Steven T. Leach

Severe Pediatric Ulcerative Colitis: A Prospective Multicenter Study of Outcomes and Predictors of Response

BACKGROUND & AIMS In a prospective study of children with severe ulcerative colitis (UC), we aimed to assess outcomes and to identify predictors of nonresponse to intravenous corticosteroids. METHODS A total of 128 children (47% males; 12.9 +/- 3.9 y) hospitalized for severe UC were enrolled from 10 pediatric centers. Clinical and laboratory data and the Pediatric UC Activity Index (PUCAI) were recorded throughout the admission. Patients were followed up for 1 year postdischarge. RESULTS Thirty-seven (29%; 95% confidence interval [CI], 22%-37%) children failed intravenous corticosteroids and received, within 10.5 +/- 6.4 days, cyclosporine (n = 1; 3%), colectomy (n = 3; 8%), or infliximab (n = 33; 89%). Several predictors were associated with intravenous corticosteroids failure, but the best model included number of stools, amount of blood, age, and new-onset disease (odds ratio [OR], 1.9; 95% CI, 1.1-3.5; OR, 2.5; 95% CI, 1.3-4.6; OR, 1.2; 95% CI, 1.04-1.36; and OR, 0.27; 95% CI, 0.1-0.7, respectively). The PUCAI, followed closely by the Travis rule, strongly predicted response when compared with other measures (Seo and Lindgren indices, C-reactive protein level, and fecal calprotectin level) (P < .001). Aiming for sensitivity on day 3, a PUCAI greater than 45 screened for patients likely to fail intravenous corticosteroids (negative predictive value, 94%; positive predictive value, 43%; P < .001). Aiming for specificity on day 5, a PUCAI score greater than 70 optimally guided implementation of salvage therapy (positive predictive value, 100%; negative predictive value, 79%; P < .001). Twenty-five of 33 children treated with infliximab responded. The overall cumulative colectomy rate was 9% and 19% by discharge and 1-year, respectively. The day 3 PUCAI score predicted response up to 1 year postdischarge (P < .001; time to salvage therapy). CONCLUSIONS The PUCAI, calculated on days 3 and 5 of steroid therapy, can identify patients requiring salvage therapy. Infliximab is an effective therapy in steroid-refractory pediatric UC.

Serum and mucosal S100 proteins, calprotectin (S100A8/S100A9) and S100A12, are elevated at diagnosis in children with inflammatory bowel disease.

Objective. Various markers characterize the complex inflammatory processes seen in chronic inflammatory bowel disease (IBD) including calprotectin, a complex of two S100 proteins, which has been evaluated and validated as a faecal marker of inflammation. However, the systemic and mucosal expression patterns of calprotectin and related S100 proteins are not well characterized in this disease. The objective of this study was to assess serum and mucosal levels of calprotectin, S100A12 and soluble receptor for advanced glycation end products (sRAGE), a putative S100 ligand, in a paediatric population with IBD. Material and methods. Children were enrolled at diagnosis of IBD, along with groups of children without IBD. Standard inflammatory markers and disease activity scores were collated. Calprotectin, S100A12 and sRAGE levels in serum and biopsy culture supernatants were measured by ELISA and tissue distribution of S100 proteins was investigated by immunohistochemistry. Results. Serum and mucosal calprotectin and S100A12 levels were increased in children with IBD as compared with non-IBD controls. Serum calprotectin levels correlated with S100A12 levels and with disease activity scores in children with IBD. sRAGE levels were not increased in IBD. S100A8, S100A9 and S100A12 were abundantly expressed throughout the lamina propria and epithelium in inflamed mucosa. In contrast, these proteins were present in the lamina propria, but not the epithelium, in non-inflamed mucosa. Conclusions. Serum calprotectin and S100A12 are increased in children with IBD and indicate disease activity. Elevated levels of these proteins are present in the colonic mucosa and may contribute to the pathogenesis of IBD. Furthermore, an imbalance between sRAGE and S100A12 may contribute to inflammatory changes present in IBD.

