Duodenal microbiota composition and mucosal homeostasis in pediatric celiac disease.

BACKGROUND: Celiac disease (CD) is an autoimmune disorder of the small intestine which is triggered by dietary gluten in genetically predisposed (HLA-DQ2/DQ8 positive) individuals. Only a fraction of HLA-DQ2/DQ8 positive individuals develop CD indicating that other factors have a role in the disorder. Several studies have addressed intestinal microbiota aberrancies in pediatric CD, but the results are inconsistent. Previously, we demonstrated that pediatric CD patients have lower duodenal expression of TLR2 and higher expression of TLR9 as compared to healthy controls (HC) indicating that microbiota may have a role in CD. METHODS: We used bacterial phylogenetic microarray to comprehensively profile the microbiota in duodenal biopsies of CD (n = 10) and HC (n = 9) children. The expression of selected mucosa-associated genes was assessed by qRT-PCR in CD and HC children and in treated CD adults (T-CD, n = 6) on gluten free diet. RESULTS: The overall composition, diversity and the estimated microbe associated molecular pattern (MAMP) content of microbiota were comparable between CD and HC, but a sub-population profile comprising eight genus-like bacterial groups was found to differ significantly between HC and CD. In HC, increased TLR2 expression was positively correlated with the expression of tight junction protein ZO-1. In CD and T-CD, the expression of IL-10, IFN-g and CXCR6 were higher as compared to HC. CONCLUSIONS: The results suggest that microbiota and altered expression of mucosal receptors have a role in CD. In CD subjects, the increased expression of IL-10 and IFN-g may have partly resulted from the increased TLR9 expression and signaling.

Expression of microbiota, Toll-like receptors, and their regulators in the small intestinal mucosa in celiac disease.

OBJECTIVES: Less than one-tenth of the carriers of the risk genes HLA-DQ2 or HLA-DQ8 develop celiac disease, suggesting that other genetic and environmental factors are important in the pathogenesis. The role of gut microbiota has been addressed previously with inconsistent findings. Our aim was to evaluate microbiota, its receptors (Toll-like receptors [TLRs]), and regulators of the TLRs in the small intestinal mucosa in celiac disease. METHODS: Microbiota was analyzed by quantitative polymerase chain reaction (total bacteria and 10 bacterial group- and species-specific primers) and gene expression of interleukin-8 (IL-8), TLR2, TLR3, TLR4, TLR5, TLR9, and regulators of TLRs, Toll-interacting protein (TOLLIP), and single immunoglobulin IL-1R-related molecule, by relative quantitative reverse transcription-polymerase chain reaction in 10 children with celiac disease (untreated celiacs), 9 children with normal small intestinal mucosa (controls), and 6 adults with celiac disease with normal small intestinal mucosa after following a gluten-free diet (treated celiacs). RESULTS: Small intestinal microbiota was comparable among controls, untreated celiacs, and treated celiacs. Expression of IL-8 mRNA, a marker of intestinal inflammation, was significantly increased in untreated celiacs as compared with treated celiacs (P=0.002) and controls (P=0.001). Expression of TLR-2 mRNA was significantly decreased in untreated (P=0.001) and treated (P=0.03) celiacs, whereas expression of TLR-9 mRNA was increased in untreated celiacs (P=0.001) as compared with controls. Expression of TOLLIP mRNA was downregulated in untreated celiacs as compared with controls (P=0.02). CONCLUSIONS: Altered gene expression of TLR2, TLR9, and TOLLIP in small intestinal biopsies in celiac disease suggests that microbiota-associated factors may be important in the development of the disease.

Proliferation and differentiation markers in snuff-induced oral mucosal lesions.

BACKGROUND: Regular use of snuff is known to cause whitish oral mucosal lesions of variable severity at the usual quid placement site. The main aim of this study was to elucidate cellular mechanisms involved in snuff-induced epithelial changes. METHODS: Expression patterns for markers of cell proliferation (PCNA, Ki-67), cell cycle regulation (p53, p21), keratin changes (pankeratin, CK18, CK19), cell stress (HSP 70) and collagen type IV in 14 snuff-induced oral mucosal lesions and 12 control samples were analyzed by immunohistochemistry (IHC). RESULTS: On light microscopy, all snuff-induced lesions were characterized by a hyperkeratinized and thickened epithelium. Some vacuolized cells, markers of cell degeneration, were frequently seen (in 9/14 of the samples) in the superficial layers in epithelia. Expression of PCNA and Ki-67 was found in a statistically significantly fewer cells in snuff-induced lesions (P < 0.001) than in the controls. This indicates that epithelia in snuff-induced lesions are not thickened as a result of increased cellular proliferation, but by protracted turnover of differentiating cells. Of cell cycle markers, p21 was found be up-regulated in 4/14 snuff-induced lesions, probably by p53-independent pathways. Only two snuff-induced lesions showed p53 positivity. However, the number of stained cells with p53 and p21 was not statistically different from that in controls. Expression of CK18, but not any alterations in CK19 expression, was seen in 5 of 14 snuff-induced lesions. Snuff also seems to stimulate the expression of collagen type IV, possibly by basal cells, as indicated by the thickened staining of the basal lamina. CONCLUSIONS: The findings of this study showing suppressed cellular proliferation and infrequent p53 dysfunction in snuff lesions may partly explain why dysplastic changes are seldom seen in mucosal lesions induced by the Scandinavian type of snuff.