Non-inflamed Sites Research Articles

A171 INTESTINAL INTERFERON-LAMBDA RECEPTOR 1 EXPRESSION AND RESPONSES ARE SIGNIFICANTLY DECREASED IN PEDIATRIC INFLAMMATORY BOWEL DISEASE PATIENTS

Abstract Background While interferon-lambdas (IFN-λs) were initially discovered for their role in antiviral immunity at mucosal barriers such as the lung and gut, there are many unanswered questions for how IFN-λs uniquely dampen inflammatory immune responses. In mouse colitis models, IFN-λs were shown to play a significant role in promoting epithelial barrier integrity and mucosal healing. Microbes present in the gut naturally induce IFN-λs, but how chronic inflammation, such as in patients with inflammatory bowel diseases (IBD), affects IFN-λ biology has not been well studied, especially in humans. Purpose Here, we tested the hypothesis that children with active, more severe IBD would present with lower intestinal IFN-λR levels and IFN-λ responses which could contribute to IBD pathology through exacerbated inflammation, decreased mucosal healing and impaired barrier function. Method We screened 14 novel antibodies to find the optimal clone that accurately stains the unique IFN-λ receptor subunit (IFN-λR1) protein in human intestinal tissue and IFN-λR1 levels were quantified by immunohistochemistry and flow cytometry in biopsy samples from children without IBD, Crohn’s disease, or ulcerative colitis (n=35 total). Fresh patient biopsies (ascending colon or terminal ileum) were also treated in media +/- IFN-λ for 24hrs using our novel ex vivo biopsy assay and changes in gene expression were quantified by RT-qPCR. Intestinal washes were also collected and microbes profiled by shotgun metagenomics. Result(s) We identified 2 new antibodies that accurately stained human cell lines and immune cells known to express IFN-λR1 protein (gut epithelial cells and B cells). We found that IFN-λ receptor (IFN-λR) levels are significantly reduced in gut epithelial and immune cells within pediatric IBD intestinal tissue, even at non-inflamed sites (p<0.01, 30-50% reduction) and this was even more pronounced when comparing moderate/severe disease compared to children with no disease activity. This led to lower IFN-λ responsiveness in IBD compared to non-IBD biopsies when investigating IFN-stimulated gene expression (p<0.05, up to 7-fold reduction). Paired patient gut microbe analyses identified specific species that correlated with changes in IFN-λ receptor expression. Conclusion(s) Together, our findings demonstrate pediatric IBD patients may be less able to induce critical IFN-λ-mediated antimicrobial responses and protective anti-inflammatory pathways. This work supports the goal to restore and promote IFN-λ responses as a novel therapeutic strategy for pediatric IBD. Please acknowledge all funding agencies by checking the applicable boxes below Other Please indicate your source of funding; Children's Hospital Research Institute of Manitoba Disclosure of Interest None Declared

P028 Inhibition of stromal glycolysis by targeting PFKFB3 decreases experimental colitis

Abstract Background Studies in fibrotic diseases revealed that glycolysis is the preferred energy source for fibroblasts. 6-phosphofructo-2-kinase/fructose-2, 6-bisphosphatase 3 (PFKFB3) has the highest kinase activity to shunt glucose toward glycolysis. Therefore inhibition of PFKFB3 has been proposed as a potential target for several cancers and inflammatory diseases. However, the metabolic status of fibroblasts in patients with inflammatory bowel disease (IBD) and the role of PFKFB3 are currently unknown. Methods Single-sample gene set enrichment analysis (ssGSEA) of GSE16879 was performed to evaluate metabolic changes in IBD. Seahorse real-time cell metabolic analysis was performed to explore the metabolic activity of fibroblasts. Next the expression of PFKFB3 in primary patient derived intestinal fibroblast was determined by quantitative PCR (qPCR) and western blot under normal and inflammatory conditions. Proliferation and migration of fibroblasts were measured using colony formation and wound healing assays. In order to evaluate the effect of the inhibition of PFKFB3 in vivo, PFK15, a specific inhibitor of PFKFB3, was intraperitoneal injected in mice with dextran sodium sulfate (DSS)-induced colitis and in the T-cell transfer model for colitis. Next to clinical parameters, the abundance of α-smooth muscle actin (α-SMA) expressing fibroblasts, immune cells (CD45) and endothelial cells (CD105) was determined by immunohistochemistry. Results The ssGSEA analysis revealed glycolysis was significantly higher in IBD patients, compared to healthy controls. Consistently, the expression of PFKFB3 was also elevated in a cohort of inflamed intestinal tissues from IBD patients compared to non-inflamed sites from the same patient or healthy controls. On the cellular levels, this analysis showed that PFKB3 expression was higher in IBD-derived stromal cells compared to healthy or non-inflamed stromal cells. In vitro PFKFB3 expression in fibroblasts was increased after the stimulation with pro-inflammatory cytokines like TNF-α and a mix of cytokines often upregulated in IBD patients: interleukin (IL)-17A, oncostatin M (OSM) and IL-1β. As for the metabolic changes, inflamed fibroblasts had a higher extracellular acidification rate and a lower oxygen consumption rate, which could be reverted by inhibition of PFKFB3 using PFK15. Furthermore, PFK15 suppressed the proliferation and migration of fibroblasts. The in vivo experiments showed that PFK15 reduced the severity of the colitis, accompanied by a reduction of the total amount of immune cells (CD45), activated fibroblasts (α-SMA) and angiogenesis (CD105). Conclusion Increased PFKFB3 expression seems to contribute the inflammation and the pathological function of fibroblasts in IBD.