Fecal S100A12 and fecal calprotectin as noninvasive markers for inflammatory bowel disease in children

Background: Fecal calprotectin is a sensitive marker for gut inflammation. Recently, we have established that a related protein, S100A12, is elevated in the feces of children with inflammatory bowel disease (IBD). This may represent a specific and sensitive disease marker. The objective was to investigate the utility of fecal S100A12, in comparison to fecal calprotectin and standard inflammatory markers, as a screening marker for IBD in children with gastrointestinal symptoms. Methods: Stool samples were obtained from 61 children presenting with gastrointestinal symptoms requiring endoscopy. Fecal S100A12, calprotectin, and serum S100A12 levels were measured and correlated to final diagnosis and standard tests (ESR, CRP, platelet count, and albumin). Results: Children diagnosed with IBD (n = 31) had elevated fecal S100A12 (median 55.2 mg/kg) and calprotectin (median 1265 mg/kg) levels compared with the children without IBD (n = 30; S100A12: median 1.1 mg/kg, P < 0.0001; calprotectin: median 30.5 mg/kg; P < 0.0001). The sensitivity and specificity of fecal S100A12 (cutoff 10 mg/kg) for the detection of IBD were both 97%, whereas fecal calprotectin (cutoff 50 mg/kg) gave a sensitivity of 100% and a specificity of 67%. Conclusions: Both fecal markers were superior to the sensitivities and specificities of any standard inflammatory test. Both fecal S100A12 and calprotectin are sensitive markers of gastrointestinal inflammation, but fecal S100A12 provided exceptional specificity in distinguishing children with IBD from children without IBD. Fecal S100A12 is a simple, noninvasive test that can be used to screen and select children warranting further invasive and laborious procedures such as endoscopy for the investigation of their gastrointestinal symptoms.

Campylobacter concisus and other Campylobacter species in children with newly diagnosed Crohn's disease

Background: Campylobacter concisus and other members of the Campylobacter genus have recently been suggested as possible etiological agents of Crohns disease (CD). To further investigate this issue we determined the prevalence of these organisms in pediatric patients newly diagnosed with CD. Methods: DNA was extracted from fecal specimens collected from 54 children with CD, 27 noninflammatory bowel disease (non‐IBD), and 33 healthy controls and subjected to polymerase chain reaction (PCR) sequencing. Results: Detection of C. concisus DNA using a newly developed PCR assay targeting the 16S rRNA gene of C. concisus showed that 65% (35/54) of fecal samples from CD children were positive, a prevalence significantly higher than that in the healthy (33%, 11/33, P = 0.008) and non‐IBD controls (37%, 10/27, P = 0.03). The prevalence of all Campylobacter DNA using genus‐specific primers in children with CD was 72% (39/54), which was significantly higher than the 30% (10/33, P = 0.0002) and 30% (8/27, P = 0.0003) observed in healthy and non‐IBD controls, respectively. Conclusions: Given the strengthening evidence for a significantly higher prevalence of C. concisus and other non‐jejuni Campylobacter species in pediatric CD, investigation into the role of these non‐jejuni Campylobacter species in the initiation of human IBD is clearly a priority. (Inflamm Bowel Dis 2009;)

Defensins and inflammation: The role of defensins in inflammatory bowel disease

Defensins are antimicrobial peptides produced at a variety of epithelial surfaces. In the intestinal tract, they contribute to host immunity and assist in maintaining the balance between protection from pathogens and tolerance to normal flora. However, attenuated expression of defensins compromises host immunity and hence may alter the balance toward inflammation. Altered defensin production is suggested to be an integral element in the pathogenesis of inflammatory bowel disease (IBD). Evidence for this is shown in Crohns disease where reduced α‐defensin levels are seen in patients with ileal disease and reduced β‐defensin levels in those with colonic involvement. Further evidence is provided by research linking nucleotide oligomerization domain 2 (NOD2) mutations and deficient defensin expression. However, alternate studies suggest that NOD2 status and defensin expression are independent, and that defensin deficiency is due to mucosal surface destruction as a result of inflammatory changes, indicating that reduced defensin expression is a symptom of the disease and not the cause. Although it is clear that defensin expression is altered in IBD, it is less clear whether defensin deficiency is implicated in the pathogenesis of IBD or is a consequence of the disease process. The aim of this article is to review the current knowledge of defensins in IBD and discuss their potential role in IBD pathogenesis.

Detection and isolation of Campylobacter species other than C. jejuni from children with Crohn's disease.

