Safety of Oats in Children with Celiac Disease: A Double-Blind, Randomized, Placebo-Controlled Trial.

OBJECTIVE: To evaluate the long-term validity and safety of pure oats in the treatment of children with celiac disease. STUDY DESIGN: This noninferiority clinical trial used a double-blind, placebo-controlled, crossover design extended over 15 months. Three hundred six children with a biopsy-proven diagnosis of celiac disease on a gluten-free diet for ≥2 years were randomly assigned to eat specifically prepared gluten-free food containing an age-dependent amount (15-40 g) of either placebo or purified nonreactive varieties of oats for 2 consecutive 6-month periods separated by washout standard gluten-free diet for 3 months. Clinical (body mass index, Gastrointestinal Symptoms Rating Scale score), serologic (IgA antitransglutaminase antibodies, and IgA anti-avenin antibodies), and intestinal permeability data were measured at baseline, and after 6, 9, and 15 months. Direct treatment effect was evaluated by a nonparametric approach using medians (95% CI) as summary statistic. RESULTS: After the exclusion of 129 patients who dropped out, the cohort included 177 children (79 in the oats-placebo and 98 in the placebo-oats group; median, 0.004; 95% CI, -0.0002 to 0.0089). Direct treatment effect was not statistically significant for clinical, serologic, and intestinal permeability variables (body mass index: median, -0.5; 95% CI, -0.12 to 0.00; Gastrointestinal Symptoms Rating Scale score: median, 0; 95% CI, -2.5 to 0.00; IgA antitransglutaminase antibodies: median, -0.02; 95% CI, -0.25 to 0.23; IgA anti-avenin antibodies: median, -0.0002; 95% CI, -0.0007 to 0.0003; intestinal permeability test: median, 0.004; 95% CI, -0.0002 to 0.0089). CONCLUSIONS: Pure nonreactive oat products are a safe dietary choice in the treatment of children with celiac disease. TRIAL REGISTRATION: ClinicalTrials.gov: NCT00808301.

Parallel synthesis and screening of peptide conjugates.

Peptide conjugates represent an emerging class of therapeutics. However, in contrast to that of small molecules and peptides, the discovery and optimization of peptide conjugates is low in throughput, resource intensive, time-consuming, and based on educated decisions rather than screening. A strategy for the parallel synthesis and screening of peptide conjugates is presented that (1) reduces variability in the conjugation steps; (2) provides a new method to rapidly and quantitatively measure conversion in crude conjugation mixtures; (3) introduces a purification step using an immobilized chemical scavenger that does not rely on protein-specific binding; and (4) is supported by robust analytical methods to characterize the large number of end products. Copper-free click chemistry is used as the chemoselective ligation method for conjugation and purification. The productivity in the generation and screening of peptide conjugates is significantly improved by applying this strategy as is demonstrated by the optimization of the anti-Angiopoietin-2 (Ang2) CovX-body, CVX-060, a peptide-antibody scaffold conjugate that has advanced in clinical trials for oncology indications.

Antiendomysium antibodies assay in the culture medium of intestinal mucosa: an accurate method for celiac disease diagnosis.

BACKGROUND: Celiac disease (CD) diagnosis is becoming more difficult as patients with no intestinal histology lesions may also be suffering from CD. AIM: To evaluate the diagnostic accuracy of antiendomysium (EmA) assay in the culture medium of intestinal biopsies for CD diagnosis. PATIENTS AND METHODS: The clinical charts of 418 patients with CD and 705 non-CD controls who had all undergone EmA assay in the culture medium were reviewed. RESULTS: EmA assay in the culture medium had a higher sensitivity (98 vs. 80%) and specificity (99 vs. 95%) than serum EmA/antibodies to tissue transglutaminase (anti-tTG) assay. All patients with CD who were tested as false-negatives for serum EmA and/or anti-tTG (32 adults and 39 children) carried the human leukocyte antigen alleles associated to CD. Furthermore, during the follow-up, four patients with negative-serum EmA/anti-tTG, normal villi architecture, and positive-EmAs in the culture medium, developed villous atrophy and underwent gluten-free diet with consequent resolution of the symptoms and complete intestinal histology recovery. CONCLUSION: EmA assay in the culture medium should be included in the diagnostic criteria for CD diagnosis in 'seronegative' patients.

A cytologic assay for diagnosis of food hypersensitivity in patients with irritable bowel syndrome.