Multiplex gene expression profile in inflamed mucosa of patients with Crohn's disease ileal localization: A pilot study.

BACKGROUNDCrohn’s disease (CD) is a complex disorder resulting from the interaction of genetic, environmental, and microbial factors. The pathogenic process may potentially affect any segment of the gastrointestinal tract, but a selective location in the terminal ileum was reported in 50% of patients.AIMTo characterize clinical sub-phenotypes (colonic and/or ileal) within the same disease, in order to identify new therapeutic targets.METHODS14 consecutive patients undergoing surgery for ileal CD were recruited for this study. Peripheral blood samples from each patient were collected and the main polymorphisms of the gene Card15/Nod2 (R702W, G908R, and 1007fs) were analyzed in each sample. In addition, tissue samples were taken from both the tract affected by CD and from the apparently healthy and disease-free margins (internal controls). We used a multiplex gene assay in specimens obtained from patients with ileal localization of CD to evaluate the simultaneous expression of 24 genes involved in the pathogenesis of the disease. We also processed surgery gut samples with routine light microscopy (LM) and transmission electron microscopy (TEM) techniques to evaluate their structural and ultrastructural features.RESULTSWe found a significant increase of Th17 (IL17A and IL17F, IL 23R and CCR6) and Th1 (IFN-γ) gene expression in inflamed mucosa compared to non-inflamed sites of 14 CD patients. DEFB4 and HAMP, two genes coding for antimicrobial peptides, were also strongly activated in inflamed ileal mucosa, suggesting the overwhelming stimulation of epithelial cells by commensal microbiota. IFN-γ and CCR6 were more expressed in inflamed mucosa of CD patients with ileal localization compared with patients with colonic localization suggesting a more aggressive inflammation process in this site. Morphological analysis of the epithelial lining of Lieberkün crypts disclosed enhanced release activity from goblet mucocytes, whereas the lamina propria contained numerous cells pertaining to various lines.CONCLUSIONWe observed that the expression of ileal genes related to Th1 and Th17 activity is strongly activated as well as the expression of genes involved in microbiota regulation.

P080 Association between intestinal mucosa and plasmatic levels of infliximab and TNFa in Chilean patients with inflammatory bowel disease