ABSTRACT The presence of Campylobacter species other than Campylobacter jejuni and antibodies to Campylobacter concisus in children were investigated. A significantly greater presence of C. concisus and higher levels of antibodies to C. concisus were detected in children with Crohns disease (CD) than in controls. Campylobacter species other than C. jejuni were isolated from intestinal biopsy specimens of children with CD.

Sustained modulation of intestinal bacteria by exclusive enteral nutrition used to treat children with Crohn's disease

Background  The use of exclusive enteral nutrition to treat paediatric Crohn’s disease (CD) is widely accepted, although the precise mechanism(s) of action remains speculative.

Faecal calprotectin, lactoferrin, M2-pyruvate kinase and S100A12 in severe ulcerative colitis: a prospective multicentre comparison of predicting outcomes and monitoring response

Objective To compare four faecal markers for their ability to predict steroid refractoriness in severe paediatric ulcerative colitis (UC). Construct validity and responsiveness to change were also assessed. Methods This was a prospective multicentre cohort study. Stool samples from 101 children (13.3±3.6 years; Pediatric UC Activity Index (PUCAI) at admission 72±12 points) were obtained at the third day of intravenous steroid therapy. Repeated samples at discharge were obtained from 24 children. Predictive validity was assessed using diagnostic utility statistics to predict steroid failure (ie, the need for salvage treatment). Concurrent validity was assessed using correlational analysis with the following constructs: PUCAI, Lindgren and Seo scores, physicians global assessment, albumin, erythrocyte sedimentation rate and C-reactive protein (CRP). Responsiveness was assessed using test utility and correlational strategies. Results Median values (IQR) were very high at baseline for all four markers (calprotectin 4215 μg/g (2297–8808); lactoferrin 212 μg/g (114–328); M2-pyruvate kinase (M2-PK) 363 U/g (119–3104); and S100A12 469 μg/g (193–1112)). M2-PK was numerically superior to the other three markers and CRP in predicting response to corticosteroid treatment (area under the receiver operating characteristic (ROC) curve 0.75 (95% CI 0.64 to 0.85; p<0.001) vs <0.65 for the others). However, it did not add to the predictive ability of the PUCAI (area under the ROC 0.81 (95% CI 0.73 to 0.89)). M2-PK also had the highest construct validity but with a modest mean correlation with all constructs (r=0.3; p<0.05). None of the markers was responsive to change (Spearmans rho correlation with change in the PUCAI <0.1; p>0.05, area under the ROC curve <0.65; p>0.05). Conclusions The four markers were greatly elevated in severe paediatric UC. Only M2-PK had good construct and predictive validity, and none was responsive to change. The PUCAI, a simple clinical index, performed better than the faecal markers in predicting outcome following a course of intravenous corticosteroids in severe UC.

Fecal S100A12: a novel noninvasive marker in children with Crohn's disease.

Background: The calcium‐binding protein S100A12 is related to calprotectin, a protein shown to be a useful marker of gut inflammation. S100A12 levels are elevated in serum and mucosa of children with inflammatory bowel disease (IBD) and may be implicated in the pathogenesis of IBD. The aims of this study were to validate an immunoassay for the detection of fecal S100A12, to assess its value as a new noninvasive marker of gut inflammation, and to investigate S100A12 levels in feces of children with IBD at diagnosis and during treatment. Materials and Methods: Feces were collected from children with active IBD at diagnosis and during treatment for IBD and from normal healthy control subjects. Fecal and serum levels of S100A12 were measured by immunoassay. Results: A sensitivity of 96% and a specificity of 92% were observed when 10 mg/kg fecal S100A12 was used as a cutoff. S100A12 levels were evenly distributed throughout fecal samples and were stable for 7 days when stored at room temperature. Fecal S100A12 was elevated in children with IBD compared with healthy control subjects, with levels closely correlated to disease activity and other serum inflammatory markers, particularly lower gut involvement. Fecal S100A12 levels fell during therapy in children entering remission with normal C‐reactive protein levels. Conclusions: Fecal S100A12 is a novel noninvasive marker that distinguishes children with active IBD from healthy control subjects with high sensitivity and specificity. Fecal S100A12 possesses characteristics that are desirable for a noninvasive disease marker and therefore is a suitable candidate marker for IBD. Further evaluation is required to examine this marker in additional contexts.

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.