Investigation of potential early Histologic markers of pediatric inflammatory bowel disease.

BACKGROUND: Early manifestations of pediatric inflammatory bowel disease (IBD) can be relatively nonspecific. Initial mucosal biopsies may not be conclusive, delaying the diagnosis until subsequent biopsies demonstrate typical histologic features of IBD. We hypothesized that certain inflammatory cell types may be utilized as early histologic indicators of IBD in children. METHODS: A retrospective analysis compared histologic findings from initially inconclusive or negative endoscopic studies in 22 patients who were subsequently diagnosed with IBD (after diagnostic endoscopy) to those of 20 comparison patients with functional abdominal pain matched for age, gender, and study type. A pediatric pathologist, blinded to study group, reviewed biopsies for histologic abnormalities. Eosinophil densities were obtained from the stomach, duodenum, and rectosigmoid areas. Immunohistochemistry (IHC) staining for tumor necrosis factor-α (TNF-α) and matrix metalloproteinase-9 (MMP-9) was performed on the stomach and rectosigmoid areas. RESULTS: Gastritis and colonic crypt distortion were present in the IBD group at a greater rate (61 % vs. 22 %, p = 0.020; 34 % vs. 4 %, p = 0.008, respectively). Peak and mean eosinophil densities in the rectosigmoid area were greater in the IBD group (17.0/hpf vs. 5.0/hpf, p = 0.0063; 12.3/hpf vs. 4.2/hpf, p = 0.0106, respectively). TNF-α and MMP-9 staining did not reveal any significant differences. CONCLUSIONS: Our data suggests that significantly greater inflammation in the stomach, crypt distortion in the colon, and eosinophilia in the rectosigmoid distinguished the IBD group from the comparison group at the time of the initial endoscopic evaluation.

Intestinal permeability in children/adolescents with functional dyspepsia.

BACKGROUND: An altered intestinal mucosal barrier has been demonstrated in subsets of patients with IBS and FAP and may be an additional biological factor contributing to symptom generation in children with FD. The objective of this study was to determine if intestinal permeability is increased in children/adolescents with functional dyspepsia (FD) and whether intestinal permeability is correlated with mucosal inflammation and/or symptoms of anxiety or depression in this population. METHODS: A sugar absorption test was performed in 19 patients with FD and 19 controls. Anxiety and depression were assessed in both groups utilizing a standard questionnaire. In FD patients, duodenal mean and peak mast cell and eosinophil densities were determined. RESULTS: Intestinal permeability as measured by the sugar absorption test did not differ between children with FD and controls. In children with FD, there was no correlation between permeability and mast cell density, eosinophil density, anxiety scores, or depression scores, respectively. CONCLUSIONS: Pediatric FD does not appear to be associated with increased small bowel intestinal permeability, however, there are some limitations to the current study. TRIAL REGISTRATION: ClinicalTrials.gov; NCT00363597.

Validation of methods to assess potential biomarkers in pediatric patients with esophageal eosinophilia.

