Background To investigate the association of active matrix-metalloproteinase-8 (aMMP-8) levels in total oral fluid (TOF), gingival crevicular fluid (GCF) and periodontal disease progression in the last 10 years in a cohort of patients with stage III/IV periodontitis. Methods 47 patients receiving steps 1 and 2 of periodontal therapy with an observation period of over 10 years were included. Levels of current aMMP-8 in TOF, GCF and serum were determined at a single point at the ten-year follow-up. Past periodontal disease progression was characterized by clinical attachment loss over 10 years. For statistical analysis, linear regression models along with binary logistic regression models to analyze the progression rate based on age, gender, smoking, diabetes, full mouth bleeding score (FMBS), and T2 aMMP-8 levels were performed. Results At T2, the median patient age was 73 years [67; 79], with a mean attachment loss of 1.5 mm [1.0; 2.1] over the observation period. aMMP-8 levels did not differ between patients with mild and moderate disease progression. In multivariate linear regression, aMMP-8 levels were not associated with mean attachment loss. In contrast, sex, non-smoking status, and FMBS showed significant associations with past progression. Classification models for GCF and serum aMMP-8 expression yielded the highest accuracy (ROC = 0.80) for the binary assessment into mild and moderate progression, although this did not differ significantly from the model based on covariates alone (ROC = 0.75). Conclusion Current aMMP-8 levels determined at a single point were not associated with past periodontal disease progression during a 10-year period. Clinical factors such as sex, non-smoking status, and FMBS showed a stronger association with past progression. Clinical Significance Biomarkers might improve the precision of periodontal diagnosis and monitoring; however, evidence is limited regarding their association with disease progression. Active-MMP-8 has gained increasing interest in recent years.

Intestinal epithelial overexpression of the Th17 cell chemoattractant CCL20 is implicated in inflammatory bowel disease and influenced by NOD2 mutations in Crohn’s disease. Vitamin D metabolites have been shown to ameliorate inflammatory bowel disease. Considering NOD2 mutations in Crohn’s disease, we investigated whether Vitamin D deficiency (serum 25-hydroxyvitamin D concentration < 20 ng/mL) increases circulating CCL20 levels in inflammatory bowel disease patients and healthy controls and whether active 1,25-dihydroxyvitamin D (calcitriol) downregulates systemic and intestinal CCL20 expression. In a cross-sectional study, serum concentrations of CCL20, 25-hydroxyvitamin D, and calcitriol were measured in 170 NOD2 -genotyped Crohn’s disease patients, 80 ulcerative colitis patients, and 60 healthy controls. Additionally, the effect of calcitriol on experimentally induced CCL20 expression was examined using human intestinal epithelial HT-29 cells. Multivariable linear regression analyses revealed that both the diagnosis of inflammatory bowel disease and vitamin D deficiency were independently associated with elevated CCL20 levels. Compared to healthy controls, Crohn’s disease patients and ulcerative colitis patients exhibited significantly higher circulating CCL20 levels. Unlike in Crohn’s disease patients, vitamin D deficiency was associated with higher CCL20 levels in healthy controls and ulcerative colitis patients, whereas the calcitriol/25-hydroxyvitamin D activation ratios were negatively correlated with serum CCL20 levels in healthy controls and ulcerative colitis patients with sufficient serum 25-hydroxyvitamin D status. Furthermore, calcitriol markedly inhibited intestinal epithelial induction of CCL20. In Crohn’s disease patients, cholecalciferol supplementation was associated with lower serum CCL20 levels, which were unaffected by NOD2 mutations. These findings suggest that although vitamin D metabolites may downregulate CCL20 expression in healthy controls and ulcerative colitis patients, this regulatory effect appears to be impaired in Crohn’s disease patients.

Oxidative stress (OS) is a common feature of many inflammatory diseases, oral pathologies, and aging processes. The impact of OS on periodontal ligament cells (PDLCs) in relation to oral pathologies, including periodontal diseases, has been investigated in different studies. However, its impact on orthodontic tooth movement (OTM) remains poorly understood. This study used an in vitro model with human PDLCs previously exposed to H2O2 to investigate the effects of OS under a static compressive force which simulated the conditions of OTM. Human PDLCs were treated with varying concentrations of H2O2 to identify sub-lethal doses that affected viability minimally. To mimic compromised conditions resembling OTM under OS, the cells were pretreated with the selected H2O2 concentrations for 24 h. Using an in vitro loading model, a static compressive force (2 g/cm2) was applied for an additional 24 h. The cell viability, proliferation, and cytotoxicity were evaluated using live/dead and resazurin assays. Apoptosis induction was assessed based on caspase-3/7 activity. The gene expression related to bone remodeling (RUNX2, TNFRSF11B/OPG, BGLAP), inflammation (IL6, CXCL8/IL8, PTGS2/COX2), apoptosis (CASP3, CASP8), and autophagy (MAP1LC3A/LC3, BECN1) was analyzed using RT-qPCR. This study suggests an altering effect of previous OS exposure on static-compression-related mechanosensing. Further research is needed to fully elucidate these mechanisms.

