Guidance on the interpretation of faecal calprotectin levels in children.

BACKGROUND: Faecal calprotectin (FCP) is a powerful tool to predict inflammatory bowel disease (IBD) in patients with gastrointestinal symptoms. In the paediatric patient population, the reference value of < 50 µg/g and the influence of age on FCP levels result in a high number of redundant investigations and specialist referrals. We assessed paediatric FCP levels, their diagnostic value and corresponding referral pathways from primary and secondary care. METHODS: We analysed two cohorts from a precisely defined catchment area: one consisted of all FCPs measured in this area (n = 2788). The second cohort-a subset of the first cohort-consisted of FCP values and corresponding clinical data from children who were referred for possible IBD to our department (n = 373). RESULTS: In the first cohort, 47% of FCP levels were > 50 µg/g, 15% were ≥ 250 µg/g. Children < 1y had significantly (p < 0.001) higher FCP than older children. In the second cohort, 6.7% of children with an FCP of < 250 µg/g (or 8.6% with an FCP of < 600 µg/g) had IBD-all featured symptoms suggestive of IBD (e.g. bloody diarrhoea, nocturnal abdominal pain, weight loss) or abnormal blood tests. 76% of patients in whom raised FCP (> 50 µg/g) was the sole reason for being referred for suspected IBD did not have IBD. CONCLUSION: Children with an FCP < 600 µg/g and without matching symptoms suggestive of IBD are unlikely to have IBD. A higher FCP reference value may provide cost-effective improvement that could avoid redundant investigations and specialist referrals. A guideline for specialist referrals is proposed.

DNA methylation analysis in the intestinal epithelium-effect of cell separation on gene expression and methylation profile.

BACKGROUND: Epigenetic signatures are highly cell type specific. Separation of distinct cell populations is therefore desirable for all epigenetic studies. However, to date little information is available on whether separation protocols might influence epigenetic and/or gene expression signatures and hence might be less beneficial. We investigated the influence of two frequently used protocols to isolate intestinal epithelium cells (IECs) from 6 healthy individuals. MATERIALS AND METHODS: Epithelial cells were isolated from small bowel (i.e. terminal ileum) biopsies using EDTA/DTT and enzymatic release followed by magnetic bead sorting via EPCAM labeled microbeads. Effects on gene/mRNA expression were analyzed using a real time PCR based expression array. DNA methylation was assessed by pyrosequencing of bisulfite converted DNA and methylated DNA immunoprecipitation (MeDIP). RESULTS: While cell purity was >95% using both cell separation approaches, gene expression analysis revealed significantly higher mRNA levels of several inflammatory genes in EDTA/DTT when compared to enzymatically released cells. In contrast, DNA methylation of selected genes was less variable and only revealed subtle differences. Comparison of DNA methylation of the epithelial cell marker EPCAM in unseparated whole biopsy samples with separated epithelium (i.e. EPCAM positive and negative fraction) demonstrated significant differences in DNA methylation between all three tissue fractions indicating cell type specific methylation patterns can be masked in unseparated tissue samples. CONCLUSIONS: Taken together, our data highlight the importance of considering the potential effect of cell separation on gene expression as well as DNA methylation signatures. The decision to separate tissue samples will therefore depend on study design and specific separation protocols.

Improvement in biomarkers of bone formation during infliximab therapy in pediatric Crohn's disease: results of the REACH study.

BACKGROUND & AIMS: Crohn's disease (CD) is associated with altered bone metabolism. This study examined changes in bone formation and resorption after infliximab induction and associations between bone biomarkers, linear growth, and disease activity (Pediatric Crohn's Disease Activity Index [PCDAI]) after 54 weeks of infliximab therapy. METHODS: One hundred twelve subjects ages 6-17 years with moderate to severe CD received infliximab induction (5 mg/kg/dose) at weeks 0, 2, and 6; week-10 responders were randomized to infliximab every 8 or every 12 weeks maintenance therapy. Serum bone-specific alkaline phosphatase (BSAP), N-terminal propeptide of type 1 collagen (P1NP), urine C-telopeptide of collagen cross-links (CTX-1), and deoxypyrodinoline (DPD) were collected at baseline and 10 weeks. PCDAI and height z-scores were assessed at baseline and at 10 and 54 weeks. RESULTS: Models were adjusted for bone age, gender, height, and steroid use. Baseline BSAP and P1NP levels were negatively associated with PCDAI (both P = .01). BSAP and P1NP increased during induction (both P < .001) and were associated with 54-week increases in height z-score (P < .05 and P < .001, respectively). Improvements in P1NP were associated with 54-week decreases in PCDAI (P = .01). CTX-1 and DPD also increased during induction (P < .001 and P = .01, respectively) but were not associated with changes in PCDAI. Changes in CTX-1 were associated with improvements in height z-score (P < .002). CONCLUSIONS: Infliximab therapy is associated with dramatic increases in BSAP and P1NP, consistent with inhibition of tumor necrosis factor-alpha effects on osteoblasts. The increases in CTX-1 and DPD likely reflect coupling of bone formation and resorption and increases in linear growth.