BACKGROUND: Inflammatory bowel disease (IBD) is characterized by chronic intestinal inflammation. Tumor Necrosis Factor a (TNFa) levels have been found to be elevated in the intestinal mucosa of patients with Crohn's Disease (CD) and Ulcerative Colitis (UC), which turned the anti-TNFa agents as one of the mainstays of the treatment. Nevertheless, some patients fail to respond or loose response, despite adequate plasmatic levels of anti-TNFa agents. The aim of this study was to determine intestinal mucosa levels of infliximab (IFX) and TNFa and its possible association with their plasmatic levels and the endoscopic activity in IBD patients. METHODS: Cross–sectional study of patients with IBD enrolled at the IBD program of Clínica Las Condes, Chile. The enrollment was done between May 2016 and January 2017. Tissue and plasmatic IFX and TNFa samples were taken the same day just before the next IFX infusion. The endoscopic activity was determined by colonoscopy using Mayo Score for UC and SES-CD/Rutgeerts for CD. Samples of mucosa were taken from colon in patients in remission and from inflamed and non-inflamed colon tissue in active patients. Mucosal levels of IFX and TNFa were normalized by total protein levels. The statistical analysis was performed using paired t-test analysis for comparison between inflamed and non-inflamed sites and an alpha value of <0.05 was considered statistically significant. RESULTS: Fourteen patients were included (9 UC and 5 CD) of which 6 were in remission and 8 with active disease (2 mild, 5 moderate and 1 severe). Plasmatic levels of IFX were higher in patients in remission compared to patients with active disease (4.488 AU/mL vs 2.378 AU/mL; P = 0.1355). In this last group, higher levels were observed in patients with mild than moderate endoscopic activity (4.785 AU/mL vs 1.552 AU/mL; P = 0.0264). Plasmatic TNFa levels were higher in patients with active disease compared to patients in remission (111.938 pg/mL vs 23.467 pg/mL; P = 0.0011), as opposed to IFX levels. Mucosal IFX levels were higher in patients with active disease vs remission, not statistically significant (6.136 ng/mL vs 3.917 ng/mL; P = 0.1186). On the contrary, mucosal TNFa levels were significantly higher in patients in remission vs active disease (65.062 pg/mL vs 36.479 pg/mL; P = 0.0330). In patients with active disease (n = 8), there was no statistical difference between the IFX level in the inflamed compared to the non-inflamed mucosa (6.136 ng/mL vs 5.000 ng/mL; P = 0.3534). TNFa in the non-inflamed mucosa was significantly higher compared to inflamed mucosa (64.846 pg/mL vs 36.479 pg/mL; P = 0.0066). In both, no difference was observed according to endoscopic activity level. The proportion of IFX/TNFα in mucosa was higher in patients with mild and moderate activity (0.182 and 0.179, respectively) than patients with severe activity (0.074). CONCLUSION(S): The discordance between plasmatic and intestinal mucosa IFX and TNFα, in patients with active disease, indicate that the inflammatory cascade might be determined by others specific inflammatory pathways. Further studies need to be performed in a larger sample of patients to better elucidate the mechanistic explanation for persistent inflammation in certain IBD patients.

Decreased Expression of Cystathionine β-Synthase Exacerbates Intestinal Barrier Injury in Ulcerative Colitis.

Endogenous H2S regulates multiple physiological and pathological processes in colon epithelial tissues. The current study investigated the role of cystathionine β-synthase [CBS], a major producer of H2S in colon epithelial cells, in the pathogenesis of ulcerative colitis [UC]-related intestinal barrier injury. The expression and DNA methylation level of CBS were investigated in inflamed and non-inflamed colon tissues collected from UC patients, and the effect of decreased CBS levels on Caco-2 monolayer barrier injury and altered status of tight junctions elicited by tumour necrosis factor/interferon [TNF/IFN] was determined. The expression of CBS and the methylation level of the CBS promoter were assessed in non-inflamed and inflamed colon epithelial tissue samples collected from UC patients. Barrier function, status of tight junction proteins and activation of the NF-κB p65-mediated MLCK-P-MLC signalling pathway were further investigated in Caco-2 monolayers. Decreased expression of CBS and elevated methylation levels of the CBS promoter were observed in inflamed sites compared with in non-inflamed sites in the colon epithelial samples from UC patients. In Caco-2 monolayers, decreased expression of CBS exacerbated TNF/IFN-induced barrier injury and altered localization of tight junction proteins. Decreased expression of CBS predisposed Caco-2 monolayers to injury elicited by TNF/IFN via augmentation of the NF-κB p65-mediated MLCK-P-MLC signalling pathway. Decreased expression of CBS propagates the pathogenesis of UC by exacerbating inflammation-induced intestinal barrier injury. Elevated methylation of the CBS promoter might be one of the mechanisms underlying the decreased expression of CBS in inflamed sites of colon epithelial tissues from UC patients.

Clinical attachment loss and molecular profile of inflamed sites before treatment.

To monitor early periodontal disease progression and to investigate clinical and molecular profile of inflamed sites by means of crevicular fluid and gingival biopsy analysis. Eighty-one samples of twenty-seven periodontitis subjects and periodontally healthy individuals were collected for the study. Measurements of clinical parameters were recorded at day -15, baseline and 2 months after basic periodontal treatment aiming at monitoring early variations ofthe clinical attachment level. Saliva, crevicular fluid and gingival biopsies were harvested from clinically inflamed and non-inflamed sites from periodontal patients and from control sites of healthy patients for the assessment of IL-10, MMP-8, VEGF, RANKL, OPG and TGF-β1 protein and gene expression levels. Baseline IL-10 protein levels from inflamed sites were higher in comparison to both non-inflamed and control sites (p<0.05). Higher expression of mRNA for IL-10, RANK-L, OPG, e TGF-β1 were also observed in inflamed sites at day -15 prior treatment (p<0.05). After the periodontal treatment and the resolution of inflammation, seventeen percent of evaluated sites still showed clinically detectable attachment loss without significant differences in the molecular profile. Clinical attachment loss is a negative event that may occur even after successful basic periodontal therapy, but it is small and limited to a small percentage of sites. Elevated inflammation markers of inflamed sites from disease patients reduced to the mean levels of those observed in healthy subjects after successful basic periodontal therapy. Significantly elevated both gene and protein levels of IL-10 in inflamed sites prior treatment confirms its modulatory role in the disease status.