BACKGROUND & AIMS: A percentage of patients with symptoms of irritable bowel syndrome (IBS) suffer from food hypersensitivity (FH) and improve on a food-elimination diet. No assays have satisfactory levels of sensitivity for identifying patients with FH. We evaluated the efficacy of an in vitro basophil activation assay in the diagnosis of FH in IBS-like patients. METHODS: Blood samples were collected from 120 consecutive patients diagnosed with IBS according to Rome II criteria. We analyzed in vitro activation of basophils by food allergens (based on levels of CD63 expression), as well as total and food-specific immunoglobulin (Ig)E levels in serum. Effects of elimination diets and double-blind food challenges were used as standards for FH diagnosis. RESULTS: Twenty-four of the patients (20%) had FH (cow's milk and/or wheat hypersensitivity); their symptom scores improved significantly when they were placed on an elimination diet. Patients with FH differed from other IBS patients in that they had a longer duration of clinical history, a history of FH as children, and an increased frequency of self-reported FH; they also had hypersensitivities to other antigens (eg, egg or soy). The basophil activation assay diagnosed FH with 86% sensitivity, 88% specificity, and 87% accuracy; this level of sensitivity was significantly higher than that of serum total IgE or food-specific IgE assays. CONCLUSIONS: A cytometric assay that quantifies basophils after stimulation with food antigens based on cell-surface expression of CD63 had high levels of sensitivity, specificity, and accuracy in diagnosing FH. This assay might be used to diagnose FH in patients with IBS-like symptoms.

Diagnostic efficacy of the ELISA test for the detection of deamidated anti-gliadin peptide antibodies in the diagnosis and monitoring of celiac disease.

BACKGROUND AND AIM: We evaluated the diagnostic performance of an ELISA test for anti-gliadin IgA and IgG antibodies, which uses synthetic deamidated gliadin peptides (anti-gliadin antibodies, AGAs) as coating; the results were compared with a test that uses extracted gliadin (AGAe). METHODS: The study was conducted on the sera of 144 patients suffering from celiac disease (CD), including 20 patients with IgA deficiency and 9 who were following a gluten-free diet (GFD), and 129 controls. RESULTS: In the 115 CD patients (without IgA deficiency), the sensitivity of AGAe IgA and IgG was 32.2 and 60.9%, whereas that of AGAs IgA and IgG was 59.1 and 72.2%. The specificity for AGAe IgA and IgG, and AGAs IgA and IgG was 93.8 and 89.9%, and 96.9% and 99.2%, respectively. Of the 20 patients with CD and IgA deficiency, 7 tested positive for AGAe IgG and 14 for AGAs IgG. The test using deamidated gliadin peptides performed better in terms of sensitivity and specificity than the AGA tests with extracted antigen. CONCLUSIONS: The very high specificity of the AGAs IgG test (99.2%) also suggests that patients who test positive with this assay require a thorough followup, even if the anti-tissue transglutaminase antibodies (anti-tTG) and anti-endomysial autoantibodies (EMA) assays are negative.

Food hypersensitivity as a cause of rectal bleeding in adults.

BACKGROUND & AIMS: Rectal bleeding and lymphonodular hyperplasia (LNH) in children can be caused by food hypersensitivity (FH). Our aim was to verify whether similar clinical and endoscopy presentations in adults can be due to FH. METHODS: Consecutive adult patients with rectal bleeding were enrolled. All underwent routine assays, colonoscopy, and histology study. RESULTS: Ten of 64 (15%) patients showed LNH as the unique sign at colonoscopy. An oligoantigenic diet resolved the rectal bleeding in 9 patients, and the reintroduction of several foods caused symptom reappearance. Double-blind placebo-controlled challenges with cow's milk and wheat protein confirmed the FH; symptoms reappeared 1-96 hours after the challenge. None of the patients were positive for IgE-mediated assays. In patients with LNH and FH, histology of the ileum and colon mucosa showed a higher number of lymphoid follicles and intraepithelial and lamina propria eosinophils compared with the other patients with rectal bleeding. CONCLUSIONS: Recurrent rectal bleeding can be caused by FH in adult patients. Endoscopic evidence of LNH characterizes these cases.

Clinical symptoms in celiac patients on a gluten-free diet.

OBJECTIVE: Persistent villous atrophy in patients with celiac disease (CD) on a gluten-free diet (GFD) is reported with increasing frequency. The aim of this study was to evaluate a possible association between persistent damage of the villi and "atypical" gastrointestinal symptoms in CD patients on a GFD. MATERIAL AND METHODS: Sixty-nine CD patients on a GFD were divided into two groups: Group A included 42 patients (6 M, 36 F, age range 17-62 years) undergoing esophagogastroduodenoscopies (EGDs) due to the presence of symptoms; Group B included 27 control patients (6 M, 21 F, age range 24-71 years) who were asymptomatic at the time of the study. Both groups underwent EGDs and a duodenal histologic study. RESULTS: Persistent endoscopic lesions were more frequent in Group A (30/42) than in Group B (12/27; p=0.01). Villous atrophy was significantly more frequent in Group A than in Group B: 85% versus 33% (p<0.0001; odds ratio (OR)=12; 95% CI 3.7-38.9). Gastrointestinal symptoms in the Group A patients were different from those present at CD diagnosis: anemia/diarrhea/weight loss in 6 cases; gastroesophageal reflux disease (GERD)-like symptoms in 12 cases; abdominal pain/constipation in 24 cases. In Group A there was no difference in gender distribution, age and duration of GFD between subjects with normal villi and those with persistent partial villous atrophy. Patients with persistent symptoms showed a higher intraepithelial eosinophil count (p=0.005) than the asymptomatic patients (p=0.01). CONCLUSIONS: Persistent intestinal villous atrophy in CD patients on a GFD is associated with gastrointestinal symptoms considered "atypical" for CD and not present at CD diagnosis.