In recent years, there has been a growing number of adult orthodontic patients with periodontal disease. The progression of periodontal disease is well-linked to oxidative stress (OS). Nevertheless, the impact of OS on orthodontic tooth movement (OTM) is not fully clarified. Therefore, we applied an OS in vitro-model utilizing H2O2 to study its effect on tension-induced mechanotransduction in human osteoblasts (hOBs). Experimental parameters were established based on cell viability and proliferation. Apoptosis detection was based on caspase-3/7 activity. Gene expression related to bone-remodeling (RUNX2, P2RX7, TNFRSF11B/OPG), inflammation (CXCL8/IL8, IL6, PTRGS2/COX2), autophagy (MAP1LC3A/LC3, BECN1), and apoptosis (CASP3, CASP8) was analyzed by RT-qPCR. IL6 and PGE2 secretion were determined by ELISA. Tension increased the expression of PTRGS2/COX2 in all groups, especially after stimulation with higher H2O2 concentration. This corresponds also to the measured PGE2 concentrations. CXCL8/IL8 was upregulated in all groups. Cells subjected to tension alone showed a general upregulation of osteogenic differentiation-related genes; however, pre-stimulation with OS did not induce significant changes especially towards downregulation. MAP1LC3A/LC3, BECN1 and CASP8 were generally upregulated in cells without OS pre-stimulation. Our results suggest that OS might have considerable impacts on cellular behavior during OTM.

Antigen presentation via major histocompatibility complex (MHC) class I and class II receptors plays a fundamental role in T cell-mediated adaptive immunity. A dysregulation of this fine-tuned recognition might result in the development of autoimmune diseases such as inflammatory bowel diseases that are characterized by chronic relapsing inflammation of the intestinal tract and a damaged intestinal epithelial barrier. While MHCII receptors are usually expressed by professional antigen presenting cells (APC) only, there is increasing evidence that non-immune cells such as intestinal epithelial cells (IEC) might express MHCII upon stimulation with IFN-γ and thus act as non-professional APC. However, little is known about other factors regulating intestinal epithelial MHC expression. Here, we identify IL-27 as an inducer of different MHCI and MHCII receptor subtypes and the invariant chain (CD74/li) in IEC via the STAT1/IRF1/CIITA axis. CIITA, MHCII, and CD74 expression was significantly increased in IEC from Crohn’s disease (CD) patients with active disease compared to controls or CD patients in remission. IEC phagocytosed and digested external antigens and apoptotic cells. IL-27 strongly stimulated antigen processing via the immunoproteasome in a IRF1-dependent manner. In co-culture experiments, antigen-primed IEC strongly enhanced lymphocyte proliferation and IL-2 secretion, dependent on direct cell-cell contact. IL-27 pretreatment of IEC significantly increased CD4+ T cell proliferation and reduced IL-2 levels in lymphocytes in coculture. In summary, we identified IL-27 as a novel regulator of IEC antigen processing and presentation via MHCI and MHCII receptors, underscoring the importance of IEC as non-professional APC.

Objectives: A current trend to simplify dental restorative procedures is toward using uni-versal chromatic light-cured resin-based composites (RBCs) designed to adapt esthetically to various clinical situations. This study offers a comparative characterization of the me-chanical and cytotoxic behavior of such materials that use different techniques to adjust their optical properties (e.g., structural color instead of pigment addition), have different filler systems but are based on a comparable organic matrix. Methods: The structural appearance of the filler systems was assessed by scanning electron microscopy. Various quasi-static and viscoelastic parameters were evaluated at clinically relevant frequencies (0.5-5 Hz) using an instrumented indentation test with a Dynamic Mechanical Analysis (DMA) module. Cytotoxicity on human gingival fibroblasts (HGF-1), when exposed to eluates from tested RBCs specimens (up to one month), was assessed using a WST-1 colorimetric proliferation assay. Multifactor analysis of variance was applied to compare the parameters of interest (Martens, Vickers, and indentation hardness; elastic and total indentation work; creep, indentation depth; storage, loss, and indentation moduli; loss factor; cell viability) between analyzed RBCs, loading frequencies, and eluate age. Results: Structural particularities of the filler systems are directly reflected in the me-chanical behavior of the analyzed materials. Changes in the filler system, necessary to achieve structural color, generally resulted in lower mechanical properties but a better ability to absorb shock. In contrast, the cytotoxicity was comparable. Significance: Based on the performed characterization, universal chromatic RBCs fits in the conventional RBCs class to expect comparable clinical behavior. (c) 2022 The Academy of Dental Materials. Published by Elsevier Inc. All rights reserved.