Comparison of the microbial community structure between inflamed and non-inflamed sites in patients with ulcerative colitis.

The gut microbiota is suggested to play an important role in the pathogenesis of ulcerative colitis (UC). However, interindividual and spatial variations hamper the identification of UC-related changes. We thus investigated paired mucosa-associated microbiota obtained from both inflamed and non-inflamed sites of UC patients and corresponding sites of non-inflammatory bowel disease (IBD) controls. Mucosal biopsies of both inflamed and non-inflamed sites were obtained from 14 patients with active UC of the left-sided or proctitis type. Paired mucosal biopsies of the corresponding sites were obtained from 14 non-IBD controls. The microbial community structure was investigated using 16S ribosomal RNA gene sequences, followed by data analysis using qiime and LEfSe softwares. Microbial alpha diversity in both inflamed and non-inflamed sites was significantly lower in UC patients compared with non-IBD controls. There were more microbes of the genus Cloacibacterium and the Tissierellaceae family, and there were less microbes of the genus Neisseria at the inflamed site when compared with the non-inflamed site in UC patients. Decreased abundance of the genera Prevotella, Eubacterium, Neisseria, Leptotrichia, Bilophila, Desulfovibrio, and Butyricimonas was evident at the inflamed site of UC patients compared with the corresponding site of non-IBD controls. Among these taxa, the genera Prevotella and Butyricimonas were also less abundant at the non-inflamed site of UC patients compared with the corresponding site in non-IBD controls. Mucosal microbial dysbiosis occurs at both inflamed and non-inflamed sites in UC patients. The taxa showing altered abundance in UC patients might mediate colonic inflammation.

P784 A comparison study of the mucosa-associated microbiota between inflamed and non-inflamed sites in ulcerative colitis patients

P784 A comparison study of the mucosa-associated microbiota between inflamed and non-inflamed sites in ulcerative colitis patients

Giardia duodenalis-induced alterations of commensal bacteria killCaenorhabditis elegans: a new model to study microbial-microbial interactions in the gut

Giardia duodenalis is the most common cause of parasitic diarrhea worldwide and a well-established risk factor for postinfectious irritable bowel syndrome. We hypothesized that Giardia-induced disruptions in host-microbiota interactions may play a role in the pathogenesis of giardiasis and in postgiardiasis disease. Functional changes induced by Giardia in commensal bacteria and the resulting effects on Caenorhabditis elegans were determined. Although Giardia or bacteria alone did not affect worm viability, combining commensal Escherichia coli bacteria with Giardia became lethal to C. elegans. Giardia also induced killing of C. elegans with attenuated Citrobacter rodentium espF and map mutant strains, human microbiota from a healthy donor, and microbiota from inflamed colonic sites of ulcerative colitis patient. In contrast, combinations of Giardia with microbiota from noninflamed sites of the same patient allowed for worm survival. The synergistic lethal effects of Giardia and E. coli required the presence of live bacteria and were associated with the facilitation of bacterial colonization in the C. elegans intestine. Exposure to C. elegans and/or Giardia altered the expression of 172 genes in E. coli. The genes affected by Giardia included hydrogen sulfide biosynthesis (HSB) genes, and deletion of a positive regulator of HSB genes, cysB, was sufficient to kill C. elegans even in the absence of Giardia. Our findings indicate that Giardia induces functional changes in commensal bacteria, possibly making them opportunistic pathogens, and alters host-microbe homeostatic interactions. This report describes the use of a novel in vivo model to assess the toxicity of human microbiota.

Myeloperoxidase level around dental implants as an indicator of an inflammatory process.

Aim:Myeloperoxidase (MPO) has been widely used as an inflammatory marker of both acute and chronic conditions. The aim of the present study was to analyze MPO found in the peri-implant sulcus fluid of implants (PISF) and gingival cervicular fluid (GCF) of natural teeth in healthy or diseased states.Material and Methods:A total of 107 dental implant sites, either healthy/noninflamed, inflamed or affected by periodontitis, were classified and GCF/PISF samples were obtained. GCF/PISF MPO was spectrophotometrically determined.Results:Both the GCF and the PISF volumes exhibited a gradual increase with gingival inflammation (P < 0.05). PISF from inflamed sites (P = 0.0001) and GCF from the gingivitis and periodontitis sites showed significantly higher total MPO levels (P < 0.05) in comparison with the noninflamed sites. The volumetric similarities of PISF and GCF in terms of response to inflammation were seen in the present study. However, some differences in PISF and GCF were observed.Conclusion:PISF may be suggested to have a considerable diagnostic potential as it exhibits the biologic changes around load-bearing endosseous dental implants.

