, including elastase (NE) and protease 3 (PR3), the extracellular activity of which is tightly controlled by endogenous inhibitors. In vitro these inhibitors are degraded by gingipains, cysteine proteases produced by Porphyromonas gingivalis. The purpose of this study was to determine the level of selected protease inhibitors in gingival crevicular fluid (GCF) in relation to periodontal infection. The GCF collected from 31 subjects (nine healthy controls, seven with gingivitis, five with aggressive periodontitis and 10 with chronic periodontitis) was analyzed for the levels of elafin and secretory leukocyte protease inhibitor (SLPI), two main tissue-derived inhibitors of neutrophil serine proteases. In parallel, activity of NE, PR3 and arginine-specific gingipains (Rgps) in GCF was measured. Finally loads of P. gingivalis, Aggregatibacter actinomycetemcomitans, Tannerella forsythia and Treponema denticola were determined. The highest values of elafin were found in aggressive periodontitis and the lowest in controls. The quantity of elafin correlated positively with the load of P. gingivalis, Ta. forsythia and Tr. denticola, as well as with Rgps activity. In addition, NE activity was positively associated with the counts of those bacterial species, but not with the amount of elafin. In contrast, the highest concentrations of SLPI were found in periodontally healthy subjects whereas amounts of this inhibitor were significantly decreased in patients infected with P. gingivalis. Periodontopathogenic bacteria stimulate the release of NE and PR3, which activities escape the control through degradation of locally produced inhibitors (SLPI and elafin) by host-derived and bacteria-derived proteases.