Background Crohn's disease (CD) is associated with defective sensing of pathogens in genetically susceptible individuals. Nucleotide-binding oligomerization domain containing 2 (NOD2) mutations in coding regions are strongly linked to CD pathogenesis. Our laboratory has reported that microRNAs (miRNAs) are differentially expressed in CD. However, miRNA regulation of NOD2 remains unknown. This study was designed to determine whether miRNAs regulate NOD2 expression as well as downstream nuclear factor kappaB activation and inflammatory responses in colonic epithelial HCT116 cells. Methods NOD2 and miRNA expression in stimulated HCT116 cells were assessed by quantitative reverse transcription–polymerase chain reaction. Regulation of NOD2 expression by miRNAs was determined by luciferase reporter construct assays and transfection of specific miRNA mimics. Regulation of NOD2 signaling and immune response by miRNAs was assessed by transfection of mimics followed by muramyl dipeptide stimulation. Results Muramyl dipeptide-induced increases in NOD2, interleukin-8, and CXCL3 expression were inversely associated with miRNA expression. Overexpression of miR-192, miR-495, miR-512, and miR-671 suppressed NOD2 expression, muramyl dipeptide-mediated NF-κB activation, and messenger RNA expressions of interleukin-8 and CXCL3 in HCT116 cells. A single-nucleotide polymorphism (rs3135500) located in the NOD2 3′-untranslated region significantly reduced miR-192 effects on NOD2 gene expression. Conclusions To our knowledge, this is the first report demonstrating that miRNAs regulate NOD2 and its signaling pathway. Four miRNAs downregulate NOD2 expression, suppress NF-κB activity, and inhibit interleukin-8 and CXCL3 messenger RNA expression. Treatment of CD with miRNAs may represent a potential anti-inflammatory therapeutic strategy in CD patients with and without NOD2 gene mutations.

Nonmelanoma skin carcinoma (NMSC) is the most frequent cancer in the USA with over 1.3 million new diagnoses a year; however due to an underappreciation of its associated mortality and growing incidence and its ability to be highly aggressive, the molecular mechanism is not well delineated. Whereas the molecular profiles of melanoma have been well characterized, those for cutaneous squamous cell carcinoma (cSCC) have trailed behind. This importance of the new staging paradigm is linked to the ability currently to better clinically cluster similar biologic behavior in order to risk-stratify lesions and patients. In this paper we discuss the trends in NMSC and the etiologies for the subset of NMSC with the most mortality, cutaneous SCC, as well as where the field stands in the discovery of a molecular profile. The molecular markers are highlighted to demonstrate the recent advances in cSCC.

METHODS: ICAM-1 expression in cultured IECs and mouse intestinal epithelium was induced by exposure to IFNc (100U/mL, 24h) and IFNcþTNFa (500ng each, 24h, ip) respectively, and analyzed by flow cytometry and immunofluorescence. The effects of neutrophil-IEC interactions, and specifically of ICAM-1 engagement by antibody crosslinking on the regulation of epithelial barrier function was examined in T84 IECs by measuring changes in transepithelial resistance (TER) and permeability to 4kDa FITC-Dextran, and by using an intestinal loop model from anesthetized mice, measuring absorption of FITC-dextran from the intestinal lumen into the circulation. RESULTS: We confirmed upregulation of ICAM-1 expression in IFNc treated T84 cells, in the intestinal epithelium of patients with IBD, and further showed induction in ICAM-1 expression in-vivo in murine small intestine pre-treated with IFNc and TNFa. In cultured IECs, expression of ICAM-1 enhanced neutrophil adhesion to the apical epithelial membrane. The majority ($60%) of apically adhered neutrophil that completed TEM were found to exhibit luminal crawling behavior, which was significantly enhanced (>33%) when T84 cells were pre-exposed to IFNc. Neutrophil interactions with apical epithelial membrane of IFNc pretreated IECs also resulted in enhanced neutrophil survival as evident by decreased apoptosis. The effects on neutrophil crawling and apoptosis were ICAM-1-dependent and were reversed by treatment with an anti-ICAM-1 function-blocking antibody. Furthermore, neutrophil interactions with the apical epithelial membrane resulted in time-dependent decrease in TER. This decrease was prevented by inhibition of neutrophil adhesion and crawling using an anti-CD11b/CD18 function-blocking antibody, suggesting that neutrophil induced effects on barrier function were due to direct engagement of ligands on epithelial surface. Experiments modeling neutrophil apical engagement using antibody-mediated crosslinking of cell surface ICAM-1 in cultured IECs and in-vivo, using murine intestinal loop model confirmed a direct role for ICAM-1 engagement in regulating epithelial barrier function. Antibody crosslinking of ICAM-1 resulted in time-dependent decrease in TER (up to 30%) and increased flux of FITC-dextran ($2.5-fold at 4 hours) in-vitro and increased flux of FITC-dextran ($1.6-fold) in-vivo. ICAM-1 mediated changes in barrier function were accompanied by actin rearrangement and dependent on MLCK. CONCLUSION(S): These studies suggest that neutrophil engagement of apically expressed ICAM-1during IBD results in signaling events that can inhibit neutrophil clearance and alter barrier. Such interactions may contribute to further recruitment of inflammatory cells and impede resolution of inflammation. Thus, targeting ICAM-1 in the intestinal epithelium may provide new/improved therapeutic approaches for treating IBD. P-209 YIBACKGROUND: Multiple genetic studies have implicated autophagy-related genes, ATG16L1 and IRGM, in the pathogenesis of Crohn's disease (CD). Functional characteriz...

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