O-015 Metabolic Alterations to the Mucosal Microbiota of Inflammatory Bowel Disease Patients are Associated with Cd4+ T Cell Homeostasis

The pathogenesis of inflammatory bowel disease (IBD) may be caused by abnormal interactions between the immune system and microbiome. Recent studies using 16S ribosomal sequencing have shown that IBD is associated with dysbiosis of the microbiota. Inflammation may alter nutrient availability to adherent mucosal bacteria and impact their metabolic function. Microbial metabolites may also regulate intestinal CD4+ T cell homeostasis. We investigated the relationship between inflammation and microbial function by inferred metagenomics of the mucosal microbiota from colonic pinch biopsies of IBD patients to characterize differences in microbial metabolic pathways between inflamed and non-inflamed biopsy sites. Institutional review board approval was obtained before involving patients in the study. Paired pinch biopsy samples of known inflammation states were analyzed from UC (23), CD (21) and controls (24) by 16S ribosomal sequencing and inferred metagenomics with comparison to pathology results and flow cytometry data. The V4 region of the 16S rRNA gene was amplified and sequenced on a MiSeq sequencer. Sequences were assigned to operational taxonomic units (OTUs) and classified taxonomically according to the Ribosomal Database Project (RDP) for use in taxonomic analysis. PICRUSt was then used with the Greengenes OTU database to generate metagenomic data, and derive relative Kyoto Encyclopedia of Genes and Genomes (KEGG) Pathway abundance information. Leukocytes were isolated from paired biopsy samples and activated with PMA/Ionomycin for intracellular cytokine (IL-22, IL-17, IFN-g, IL-4 and TNFa) as well as nuclear antigen (Foxp3) analysis by multi-color flow cytometry on a BD LSRII. Active inflammation was defined by neutrophil infiltration into the epithelium, in the setting of epithelial cell damage. Carriage of metabolic pathways in the mucosal microbiota was relatively stable among IBD patients despite large variations in individual bacterial community structures (Fig. 1). However, microbial function was significantly altered in inflamed tissue of UC patients, with a reduction in carbohydrate and nucleotide metabolism in favor of increased lipid and amino acid metabolism (Fig. 2). These differences were not observed in samples from CD patients. In CD patients, microbial lipid and carbohydrate metabolism was tightly correlated with frequency of CD4+Foxp3+ Tregs, whereas in UC patients lipid and carbohydrate metabolism was correlated with frequency of CD4+IL-22+ (TH22) cells (Fig. 3). Metabolic pathways of the mucosal microbiota in CD do not vary as much as UC with inflammation state, indicating a more systemic perturbation of host-bacteria interactions in CD compared to more localized dysfunction in UC. The alterations in metabolic pathways correlate specifically with frequency of Tregs during CD, but with TH22 cells during UC. Alterations to metabolic pathways of the mucosal microbiota may affect the production of metabolites that can regulate intestinal CD4+ T cell populations and inflammatory responses of the gut.

PWE-108 Assessment of the Mucosal Microbiota in Inflammatory Bowel Disease

IntroductionGreat progress has been made in understanding the pathophysiology of Crohn’s disease (CD) and ulcerative colitis (UC), and it is now widely believed that dysregulation of the relationship between the...

Gingival crevicular fluid levels of interferon‐γ, but not interleukin‐4 or ‐33 or thymic stromal lymphopoietin, are increased in inflamed sites in patients with periodontal disease