Desquamative enteropathy and pyloric atresia without skin disease caused by a novel intracellular beta4 integrin mutation.

BACKGROUND: Mutations in alpha6 or beta4 integrins (ITGA6, ITGB4) are known to cause junctional epidermolysis bullosa with pyloric atresia (JEB-PA), often lethal in infancy through skin desquamation. There is 1 report of pyloric atresia associated with a desquamatory enteropathy but without skin disease, of unknown molecular basis. PATIENTS AND METHODS: We report 2 Kuwaiti siblings with pyloric atresia and life-threatening intestinal desquamation without significant skin abnormality. The older sibling died of intractable diarrhoea, and the younger sibling suffered episodes of massive protein-losing enteropathy, triggered by viral infections, in addition to obstructive uropathy. Mutation analysis was performed for ITGA6 and ITGB4 and expression of ITGA6 and ITGB4 protein was examined in skin and intestinal biopsies. Her serum also was incubated with normal intestine. RESULTS: We identified a novel mutation in ITGB4, with homozygous deletion of a single residue (isoleucine 1314) within the intracellular plectin-binding domain. Expression of ITGA6 and ITGB4 within skin, duodenal, and colonic epithelium was normal or minimally reduced, in contrast to previous reports. Biopsies taken during relapse showed accumulation of immunoglobulin G and C1q within intestinal basement membrane, whereas immunoglobulin G from her serum bound to basement membrane of normal small intestine. Immunomodulatory therapy induced significant improvement following relapses. CONCLUSIONS: ITGB4 mutation may induce a desquamative enteropathy in infancy without significant skin disease. A history of pyloric atresia is important in infants with severe chronic diarrhoeal disease and should prompt investigation for JEB-PA associated mutations. Acquired immune responses may exacerbate primary genetic disorders of epithelial adhesion and immunomodulatory therapy may be beneficial.

Natural history of paediatric inflammatory bowel diseases over a 5-year follow-up: a retrospective review of data from the register of paediatric inflammatory bowel diseases.

OBJECTIVES: The natural history of paediatric inflammatory bowel diseases (IBDs) is poorly understood. We aim to describe the disease course in this cohort and generate prognostic information for patients and clinicians. MATERIALS AND METHODS: Patient records from 6 tertiary paediatric gastroenterology centres were reviewed to generate data concerning original diagnosis, change in diagnosis, family history, surgical interventions, growth, and presence of extragastrointestinal manifestations. RESULTS: Data were collected on 116 children with Crohn disease (CD), 74 with ulcerative colitis (UC), and 20 with indeterminate colitis (IC), followed for a mean period of 3.42, 3.3, and 2.9 years from date of diagnosis, respectively. A male predominance is demonstrated in CD. Revision of diagnosis in patients with IC is mainly to UC, with most children receiving a definitive diagnosis within 2 years of initial presentation. Of the children with UC, 17.6% underwent 1 or more major operations with a median time to surgery of 1.92 years. Of children with CD, 11.6% underwent 1 or more major intraabdominal procedures with a median time to surgery of 1.83 years. We recorded a positive family history in 2.7%, 8.2%, and 10% of cases for CD, UC, and IC, respectively. For both boys and girls with CD, but only for boys with UC, height standard deviation score became more negative over time. CONCLUSIONS: This retrospective study quantifies certain distinctions between IBDs diagnosed in paediatric and adult populations. We document a trend toward male predominance in children with CD. We also note impaired linear growth in children with CD, whereas it appears maintained in girls with UC. We also have recorded a low incidence of IBDs in the families of this cohort and suggest that environmental influences may be of greater importance. We document that major intraabdominal surgery may be required in about 15% of patients with either UC or CD within 2 years of diagnosis, and that the majority of those diagnosed initially with IC will be reclassified as either UC or CD within 2 years.

