Intestinal inflammation and impact on growth in children with cystic fibrosis.

OBJECTIVE: The aim of the study was to evaluate and compare faecal markers of intestinal inflammation in children with cystic fibrosis (CF), and determine whether intestinal inflammation adversely affects the nutritional phenotype. METHODS: Faecal samples for markers of intestinal inflammation, calprotectin, S100A12, and osteoprotegerin, were collected from children with CF, healthy controls (HCs), and Crohn disease (CD). Associations between inflammatory markers and clinical and nutritional indices were determined in subjects with CF. RESULTS: Twenty-eight children with CF (mean [standard deviation (SD)] 8.4 [3.3] years old, 22 pancreatic insufficient [PI]), 47 HC, and 30 CD were recruited. Mean (SD) faecal calprotectin in CF (94.3 [100.6] mg/kg) was greater than HC (26.7 [15.4] mg/kg, P < 0.0001), but lower than CD (2133 [2781] mg/kg, P = 0.0003). Abnormal faecal calprotectin was found in subjects only with PI (17/22 (77%), P = 0.001). There was no difference in faecal mean (SD) S100A12 (0.8 [0.9] vs 1.5 [2.2] mg/kg, P = 0.14) and osteoprotegerin concentrations (72.7 [52.2] vs 62.5 [0.0] pg/mL, P = 0.2) between CF and HC. Patients with CD had significantly elevated S100A12 and osteoprotegerin compared with CF and HC. Faecal calprotectin inversely correlated with both weight (r = -0.5, P = 0.003) and height z scores (r = -0.6, P = 0.002) in CF. CONCLUSIONS: The pattern of intestinal inflammation in CF is unique and distinct from inflammatory bowel disease, with elevated faecal calprotectin but normal faecal S100A12 and osteoprotegerin concentrations. The severity of intestinal inflammation, based on faecal calprotectin, significantly correlates with poor growth.

Osteoprotegerin exerts its pro-inflammatory effects through nuclear factor-κB activation.

BACKGROUND: Osteoprotegerin (OPG), a soluble member of the tumor necrosis factor (TNF) receptor super-family, is a key factor inhibiting the differentiation and activation of osteoclasts. It has recently been implicated as a disease marker for inflammatory bowel disease (IBD) yet its role in the intestinal epithelial inflammatory response remains unknown. AIM: The primary objective of this study was to investigate whether OPG has a role in intestinal inflammation and a potential role in IBD pathogenesis. METHODS: Caco-2 and HT-29 cells were grown in vitro to confluence on culture-permeable supports and then co-cultured with either TNF-α or OPG. After exposure to either TNF-α or OPG, interleukin (IL)-8 protein and mRNA levels were evaluated. Ussing chamber, western blotting, real-time polymerase chain reaction, and immunofluorescence were used to further investigate the effect of OPG on intestinal barrier integrity and function. RESULTS: Similar to TNF-α, treatment of monolayers with OPG caused increased monolayer permeability and diminished tight junction function and integrity, with loss of tight junction proteins from cell membranes. This was accompanied by elevated IL-8 protein and gene levels (P < 0.05). Western blotting also revealed that OPG, similar to TNF-α, induced NF-κB activation, as shown by inhibition of NF-κB kinase subunit-α phosphorylation. CONCLUSIONS: These results indicate that OPG has pro-inflammatory properties because it induces gut barrier dysfunction and secretion of other pro-inflammatory cytokines. These results also provide evidence that OPG is likely to exert its pro-inflammatory effects through NF-κB activation and may potently contribute to IBD pathogenesis.

Host attachment, invasion, and stimulation of proinflammatory cytokines by Campylobacter concisus and other non-Campylobacter jejuni Campylobacter species.