To investigate the hypothesis that levels of interferon (IFN)-γ and interleukin (IL)-4, as well as the newer cytokines IL-33 and thymic stromal lymphopoietin (TSLP), in gingival crevicular fluid (GCF) samples differ from sites of patients at various clinical stages of periodontal disease and controls. Periodontal diseases result from the complex interplay between pathogenic bacteria and the host's immune responses. Several inflammatory mediators, such as IFN-γ and IL-4, have been detected in GCF samples in patients with periodontitis, but the results are mostly contradicting due to the lack of uniformity and collection of sites and methods of analysis. GCF samples were collected from sites with different clinical characteristics (healthy, gingivitis and periodontitis sites) from periodontally healthy ( n=14), plaque-induced gingivitis (n=17) and chronic periodontitis (n=11) subjects. The GCF samples were analyzed for the frequency of detection and levels of IFN-γ, IL-4, IL-33 and TSLP using a multiplex bead immunoassay. Inflamed sites in both patients with plaque-induced gingivitis and chronic periodontitis showed statistically significantly higher volume of GCF compared to non-inflamed sites in all patients. IFN-γ could be detected in about 50-70% of the samples analyzed and at significantly higher levels in sites with periodontitis compared to healthy sites in patients with chronic periodontitis (p=0.035). We also show a statistically significant decrease of IFN-? in healthy sites of patients with chronic periodontitis as compared to gingivitis sites in patients with plaque-induced gingivitis (p=0.047). Only some of the GCF samples showed detectable levels for IL-4 and TSLP, while IL-33 was below the detection level in all samples collected. These results suggest that IFN-γ levels in GCF depend on the clinical stage of the site and not on the disease stage of the patient, but need to be expanded to a greater number of subjects and additional analysis of corresponding gingival tissue biopsies for cytokine gene expression.

Expression of peptidylarginine deiminase‐2 and ‐4, citrullinated proteins and anti‐citrullinated protein antibodies in human gingiva

The presence of citrullinated proteins, and peptidylarginine deiminase types -2 (PAD-2) and -4 (PAD-4) in periodontal tissues, determine the presence of anti-cyclic citrullinated protein antibodies (anti-CCP) in gingival crevicular fluid (GCF) and compare the expression of these proteins between inflamed and non-inflamed sites. Tissue sections were stained using antibodies against citrullinated proteins, PAD-2 and PAD-4. RT-PCR was performed to investigate PAD-2 and PAD-4 mRNA in inflamed and non-inflamed gingival tissues. Anti-CCP antibodies in gingival crevicular fluid were detected by ELISA. Citrullinated proteins, PAD-2 and PAD-4 were detected in gingiva. There was a correlation between inflammation and expression of these proteins. mRNAs for PAD-2 and PAD-4 were detected in both inflamed and non-inflamed gingival tissues. Antibodies to CCP were found mostly in the GCF of individuals with periodontitis. PAD-2 and PAD-4 (protein and mRNA) as well as citrullinated proteins are present in inflamed gingiva, and anti-CCP antibodies can be detected in the GCF of some patients. Tissue expression of citrullinated proteins and PAD increased with the severity of inflammation. The presence of anti-CCP antibodies in GCF was almost exclusive to a subset of patients with periodontitis. Increased expression of these proteins in inflamed gingiva lends support to the notion that periodontal inflammation contributes to the inflammatory burden in a similar way to rheumatoid arthritis.

Comparative Analysis of Gingival Wetness at Natural Teeth and Dental Implant Sites

"Gingival Wetness (GW)," which is the thickness of residual saliva on gingiva, functions as a moisture retainer and a protective barrier for microbial colonization. Whether dental implant (DI) treatment affects GW scores and whether gingival inflammation has the capacity to alter GW remains unknown. Thus, this study was designed to evaluate the potential impact of DI treatment on GW. A total of 118 DIs and natural teeth (NT) sites were comparatively analyzed. Clinical periodontal and periimplant status were determined. Sites were classified into 2 subgroups based on the clinical inflammatory status as inflamed or noninflamed. GW was measured by standardized paper strips and quantified by micromoisture meter. Gingival index, clinical attachment loss, plaque index, and gingival bleeding time index scores were generally lower at DI sites. Significant differences were seen in clinical attachment loss, plaque index, and gingival bleeding time index in inflamed sites, presenting higher scores for NT. Comparable values were observed for DI and NT regarding GW for all sites. No differences were noticed in GW scores between the noninflamed NT and DI sites and also wheninflamed sites were concerned. DI treatment does not seem to result in any apparent reduction in GW. Thus, it can be speculated that similar protective processes may occur at dental implant and NT sites. Furthermore, GW does not seem to depend on the local inflammatory status of the soft tissues.