Functionalized graphene and hydroxyapatite fillers are proposed as reinforcing particles in light-cured adhesives for dental applications. Silica-silver-graphene (SiO2-Ag-Gr), silver-doped hydroxyapatite (HA-Ag), graphene and silver doped hydroxyapatite (HA-Ag-Gr), and regular silica (SiO2) as inorganic powders, together with bis-GMA (2,2-bis[4-(2-hydroxy-3-methacryloxypropoxy) oligomers, as main components of the organic matrix, were synthesized. Light field transmission electron microscopy and electron diffraction proved the successful synthesis of the powders. The experimental adhesives showed a positive influence on local biocompatibility up to 6 months in an in-vitro simulation of clinically relevant environmental conditions and aging. Bonding to human dentin reveal a particular fracture pattern with preferential adhesion to the restorative material in contrast to the gold standard adhesive used as reference, which adheres preferentially to the tooth structure. Bond strength was slightly lower, initial bond reliability slightly improved, and the morphology of interaction with tooth structure similar to the reference. Functionalized graphene and hydroxyapatite fillers in dental adhesives have demonstrated their potential for use in dental applications.

INTRODUCTION: Iron deficiency and vitamin D deficiency are common comorbidities in inflammatory bowel disease (IBD). Accumulating evidence indicates that active 1,25-dihydroxyvitamin D (1,25(OH)D) may enhance iron absorption by suppressing hepcidin. We investigated the influence of vitamin D on iron metabolism in patients with IBD and on the expression of genes facilitating intestinal epithelial iron absorption. METHODS: Iron parameters and serum levels of 25-hydroxyvitamin D (25(OH)D), 1,25(OH)D, and hepcidin were measured in 104 adult patients with IBD (67 with Crohn's disease and 37 with ulcerative colitis). Genes involved in iron absorption were tested for induction by 1,25(OH)D in Caco-2 cells, which resemble the small intestinal epithelium. RESULTS: In multiple regression models controlling for age, sex, body mass index, smoking status, disease activity, and C-reactive protein levels, low 25(OH)D levels were associated with iron deficiency in patients with IBD (β [SE] = −0.064 [0.030], P = 0.029). Vitamin D sufficiency was associated with increased levels of ferritin (β [SE] = 0.25 [0.11], P = 0.024) and transferrin saturation (β [SE] = 8.41 [4.07], P = 0.044). Higher 1,25(OH)D:25(OH)D ratios were associated with lower hepcidin levels (β [SE] = −4.31 [1.67], P = 0.012). Especially in Crohn's disease, increased 1,25(OH)D correlated with higher transferrin saturation (β [SE] = 0.43 [0.18], P = 0.027). Furthermore, 1,25(OH)D strongly induced the expression of the ferroxidase ceruloplasmin in Caco-2 cells. DISCUSSION: Low vitamin D levels in IBD correlate with iron deficiency. Vitamin D may ameliorate iron deficiency, potentially by downregulating hepcidin and upregulating ceruloplasmin, enhancing intestinal iron absorption.