BACKGROUND: Campylobacter concisus and other non-Campylobacter jejuni Campylobacter species have been implicated in the initiation of gastrointestinal diseases. In the present study, we investigated the interaction between these bacteria and the human intestinal epithelium and immune cells. METHODS: The ability of C. concisus, Campylobacter showae, Campylobacter hominis, and Bacteroides ureolyticus to invade epithelial cells was examined using scanning electron microscopy and gentamicin protection assays. Proinflammatory cytokines generated by epithelial and immune cells in response to these bacteria were determined by enzyme-linked immunosorbent assay. Ussing Chamber, immunofluorescent stain, and Western blot were used to further elucidate the impact of C. concisus on intestinal barrier integrity and functions. RESULTS: Attachment of non-C. jejuni Campylobacter species to Caco-2 or HT-29 cells was mediated by flagellum-dependent and/or -independent processes. C. concisus was able to invade Caco-2 cells, generate a membrane-ruffling effect on the epithelial surface on entry, and damage epithelial barrier functions by preferential attachment to the cell-cell junctions. Proinflammatory cytokine profiles exhibited by epithelial cells, monocytes, and macrophages in response to C. concisus and other non-C. jejuni Campylobacter species were species and strain specific. CONCLUSIONS: These findings demonstrate that C. concisus and other non-C. jejuni Campylobacter species may play a role in initiating gastrointestinal diseases.

Development and assessment of a modified Pediatric Crohn Disease Activity Index.

The Pediatric Crohn Disease Activity Index (PCDAI) is an established and validated measure of disease activity in children with Crohn disease. However, its use in the research setting can be limited because of ambiguity of the subjective and anthropometric components of the index. Here we propose and evaluate a modified PCDAI (Mod PCDAI) consisting of the laboratory measures of the PCDAI plus C-reactive protein. This Mod PCDAI can provide an indication of disease activity because it correlates with the PCDAI, physicians' global assessment, and fecal calprotectin, and therefore may provide a suitable alternative to the PCDAI when required.

The major pathway by which polymeric formula reduces inflammation in intestinal epithelial cells: a microarray-based analysis.

Nutritional therapy is well established as a means to induce remission in active Crohn's disease (CD). Evidence indicates that exclusive enteral nutrition (EEN) therapy for CD both alters the intestinal microbiota and directly suppresses the inflammatory response in the intestinal mucosa. However, the pathway(s) through which EEN suppresses inflammation is still unknown. Therefore, the aim of the current study was to use microarray technology to investigate the major pathway by which polymeric formula (PF) alters inflammatory processes in epithelial cells in vitro. HT-29 cells were grown to confluence and then co-cultured with tumour necrosis factor (TNF)-α (100 ng/ml) for 5 h in the presence or absence of PF, as used for EEN. Following incubation, RNA was extracted and subjected to polymerase chain reaction (PCR) and microarray analysis. Enzyme-linked immunosorbent assays were employed to evaluate cytokine protein levels. Neither TNF-α nor PF had a toxic effect on cells over the experimental period. Microarray analysis showed that PF modulated the expression of genes specifically linked to nuclear factor (NF)-κB, resulting in downregulation of a number of genes in this pathway. These findings were further confirmed by real-time PCR of selected dysregulated genes as well as reduced expression of IL-6 and IL-8 proteins following PF treatment. The results arising from this study provide evidence that PF alters the inflammatory responses in intestinal epithelial cells through modulation of the NF-κB pathway.

Paediatric inflammatory bowel disease: a mechanistic approach to investigate exclusive enteral nutrition treatment.

The inflammatory bowel diseases (IBD) include Crohn's disease (CD) and ulcerative colitis. The disease may present at any age although the peak of presentation is the second and third decades of life. The incidences of these diseases are increasing around the world with the age of presentation getting younger. At present CD is incurable with colectomy being the treatment for severe UC. Although several pharmacological approaches are used to modulate the inflammatory response in IBD, few lead to histological healing and most have side effects. An alternative approach is to use enteral formulae given exclusively (EEN) to treat IBD. EEN requires the consumption of an elemental or polymeric formula, with the exclusion of all other nutrients, for a period of up to 12 weeks. The introduction of EEN as a therapeutic option for IBD was through prudent observation; however, EEN has become an established and reliable option for the treatment of paediatric IBD. Despite this, the mechanisms through which EEN induces disease remission are unknown and remain hypothetical. This review will discuss recent research into EEN both describing clinical features of EEN therapy and discussing the most up-to-date understanding of the mechanisms through which EEN may be reducing intestinal inflammation and inducing disease remission.

Inflammatory bowel disease therapies and gut function in a colitis mouse model.

