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Background: Fecal high mobility group box 1 (HMGB1) has been suggested to be a novel noninvasive biomarker of gut inflammation. We aimed to assess the reliability of fecal HMGB1, compared with fecal calprotectin (FC), in detecting intestinal inflammation in pediatric and adult patients with inflammatory bowel disease (IBD) and to evaluate the accuracy of HMGB1 in identifying patients with IBD in clinical and endoscopic remission who still have histologic features of inflammation., Methods: Stool samples from 85 children with IBD (49 Crohn's disease [CD] and 36 ulcerative colitis [UC] and 119 adults [57 Crohn's disease and 62 ulcerative colitis]) were analyzed for the study. Age-matched healthy subjects were used as controls. Fecal HMGB1 and fecal calprotectin were detected through western blot and ELISA, respectively., Results: Fecal HMGB1 expression was significantly increased in pediatric and adult patients with Crohn's disease and ulcerative colitis and strongly correlated with the disease severity. Fecal calprotectin and HMGB1 significantly correlated in pediatric (r: 0.60, P < 0.001) and adult (r: 0.72, P < 0.001) IBD patients. Moreover, in patients with clinical and endoscopic remission only fecal HMGB1 showed a strong match with the degree of histological scores of inflammation (CGHAS/IGHAS for Crohn's disease and Geboes Score for ulcerative colitis)., Conclusions: Fecal HMGB1 is confirmed to be a reliable biomarker of intestinal inflammation; indeed, it significantly correlates with fecal calprotectin in pediatric and adult IBD patients. Moreover, only fecal HMGB1 identifies histologic inflammation in subjects with IBD in clinical and endoscopic remission.

Background: Large evidence supports the role of microRNAs as new important inflammatory mediators by regulating both the adaptive and innate immunity. In the present study, we speculated that miR-320 controls NOD2 (nucleotide-binding oligomerization domain) expression, because it contains multiple binding sites in the 3'-untranslated region of the gene. NOD2, the first gene associated to increased susceptibility to Crohn's disease, is a cytosolic receptor that senses wall peptides of bacteria and promotes their clearance through initiation of a proinflammatory transcriptional program. This study aims at demonstrating that NOD2 is a target of miR-320 as well as investigating the role of inflammation in modulating the miR-320 control on NOD2 expression and analyzing miR-320 expression in intestinal biopsies of children with inflammatory bowel disease., Methods: The colonic adenocarcinoma cell line HT29 was used to assess the miR-320-mediated regulation of NOD2 expression. MiR-320 and NOD2 expression were analyzed in mucosal samples of 40 children with inflammatory bowel disease., Results: During inflammation, NOD2 expression is inversely correlated with miR-320 expression in vitro and ex vivo. Exogenous miR-320 transfection in HT29 cells leads to a significant decrease of NOD2 expression, whereas the miR-320 inhibitor transfection leads to increase of NOD2 expression, nuclear translocation of nuclear factor κB, and activation of downstream cytokines., Conclusions: We show for the first time that NOD2 expression is under the control of miR-320. We also show in vitro and ex vivo that inflammation induces a decrease of miR-320 and the latter correlates negatively with NOD2 expression.

Background: Noninvasive biomarkers of high- and low-grade intestinal inflammation and of mucosal healing (MH) in patients with inflammatory bowel disease are currently lacking. We have recently shown that fecal high mobility group box 1 (HMGB1) protein is a novel biomarker of gut inflammation. We aimed at investigating in a mouse model if HMGB1 was able to foresee both a clinically evident and a subclinical gut inflammation and if its normalization indicated MH. We also aimed at confirming the results in patients with Crohn's disease (CD) and ulcerative colitis., Methods: C57BL6/J mice were treated with increasing doses of dextran sodium sulphate to induce colitis of different severity degrees; 28 with CD, 23 with ulcerative colitis, and 17 controls were also enrolled. Fecal HMGB1 was analyzed by enzyme-linked immunosorbent assay and immunoblotting., Results: Fecal HMGB1 increased by 5-, 11-, 18-, and 24-folds with dextran sodium sulphate doses of 0.25%, 0.50%, 1%, and 4%, respectively, showing that the protein detected a high-grade and a subclinical inflammation. After a recovery time of 4-week posttreatment, HMGB1 returned to control levels, paralleling MH. In patients, fecal HMGB1 significantly correlated with endoscopic indexes (Simple Endoscopic Score for Crohn's Disease [SES-CD], endoscopic Mayo subscore), but not with the disease activity indexes (Crohn's disease Activity Index, partial Mayo score)., Conclusions: Fecal HMGB1 is a robust noninvasive biomarker of clinically overt and subclinical gut inflammation; it can also be a surrogate marker of MH. We suggest the use of fecal HMGB1 to monitor the disease course and assess therapy outcomes in inflammatory bowel disease.