High-throughput clone library analysis of the mucosa-associated microbiota reveals dysbiosis and differences between inflamed and non-inflamed regions of the intestine in inflammatory bowel disease

BackgroundThe gut microbiota is thought to play a key role in the development of the inflammatory bowel diseases Crohn's disease (CD) and ulcerative colitis (UC). Shifts in the composition of resident bacteria have been postulated to drive the chronic inflammation seen in both diseases (the "dysbiosis" hypothesis). We therefore specifically sought to compare the mucosa-associated microbiota from both inflamed and non-inflamed sites of the colon in CD and UC patients to that from non-IBD controls and to detect disease-specific profiles.ResultsPaired mucosal biopsies of inflamed and non-inflamed intestinal tissue from 6 CD (n = 12) and 6 UC (n = 12) patients were compared to biopsies from 5 healthy controls (n = 5) by in-depth sequencing of over 10,000 near full-length bacterial 16S rRNA genes. The results indicate that mucosal microbial diversity is reduced in IBD, particularly in CD, and that the species composition is disturbed. Firmicutes were reduced in IBD samples and there were concurrent increases in Bacteroidetes, and in CD only, Enterobacteriaceae. There were also significant differences in microbial community structure between inflamed and non-inflamed mucosal sites. However, these differences varied greatly between individuals, meaning there was no obvious bacterial signature that was positively associated with the inflamed gut.ConclusionsThese results may support the hypothesis that the overall dysbiosis observed in inflammatory bowel disease patients relative to non-IBD controls might to some extent be a result of the disturbed gut environment rather than the direct cause of disease. Nonetheless, the observed shifts in microbiota composition may be important factors in disease maintenance and severity.

Airway Epithelial Inflammation-induced Endoplasmic Reticulum Ca2+ Store Expansion Is Mediated by X-box Binding Protein-1

Inflamed cystic fibrosis (CF) human bronchial epithelia (HBE), or normal HBE exposed to supernatant from mucopurulent material (SMM) from CF airways, exhibit endoplasmic reticulum (ER)/Ca(2+) store expansion and amplified Ca(2+)-mediated inflammation. HBE inflammation triggers an unfolded protein response (UPR) coupled to mRNA splicing of X-box binding protein-1 (XBP-1). Because spliced XBP-1 (XBP-1s) promotes ER expansion in other cellular models, we hypothesized that XBP-1s is responsible for the ER/Ca(2+) store expansion in inflamed HBE. XBP-1s was increased in freshly isolated infected/inflamed CF in comparison with normal HBE. The link between airway epithelial inflammation, XBP-1s, and ER/Ca(2+) store expansion was then addressed in murine airways challenged with phosphate-buffered saline or Pseudomonas aeruginosa. P. aeruginosa-challenged mice exhibited airway epithelial ER/Ca(2+) store expansion, which correlated with airway inflammation. P. aeruginosa-induced airway inflammation triggered XBP-1s in ER stress-activated indicator (ERAI) mice. To evaluate the functional role of XBP-1s in airway inflammation linked to ER/Ca(2+) store expansion, control, XBP-1s, or dominant negative XBP-1 (DN-XBP-1) stably expressing 16HBE14o(-) cell lines were used. Studies with cells transfected with an unfolded protein response element (UPRE) luciferase reporter plasmid confirmed that the UPRE was activated or inhibited by expression of XBP-1s or DN-XBP-1, respectively. Expression of XBP-1s induced ER/Ca(2+) store expansion and potentiated bradykinin-increased interleukin (IL)-8 secretion, whereas expression of DN-XBP-1 inhibited bradykinin-dependent IL-8 secretion. In addition, expression of DN-XBP-1 blunted SMM-induced ER/Ca(2+) store expansion and SMM-induced IL-8 secretion. These findings suggest that, in inflamed HBE, XBP-1s is responsible for the ER/Ca(2+) store expansion that confers amplification of Ca(2+)-dependent inflammatory responses.

Enteric alpha-2 adrenoceptors: Pathophysiological implications in functional and inflammatory bowel disorders

The gastrointestinal tract is innervated by extrinsic noradrenergic nerves which regulate various digestive functions, including mucosal secretions, bowel propulsion and gut sensations, via activation of α 2-adrenoceptors. These receptors are mostly involved in the prejunctional modulation of enteric neurotransmission, but they act also at extra-neural postjunctional sites. α 2-Adrenoceptor population consists of distinct subtypes, designated as α 2A, α 2B and α 2C, endowed with different physiological and pharmacological properties, and the attempts to classify α 2-adrenoceptors at gastrointestinal level have indicated a large predominance of α 2A subtypes. Studies in humans have shown a favourable influence of α 2-adrenoceptor activation on colonic tone and sensation, and there is clinical evidence indicating that α 2-agonists can improve intestinal functions and induce a satisfactory relief of symptoms in patients with irritable bowel syndrome. In addition, genetic investigations have highlighted significant associations of α 2-adrenoceptor gene polymorphisms with constipation and somatic symptoms in functional disorders of lower digestive tract. Post-operative ileus is a common surgical complication characterized by severe alteration of gut motility, resulting mainly from neurogenic and inflammatory mechanisms. Experiments in models of post-operative ileus have demonstrated an intense expression of α 2-adrenoceptors in monocytes recruited into the intestinal muscularis, and provided consistent evidence that these receptors promote post-operative gut dysfunctions by hampering enteric neurotransmission and contributing to local inflammatory reaction. Changes in the enteric nervous system are being increasingly recognized also as major determinants of digestive symptoms associated with bowel inflammation. In this regard, studies based on functional and molecular approaches concur in suggesting that the expression of enteric α 2-adrenoceptors is up-regulated in the presence of intestinal inflammation, and that α 2-mediated mechanisms are responsible for gut motor alterations occurring at both inflamed and non-inflamed sites. The present review discusses pathophysiological implications of enteric α 2-adrenoceptors, in the attempt to highlight potential therapeutic applications for drugs targeted on these receptors.