OBJECTIVE:The aim of the study was to determine the effects of ultra-fast (3 s) light-curing on the viscoelastic behaviour at clinically relevant frequencies, and cell toxicity, in a resin-based composite (RBC) with reversible addition-fragmentation-chain transfer (RAFT) mediated polymerization. METHODS:Three different protocols were used to cure cylindrical samples (height = 4 mm, ϴ = 5 mm), including ultra-fast (3s) cure with high radiant emittance, 10 s and 20 s cure with moderate radiant emittance. The properties of the light curing device were evaluated in all curing protocols by spectrophotometry up to an exposure distance of 10 mm. The light transmission through the samples was determined in real-time with the same spectrophotometer. Absorbance was calculated as a function of wavelength. The quasi-static (indentation hardness/HIT, indentation modulus/EIT) and viscoelastic (storage modulus/E', loss modulus/E″, loss factor/tan δ) material behavior was determined in an instrumented indentation test with a DMA (Dynamic Mechanical Analysis) module for 10 frequencies (0.5-5 Hz) by profiling the center of the samples in 330 μm steps from top to bottom. Cellular toxicity on human gingival fibroblast (HGF-1) was assessed using a WST-1 colorimetric assay after incubation time of up to 3 months. One and multiple-way analysis of variance (ANOVA) with Tukey honestly significant difference (HSD) post-hoc tests (α = 0.05) were applied. RESULTS:The irradiance transmitted through a 4 mm high sample was less than 7% of the incident irradiance, and the absorbance was similar for all curing protocols, showing a decrease with wavelength. Similar quasi-static and viscoelastic parameters were observed regardless of the curing protocol. HIT increased slightly and EIT, E', E″ and tan δ decreased with frequency. Occasionally, slightly higher confidence intervals were observed for the ultra-fast curing group, which were related to a potential accumulation of stress. The curing protocol had no effect on cell viability (p = 0.326) but the eluate age (p < 0.001, ηP2 = 0.879) did. None of the groups showed cell toxicity at any point in time with respect to the corresponding negative control. CONCLUSIONS:The ultra-fast curing with high irradiance induced no cell toxicity and an equivalent viscoelastic behavior as with conventional curing protocols in a RAFT-modified RBC.

Background NOD2variants are the strongest genetic predictors for susceptibility to Crohn's disease (CD). However, the clinical value ofNOD2on an individual patient level remains controversial. We aimed to define the predictive power of the majorNOD2mutations regarding complicated CD in a large single center cohort. Methods 1076 CD patients were prospectively genotyped for the three common CD-associatedNOD2mutations rs2066844, rs2066845, and rs2066847, followed by detailed genotype-phenotype analyses. Results Overall, 434 CD patients (40.3%) carried at least one of the three mainNOD2mutations. A significantly higher minor allele frequency (15.6%) of theNOD2frameshift mutation p.Leu1007fsX1008 (rs2066847) was seen in patients with aggressive disease compared to 8.2% in patients with mild disease (p = 2.6 x 10(-5)). Moreover, a total of 54 CD patients (5.0%) were homozygous for thisNOD2frameshift mutation. 100% of these patients had ileal disease compared to 82% ofNOD2wild-type carriers (p<0.0001). In homozygous carriers of theNOD2frameshift mutation, 87% presented with ileal stenosis, 68.5% had fistulas, and 72.2% required CD-related surgery despite immunosuppressive therapy in 87% of these patients. All homozygous carriers of the 1007fs mutation who were active smokers had ileal stenosis and required CD-related surgery. Conclusion Homozygosity for Leu1007fsX1008 is an excellent biomarker for predicting complicated CD on an individual patient level. Active smoking and homozygosity for this mutation is associated with a 100% risk for developing ileal stenosis requiring CD-related surgery. In these patients, smoking cessation and early initiation of immunosuppressive strategies may be beneficial.

BACKGROUND:A previous study suggested an association of the single nucleotide polymorphism (SNP) rs72796353 (IVS4+10 A>C) in the NOD2 gene with susceptibility to Crohn's disease (CD). However, this finding has not been confirmed. Given that NOD2 variants still represent the most important predictors for CD susceptibility and phenotype, we evaluated the association of rs72796353 with inflammatory bowel disease (IBD) susceptibility and the IBD phenotype.METHODOLOGY:Genomic DNA from 2256 Caucasians, including 1073 CD patients, 464 patients with ulcerative colitis (UC), and 719 healthy controls, was genotyped for the NOD2 SNP rs72796353 and the three main CD-associated NOD2 mutations rs2066844, rs2066845, and rs2066847. Subsequently, IBD association and genotype-phenotype analyses were conducted.RESULTS:In contrast to the strong associations of the NOD2 SNPs rs2066844 (p=3.51 x 10(-3)), rs2066845 (p=1.54 x 10(-2)), and rs2066847 (p=1.61 x 10(-20)) with CD susceptibility, no significant association of rs72796353 with CD or UC susceptibility was found. However, in CD patients without the three main CD-associated NOD2 mutations, rs72796353 was significantly associated with the development of perianal fistulas (p=2.78 x 10(-7), OR 5.27, [95% CI 2.75-10.12] vs. NOD2 wild-type carriers).CONCLUSION/SIGNIFICANCE:Currently, this study represents the largest genotype-phenotype analysis of the impact of the NOD2 variant rs72796353 on the disease phenotype in IBD. Our data demonstrate that in CD patients the IVS4+10 A>C variant is strongly associated with the development of perianal fistulas. This association is particularly pronounced in patients who are not carriers of the three main CD-associated NOD2 mutations, suggesting rs72796353 as additional genetic marker for the CD disease behaviour.