Background: Crohn's disease (CD) and ulcerative colitis (UC), known as inflammatory bowel diseases (IBD), are characterized by an abnormal immunological response to commensal bacteria colonizing intestinal lumen and mucosa. Among the latter, strains of adherent-invasive Escherichia coli (AIEC), capable of adhering to and invading epithelium, and to replicate in macrophages, have been described in CD adults. We aimed at identifying and characterizing AIEC strains in pediatric IBD., Methods: In all, 24 CD children, 10 UC, and 23 controls were investigated. Mucosal biopsies, taken during colonoscopy, were analyzed for the presence of AIEC strains by an adhesive-invasive test. Protein expression of the specific AIEC receptor, the carcinoembryonic antigen-related cell adhesion molecule 6 (CEACAM6), was evaluated by western blot and immunohistochemistry, while tumor necrosis factor alpha (TNF-α) and interleukin (IL)-8 mRNA expression was detected by real-time polymerase chain reaction (PCR), after bacterial infection. Transmission electron microscopy and trans-epithelial electric resistance assays were performed on biopsies to assess bacteria-induced morphological and functional epithelial alterations., Results: Two bacterial strains, EC15 and EC10, were found to adhere and invade the Caco2 cell line, similar to the well-known AIEC strain LF82 (positive control): they upregulated CEACAM6, TNF-α, and IL-8 gene/protein expression, in vitro and in cultured intestinal mucosa; they could also survive inside macrophages and damage the epithelial barrier integrity. Lesions in the inflamed tissues were associated with bacterial infection., Conclusions: This is the first study showing the presence of adhesive-invasive bacteria strains in the inflamed tissues of children with IBD. Collective features of these strains indicate that they belong to the AIEC spectrum, suggesting their possible role in disease pathogenesis., (Copyright © 2011 Crohn's & Colitis Foundation of America, Inc.)

Background: NOD2 is an intracellular protein involved in host recognition of specific bacterial molecules and is genetically associated with several inflammatory diseases, including Crohn's disease (CD). NOD2 stimulation activates the transcription factor, NF-kappaB, through RIP2, a caspase-recruitment domain-containing kinase. NOD2/RIP2 signaling also mediates the activation of antimicrobial peptides such as human alpha-defensin 5 (HD-5) and human alpha-defensin 6 (HD-6), both produced by Paneth cells. The present study is aimed at describing the downstream events triggered specifically by NOD2 induction in order to demonstrate that the protein, other than overexpressed, is also physiologically associated with RIP2 and Erbin in the bioptic intestinal inflamed specimens of children affected by CD., Methods: Fifteen children with CD and 10 children used as controls were entered in the study. Mucosal biopsy specimens were taken during endoscopy and mRNA and protein expressions were detected by using real-time polymerase chain reaction and Western blot., Results: NOD2 is able to form an immunocomplex with the kinase RIP2. As compared to controls, in the inflamed mucosa of patients both mRNA and protein expression levels of RIP2 are increased, and its active phosphorylated form is overexpressed., Conclusions: In this study we provide for the first time ex vivo evidence of physiologically relevant protein interactions with NOD2, which are able to trigger the innate immune response in intestinal mucosal specimens of children with CD.

Background: Recent advances in the pathogenesis of Crohn's disease (CD) have suggested that an aberrant innate immune response initiates the cascade of events leading to T-cell activation and to disease development. NOD2 protein, which is mainly expressed by innate immunity cells, appears to play a key role against bacteria by triggering a host defense response through the activation of the transcriptor factor NF-kappaB and a consequent proinflammatory cytokine production. The present study was aimed at investigating the expression and activity of NOD2, NF-kappaB, and of 2 proinflammatory cytokines, TNFalpha and IL-1beta, in mucosal biopsies of CD affected children compared to healthy controls., Methods: In all, 22 children with active CD and 10 matched controls were entered in the study. mRNA and protein expressions were detected using reverse-transcriptase polymerase chain reaction (RT-PCR) and Western blot; NF-kappaB binding activity was assessed by electromobility gel shift assay (EMSA)., Results: NOD2 and IL-1beta mRNAs were upregulated in CD children. Protein levels of NOD2, TNFalpha, and nuclear NF-kappaB, as well as the binding activity of NF-kappaB to a consensus DNA sequence, were significantly increased in inflamed mucosa of patients as compared to controls. Moreover, NF-kappaB activity was strongly upregulated in patients also when bound to the NOD2 promoter site. No difference was seen between patients and controls when NF-kappaB binding activity was determined in the uninflamed tissue., Conclusions: This study suggests that altered mechanisms regulating NOD2 induction, NF-kappaB activation and cytokine production may contribute to dysregulate the innate immune response underlying pediatric CD.