AIM: To validate methods for determining mast cell density, extracellular major basic protein content, and presence of fibrosis in esophageal eosinophilia. METHODS: Twenty specimens with > 20 eosinophils/high-power field (hpf) classified as high eosinophil density (HE) and 20 specimens with < 5 eosinophils/hpf classified as low esophageal density (LE) were identified. All 40 specimens underwent immunohistochemical staining and trichrome staining. Mast cell density, extracellular major basic protein (MBP) density, and presence of subepithelial fibrosis were assessed in a standardized manner. All specimens were evaluated by two separate observers and by a single observer on two separate occasions to evaluate reproducibility of the methods. RESULTS: A strong inter-observer correlation was noted for both peak and mean mast cell counts (r = 0.725, P < 0.0001 and r = 0.823, P < 0.0001). A strong intra-observer correlation also was noted for both peak and mean mast cell counts (r = 0.752, P < 0.0001 and r = 0.878, P < 0.0001). A very strong inter-observer correlation was noted for both peak (τ = 0.867, P < 0.0001) and mean extracellular MBP densities (r = 0.925, P < 0.0001). A very strong intra-observer correlation was noted for both peak (τ = 0.875; P < 0.0001) and mean extracellular MBP densities (r = 0.956, P < 0.0001). Excellent inter-rater reliability was found for fibrosis (κ = 0.887). Mast cell and MBP densities, as well as presence of fibrosis, were significantly increased in HE vs LE. The HE group had significantly higher intraepithelial mast cell peak (29.35 ± 21.61 vs 12.45 ± 8.26, P = 0.002) and mean (19.84 ± 15.81 vs 6.35 ± 4.5, P = 0.001) densities than the LE group. The HE group had significantly higher peak extracellular MBP (2.35 ± 0.67 vs 0.45 ± 0.61, P < 0.001) and mean extracellular MBP (1.95 ± 0.76 vs 0.20 ± 0.29, P < 0.0001) densities than the LE group. Seventy-three percent of patients with HE (11/15) had fibrosis, whereas only 10% of patients with LE (1/10) had fibrosis (P < 0.01). MBP performed the best in predicting classification of HE vs LE, with mean MBP demonstrating 100% sensitivity and 95% specificity at the optimal cut point. CONCLUSION: This study provides methodology and proof-of-concept for future evaluation of these biomarkers for differentiating esophageal eosinophilic diseases such as reflux esophagitis and eosinophilic esophagitis.

Mast cell activation and clinical outcome in pediatric cholelithiasis and biliary dyskinesia.

BACKGROUND: The current study was undertaken to determine the degree of activation of gallbladder mucosal mast cells, whether mast cell (MC) density or activation differ between patients with and without a positive clinical response to cholecystectomy, and whether either density or activation correlate with gallbladder emptying. RESULTS: Fifteen biliary dyskinesia (BD) and 13 symptomatic cholelithiasis (CL) patients undergoing cholecystectomy were prospectively enrolled. Gallbladder wall MC density (by immunohistochemistry) and activation (by electron microscopy) were determined. Clinical response was evaluated 30 days post-cholecystectomy on a 5-point Likert-type scale. A complete or nearly complete clinical response was seen in 100% of CL and in 87% of BD patients. The overall degranulation indices were 49.4 ± 18.7% for CL patients and 44.2 ± 16.8% for BD patients. Neither MC density nor activation correlated with the gallbladder ejection fraction. A complete clinical response was associated with lower epithelial MC density. CONCLUSION: Cholecystectomy is efficacious in relieving pain in both CL and BD patients. BD and CL are associated not only with increased MC density but a moderate to high degree of MC activation. A possible relationship between MC density and outcome for BD warrants further investigation.

Symptoms and subtypes in pediatric functional dyspepsia: relation to mucosal inflammation and psychological functioning.

OBJECTIVES: The aim of the present study was to explore relations between antral or duodenal inflammatory cells and aspects of psychological functioning with clinical symptom presentation in children with functional dyspepsia (FD), as well as to determine whether histologic inflammation and/or psychopathology are differentially associated with FD subtypes as defined by the Rome II and Rome III criteria. PATIENTS AND METHODS: One hundred pediatric patients with dyspepsia completed a standardized history and physical examination at initial evaluation. Patients and parents also completed a measure of psychological functioning. Subsequently, 63 of these patients underwent upper endoscopy with biopsy (4 patients excluded from analysis because of mucosal disease). Inflammatory cells in the mucosa of stomach and duodenum were enumerated. Associations between specific symptoms and FD subtypes with inflammatory cell densities and anxiety, depression, and somatization scores were examined. RESULTS: Rome III subtypes were more robustly related to differences in mast cell densities and scores on psychologic subscales than was true for Rome II subtypes. At the individual symptom level, having pain wake the patient from sleep was associated with higher duodenal mast cell density. Bloating was associated with lower levels of general antral inflammation, as well as higher self-reported levels of anxiety and somatization. Early satiety and bothersome postprandial fullness also were associated with higher levels of self-reported anxiety and depression. CONCLUSIONS: The present study provides preliminary evidence for a relation between clinical presentation, specific types of inflammatory cell infiltrates, and aspects of psychological functioning in children with FD. Rome III subtyping, adopted for adult dyspepsia, may be relevant to the pediatric population.