Massive gastrointestinal haemorrhage in isolated intestinal Henoch-Schonlein purpura with response to intravenous immunoglobulin infusion.

There is increasing recognition that Henoch-Schonlein purpura may present in an atypical form in which gastrointestinal symptoms may predominate, and classic cutaneous changes may be delayed or absent. This may lead to significant diagnostic delay. We report the case of a 9-year-old girl who presented acutely with life-threatening gastrointestinal haemorrhage from multiple intestinal sites, with no skin rash and only mild evidence of renal involvement. Henoch-Schonlein purpura was confirmed by finding IgA deposition on vessels within gastric and duodenal mucosa, while immunohistochemistry also identified dense focal T cell infiltration in gastric mucosa and within duodenal epithelium. After initial stabilisation, the patient became shocked due to further gastrointestinal haemorrhage. Isotope bleeding scan identified multiple bleeding sites. Her endoscopically confirmed gastritis was sufficiently severe to preclude corticosteroids, and she was thus treated with intravenous immunoglobulin. This therapy induced prompt and sustained resolution of symptoms, and she has remained well since. Our patient's response concords with previous reports in corticosteroid-resistant cases to suggest that severe intestinal Henoch-Schonlein purpura may respond preferentially to intravenous immunoglobulin (IVIG) therapy. In severe cases where there is significant gastritis, IVIG provides an effective alternative to corticosteroids that may be employed as first-line therapy.

Focal-enhanced gastritis in regressive autism with features distinct from Crohn's and Helicobacter pylori gastritis.

BACKGROUND: Immunohistochemistry allowed recent recognition of a distinct focal gastritis in Crohn's disease. Following reports of lymphocytic colitis and small bowel enteropathy in children with regressive autism, we aimed to see whether similar changes were seen in the stomach. We thus studied gastric antral biopsies in 25 affected children, in comparison to 10 with Crohn's disease, 10 with Helicobacter pylori infection, and 10 histologically normal controls. All autistic, Crohn's, and normal patients were H. pylori negative. METHODS: Snap-frozen antral biopsies were stained for CD3, CD4, CD8, gammadelta T cells, HLA-DR, IgG, heparan sulphate proteoglycan, IgM, IgA, and C1q. Cell proliferation was assessed with Ki67. RESULTS: Distinct patterns of gastritis were seen in the disease states: diffuse, predominantly CD4+ infiltration in H. pylori, and focal-enhanced gastritis in Crohn's disease and autism, the latter distinguished by striking dominance of CD8+ cells, together with increased intraepithelial lymphocytes in surface, foveolar and glandular epithelium. Proliferation of foveolar epithelium was similarly increased in autism, Crohn's disease and H. pylori compared to controls. A striking finding, seen only in 20/25 autistic children, was colocalized deposition of IgG and C1q on the subepithelial basement membrane and the surface epithelium. CONCLUSIONS: These findings demonstrate a focal CD8-dominated gastritis in autistic children, with novel features. The lesion is distinct from the recently recognized focal gastritis of Crohn's disease, which is not CD8-dominated. As in the small intestine, there is epithelial deposition of IgG.

Isolated neonatal swallowing dysfunction: a case series and review of the literature.

Our purpose was to describe the natural history of isolated neonatal swallowing dysfunction (INSD). Nine infants with INSD are described. Eight presented within two weeks of birth. Symptoms included choking and cyanotic spells with feeds, recurrent aspiration, apnea, stridor, and vomiting. Three had nonspecific neurological abnormalities and were diagnosed later in life with underlying disorders (myotonic dystrophy, CHARGE association, velocardiofacial syndrome). All required tube feeding. Six tolerated nasogastric feedings and received a gastrostomy tube. Three failed nasogastric feeds and required jejunal feedings. The gastrostomy tube was removed in 7/9 at a mean age of 37 +/- 9 months. In conclusion, INSD has a good long-term prognosis. The presence of minor neurological abnormalities at presentation suggests another underlying disorder. Nasogastric feeding followed by a gastrostomy is recommended in those without gastroesophageal reflux. Jejunal feedings are necessary in some. While most improve over time, they may need nutritional support for 3 years or more.