Background:In contrast to anti-TNF-&agr; antibodies, anti-IL-17A antibodies lacked clinical efficacy in a trial with patients suffering from Crohn's disease. We therefore analyzed how IL-17A modulates the inflammatory response elicited by TNF-&agr; in intestinal epithelial cells (IEC). Methods:Target mRNA levels in IEC and colonic biopsies were assessed by RNA microarray and quantitative real-time PCR. Signaling pathways were analyzed using receptor neutralization and pharmacological inhibitors. Target protein levels were determined by immunoblotting. Results:Microarray analysis demonstrated that IL-17A alone is a weak inducer of gene expression in IEC (29 regulated transcripts), but significantly affected the TNF-&agr;–induced expression of 547 genes, with strong amplification of proinflammatory chemokines and cytokines (>200-fold increase of CCL20, CXCL1, and CXCL8). Interestingly, IL-17A differentially modulated the TNF-&agr;–induced expression of several inflammatory bowel disease susceptibility genes in IEC (increase of JAK2 mRNA, decrease of FUT2, ICAM1, and LTB mRNA). Negative regulation of ICAM-1 by IL-17A was verified on protein level. The significance of these findings is emphasized by inflamed lesions of patients with inflammatory bowel disease demonstrating significant correlations (P < 0.01, Rho, 0.57–0.85) for JAK2, ICAM1, and LTB mRNA with IL17A and TNF mRNA. Conclusions:Our study demonstrates the modulation of inflammatory bowel disease susceptibility gene mRNA in IEC as a novel important property of IL-17A. Given the weak impact of sole IL-17A stimulation on IEC target gene expression, our study provides an important explanation for the lack of clinical efficacy of sole IL-17A neutralization, but suggests a beneficial effect of combined IL-17A/TNF-&agr; that is currently in clinical development.

Background Very recently, a sub-analysis of genome-wide association scans revealed that the non-coding single nucleotide polymorphism (SNP) rs12212067 in the FOXO3A gene is associated with a milder course of Crohn's disease (CD) (Cell 2013;155:57–69). The aim of our study was to evaluate the clinical value of the SNP rs12212067 in predicting the severity of CD by correlating CD patient genotype status with the most relevant complications of CD such as stenoses, fistulas, and CD-related surgery. Methodology/Principal Findings We genotyped 550 CD patients for rs12212067 (FOXO3A) and the three common CD-associated NOD2 mutations rs2066844, rs2066847, and rs2066847 and performed genotype-phenotype analyses. Results No significant phenotypic differences were found between the wild-type genotype TT of the FOXO3A SNP rs12212067 and the minor genotypes TG and GG independently from NOD2 variants. The allele frequency of the minor G allele was 12.7%. Age at diagnosis, disease duration, body mass index, surgery rate, stenoses, fistula, need for immunosuppressive therapy, and disease course were not significantly different. In contrast, the NOD2 mutant p.Leu1007fsX1008 (rs2066847) was highly associated with penetrating CD (p = 0.01), the development of fistulas (p = 0.01) and stenoses (p = 0.01), and ileal disease localization (p = 0.03). Importantly, the NOD2 SNP rs2066847 was a strong separator between an aggressive and a mild course of CD (p = 2.99×10−5), while the FOXO3A SNP rs12212067 did not separate between mild and aggressive CD behavior in our cohort (p = 0.35). 96.2% of the homozygous NOD2 p.Leu1007fsX1008 carriers had an aggressive disease behavior compared to 69.3% of the patients with the NOD2 wild-type genotype (p = 0.007). Conclusion/Significance In clinical practice, the NOD2 variant p.Leu1007fsX1008 (rs2066847), in particular in homozygous form, is a much stronger marker for a severe clinical phenotype than the FOXO3A rs12212067 SNP for a mild disease course on an individual patient level despite its important impact on the inflammatory response of monocytes.

expression.HBO therapy inhibited the acute distal colitis-induced up-regulation of HIF-1α and its downstream iNOS and myeloperoxidase activity, as well as producing diminished COX-2 levels.CONCLUSIONS: The results indicate that HBO therapy attenuates the severity of acute distal colitis through the down-regulation of the expression of