Useful biomarkers in pediatric eosinophilic duodenitis and their existence: a case-control, single-blind, observational pilot study.

OBJECTIVES: The aim of this study was to determine whether screening for food hypersensitivity could be a clinically useful biomarker for eosinophilic duodenitis in the pediatric population. PATIENTS AND METHODS: Twenty-two patients with functional dyspepsia and 19 controls with no significant history of gastrointestinal or allergic disorders were enrolled. Participants underwent skin prick, atopy patch, and serum-specific (S)-IgE, -IgG, and -IgG4 testing to corn, wheat, soy, peanut, milk, and egg. Participants in the patient group also underwent endoscopy with biopsies as part of standard care. RESULTS: Three participants in the patient group did not exhibit duodenal eosinophilia on biopsy and were excluded from data analyses. The patient group consisted of 13 females and 6 males, 8 to 17 years of age. The control group consisted of 10 females and 9 males, 8 to 17 years of age. Seven patients had at least 1 positive reaction to food by skin prick, atopy patch, or SIgE testing compared with 7 controls; odds ratio 1; 95% confidence interval 0.3 to 3.7. Receiver operating characteristics curves showed SIgG and SIgG4 performed poorly or no better than chance for predicting group assignment. CONCLUSIONS: Allergy screening for the foods tested was not useful as a biomarker for eosinophilic duodenitis in this small study. A higher rate of positive reactions to patch testing was observed in the control group than previous studies have reported. The incidence of a positive food patch test in nonselected subjects needs further investigation. Method standardization and establishment of reference intervals are needed for atopy patch tests, SIgG, and SIgG4 to better evaluate the clinical value of these measures.

Montelukast in the treatment of duodenal eosinophilia in children with dyspepsia: effect on eosinophil density and activation in relation to pharmacokinetics.

BACKGROUND: We have previously demonstrated the clinical efficacy of montelukast in a randomized double-blind controlled cross-over trial in patients with dyspepsia in association with duodenal eosinophilia. The mechanism of this clinical response is unknown but could involve a decrease in eosinophil density or activation. METHODS: Twenty-four dyspeptic patients 8-17 years of age underwent initial blood sampling and endoscopy with biopsy. Eighteen of these patients had elevated duodenal eosinophil density and underwent repeat blood sampling and endoscopy following 21 days of therapy with montelukast (10 mg/day). The following were determined: global clinical response on a 5-point Lickert-type scale, eosinophil density utilizing H & E staining, eosinophil activation determined by degranulation indices on electron microscopy, and serum cytokine concentrations. On day 21, pharmacokinetics and duodenal mucosal drug concentrations were determined. RESULTS: Eighty-three percent of the patients had a positive clinical response to montelukast with regard to relief of pain with 50% having a complete or nearly complete clinical response. The response was unrelated to systemic drug exposure or to mucosal drug concentration. Other than a mild decrease in eosinophil density in the second portion of the duodenum, there were no significant changes in eosinophil density, eosinophil activation, or serum cytokine concentrations following treatment with montelukast. Pre-treatment TNF-alpha concentration was negatively correlated with clinical response. CONCLUSION: The short-term clinical response to montelukast does not appear to result from changes in eosinophil density or activation. Whether the effect is mediated through specific mediators or non-inflammatory cells such as enteric nerves remains to be determined. TRIAL REGISTRATION: ClinicalTrials.gov; NCT00148603.