The bacteriology of biopsies differs between newly diagnosed, untreated, Crohn's disease and ulcerative colitis patients

The bacterial community (microbiota) that inhabits the gut of humans appears to be an important source of antigens that drive the chronic immunological processes characteristic of Crohn's disease (CD) and ulcerative colitis (UC). Most of the members of the microbiota have not yet been cultured, but nucleic-acid-based methods of detection and enumeration can provide information about the community. This investigation used these methods to obtain information about the bacteria associated with mucosal surfaces in the gut of 20 CD and 15 UC patients. Biopsies were collected from inflamed and non-inflamed sites in the intestines of newly diagnosed, untreated patients. Biopsies were also collected from several intestinal sites of 14 healthy subjects. The bacterial collections associated with the biopsies were analysed by generating PCR/denaturing gradient gel electrophoresis (DGGE) profiles, the preparation of 16S rRNA gene clone libraries, and qualitative PCR to detect specific groups of bacteria. The total numbers of bacteria associated with the biopsies were determined by real-time quantitative PCR. DGGE profiles generated from the terminal ileum and various colonic regions were characteristic of each individual but differed between subjects. DGGE profiles and 16S rRNA gene libraries showed that the bacteria associated with inflamed and non-inflamed tissues did not differ. UC patients had more bacteria associated with biopsies than did CD patients (P<0.01). Statistical analysis of the composition of 16S rRNA gene libraries showed that the bacterial collections in UC and CD patients differed (P<0.05). Unclassified members of the phylum Bacteroidetes were more prevalent in CD than in UC patients. Therefore, the types and numbers of bacteria associated with biopsy samples were distinctly different for UC and CD patients. The observations made in this study should permit targeting of specific bacteriological abnormalities in investigations of the pathogenesis of inflammatory bowel diseases and provide targets for medical interventions.

Clinical course of chronic periodontitis. I. Role of gingivitis.

The purpose of this study was to determine the influence of long-standing gingival inflammation on periodontal attachment loss. On the basis of repeated examinations, the present report describes the influence of gingival inflammation on the initiation of periodontitis from 16 to 59 years of age. The data originated from a 26-year longitudinal study of Norwegian males, who practiced daily oral home care and received state-of-the-art dental care. The initial examination included 565 individuals. Subsequent examinations took place in 1971, 1973, 1975, 1981, 1988 and 1995. Thus, the study covers the age range of 16-59 years. All tooth sites were divided into four categories according to their history of gingival inflammation over the entire observation period: sites always scoring GI = 0, GI = 1 and GI = 2 sites (GI = gingival index). Sites disclosing various GI scores at different observation periods were not considered. The mean cumulative attachment loss for non-inflamed (GI = 0) sites in individuals approaching 60 years of age was 1.94 mm. Sites always scoring GI = 1 yielded 2.42 mm, and sites that always scored GI = 2 exhibited 3.31 mm of periodontal attachment loss. At interproximal sites of all three groups where gingival trauma was assumed to be minimal or non-existent, only very few sites expressed attachment loss due to gingival recession (2-4%). At interproximal sites always scoring GI = 0, 20% loss of attachment was in the form of pocket formation by 59 years of age. The GI = 1 and the GI = 2 cohorts exhibited attachment loss with pocket formation in 28% and 54%, respectively. This study has shown that, as men approach 60 years of age, gingival sites that throughout the 26 years of observation bled on probing had approximately 70% more attachment loss than sites that were consistently non-inflamed (GI = 0). Before 40 years of age, there was a slight increase in periodontal attachment loss due to pocket formation, but after this, the frequency increased significantly. Loss of attachment due to gingival recession was very small in all three groups. The fact that sites with non-inflamed gingiva also exhibited some loss of attachment and pocket formation may be explained by fluctuation in the variations of tissue status during long observation intervals combined with the presence of subclinical inflammation.