BACKGROUND: Exclusive enteral nutrition (EEN) is a well-established approach to the management of Crohn's disease. Aim. To determine effects of EEN upon inflammation and gut barrier function in a colitis mouse model. METHODS: Interleukin-10-deficient mice (IL-10(-/-)) were inoculated with Helicobacter trogontum and then treated with EEN, metronidazole, hydrocortisone, or EEN and metronidazole combination. Blood and tissue were collected at 2 and 4 weeks with histology, mucosal integrity, tight junction integrity, inflammation, and H. trogontum load evaluated. RESULTS: H. trogontum induced colitis in IL-10(-/-) mice with histological changes in the cecum and colon. Elevated mucosal IL-8 mRNA in infected mice was associated with intestinal barrier dysfunction indicated by decreased transepithelial electrical resistance and mRNA of tight junction proteins and increased short-circuit current, myosin light chain kinase mRNA, paracellular permeability, and tumor necrosis factor- α and myeloperoxidase plasma levels (P < 0.01 for all comparisons). EEN and metronidazole, but not hydrocortisone, treatments restored barrier function, maintained gut barrier integrity, and reversed inflammatory changes along with reduction of H. trogontum load (versus infected controls P < 0.05). CONCLUSION: H. trogontum infection in IL-10(-/-) mice induced typhlocolitis with intestinal barrier dysfunction. EEN and metronidazole, but not hydrocortisone, modulate barrier dysfunction and reversal of inflammatory changes.

Differential effects of nutritional and non-nutritional therapies on intestinal barrier function in an in vitro model.

BACKGROUND: Diminished intestinal epithelial barrier function contributes to the pathogenesis of Crohn's disease. Clinical and experimental studies propose that increased tumor necrosis factor (TNF)-α promotes barrier dysfunction. The aim of this study was to investigate the effects of nutritional and other therapies upon intestinal barrier function in the presence of TNF-α in an in vitro model. METHODS: Caco-2 monolayers were grown to confluence on membrane supports and then exposed to TNF-α in the presence of polymeric formula, hydrocortisone or infliximab. Monolayer permeability was evaluated by measuring epithelial resistance, short-circuit current and horseradish peroxidase flux in an Ussing chamber. Tight junction and myosin II regulatory light-chain kinase gene expression was analysed by real-time PCR, with protein expression and localization analysed by Western blot and immunofluorescence. RESULTS: TNF-α increased monolayer permeability and diminished tight junction integrity. However both polymeric formula and infliximab completely abrogated the effects of TNF-α. These monolayers displayed unchanged permeability and tight junction integrity compared to untreated cells (media-no-TNF-α controls). In contrast, hydrocortisone only partially abrogated the effects of TNF-α, with these monolayers having increased permeability and altered tight junction integrity compared to media-no-TNF-α controls. CONCLUSIONS: Both polymeric formula and infliximab completely prevent epithelial barrier dysfunction in the presence of TNF-α, whereas hydrocortisone partially prevents barrier dysfunction. These results provide evidence that superior mucosal healing can be achieved with both polymeric formula and infliximab compared to hydrocortisone.

Osteoprotegerin in pediatric Crohn's disease and the effects of exclusive enteral nutrition.

BACKGROUND: Osteoprotegerin (OPG) may have proinflammatory roles in addition to its contribution to the maintenance of bone mass. Exclusive enteral nutrition (EEN) is an established therapy for the induction of remission in Crohn's disease (CD). The aims of this study were to ascertain serum, fecal, and mucosal expression of OPG in children with CD and to investigate the effects of EEN on OPG expression. METHODS: OPG was measured by enzyme-linked immunosorbent assay in serum, mucosal, and fecal samples collected from children with CD and controls. Fecal and Serum OPG was measured prior to and following 6-8 weeks of EEN therapy. RESULTS: Children with CD (n=82) and controls (n=45) were included. Mucosal and fecal OPG levels were elevated in CD compared to controls (P=0.018 and P<0.0001, respectively). Serum OPG was elevated in children with severe CD (P=0.005). Serum and fecal OPG levels dropped significantly following EEN therapy (P=0.0001 and P=0.002, respectively). CONCLUSIONS: Increased serum and fecal OPG are seen in active CD and likely originate from the inflamed gut. Fecal and serum OPG decrease following EEN therapy. Further investigation of OPG and related proteins in the setting of IBD is now required.