Colonic lymphoid nodular hyperplasia in children: relationship to food hypersensitivity.

BACKGROUND & AIMS: The clinical significance of lymphoid nodular hyperplasia (LNH) of the lower gastrointestinal tract is unclear. The aim of this study was to define the frequency and clinical significance of LNH in pediatric patients undergoing colonoscopy. METHODS: Two hundred forty-five children (101 male, 144 female; median age, 8.5 years) for whom colonoscopy had been indicated were evaluated during a 3-year period. Apart from ileocolonoscopy with biopsy, all patients underwent routine biochemistry, serum total and specific IgE, and/or skin prick tests for food allergens. Patients with LNH underwent elimination diet and subsequent food challenges. RESULTS: LNH was observed in 73 of 245 (30%) consecutive colonoscopies. LNH was the only abnormal finding in 52 of the 73 cases (71%). In 43 of these 52 patients a diagnosis of cow's milk or multiple food hypersensitivity was made. Food allergy was significantly more common than in patients without LNH (83% vs 31%; P < .0001). The patients with LNH and food hypersensitivity presented hematochezia (P < .0001), elevated serum anti-beta-lactoglobulin IgG (P < .0001), anemia (P < .005), and failure to thrive (P < .03) more frequently than those without LNH. In the LNH patients histologic examination showed a higher number of lymphoid follicles throughout the colon and the terminal ileum and an increased number of lamina propria and intraepithelial eosinophils. CONCLUSIONS: The presence of LNH in the colon and/or terminal ileum is a frequent finding in symptomatic children undergoing colonoscopy. Unless associated with other specific endoscopic or histologic lesions, LNH is related to a condition of delayed-type food hypersensitivity.

A prospective, double-blind, placebo-controlled trial to establish a safe gluten threshold for patients with celiac disease.

BACKGROUND: Treatment of celiac disease (CD) is based on the avoidance of gluten-containing food. However, it is not known whether trace amounts of gluten are harmful to treated patients. OBJECTIVE: The objective was to establish the safety threshold of prolonged exposure to trace amounts of gluten (ie, contaminating gluten). DESIGN: This was a multicenter, double-blind, placebo-controlled, randomized trial in 49 adults with biopsy-proven CD who were being treated with a gluten-free diet (GFD) for > or =2 y. The background daily gluten intake was maintained at < 5 mg. After a baseline evaluation (t0), patients were assigned to ingest daily for 90 d a capsule containing 0, 10, or 50 mg gluten. Clinical, serologic, and histologic evaluations of the small intestine were performed at t0 and after the gluten microchallenge (t1). RESULTS: At t0, the median villous height/crypt depth (Vh/Cd) in the small-intestinal mucosa was significantly lower and the intraepithelial lymphocyte (IEL) count (x 100 enterocytes) significantly higher in the CD patients (Vh/Cd: 2.20; 95% CI: 2.11, 2.89; IEL: 27; 95% CI: 23, 34) than in 20 non-CD control subjects (Vh/Cd: 2.87; 95% CI: 2.50, 3.09; IEL: 22; 95% CI: 18, 24). One patient (challenged with 10 mg gluten) developed a clinical relapse. At t(1), the percentage change in Vh/Cd was 9% (95% CI: 3%, 15%) in the placebo group (n = 13), -1% (-18%, 68%) in the 10-mg group (n = 13), and -20% (-22%, -13%) in the 50-mg group (n = 13). No significant differences in the IEL count were found between the 3 groups. CONCLUSIONS: The ingestion of contaminating gluten should be kept lower than 50 mg/d in the treatment of CD.

Relationship between CARD15, SLC22A4/5, and DLG5 polymorphisms and early-onset inflammatory bowel diseases: an Italian multicentric study.

BACKGROUND: Inflammatory bowel disease (IBD) has been associated with several polymorphisms in genes likely involved in innate immune responses and integrity of epithelial mucosal barrier. A major role in adult Crohn's disease (CD) has been defined for 3 polymorphisms in the CARD15 gene, whereas variants in the SLC22A4, SLC22A5, and DLG5 genes could have a minor contribution to IBD susceptibility. METHODS: We analyzed a panel of 6 polymorphisms within these genes in 227 Italian early-onset IBD patients (134 CD, 93 ulcerative colitis [UC]; age at diagnosis

Anti-transglutaminase antibody assay of the culture medium of intestinal biopsy specimens can improve the accuracy of celiac disease diagnosis.

BACKGROUND: We measured anti-transglutaminase (anti-tTG) antibody in the culture medium of intestinal biopsy specimens from patients with suspected celiac disease (CD) and evaluated the relationship between antibody production and severity of intestinal mucosal damage. METHODS: We performed diagnostic testing for CD on 273 consecutive patients. In addition to routine histologic evaluation of duodenal biopsy specimens, we assayed anti-tTG antibodies in serum and in the culture medium of duodenal biopsy specimens. RESULTS: CD was diagnosed in 191 of the 273 patients. Sensitivity and specificity of the serum anti-endomysium (EmA) and anti-tTG assays were 83% and 85% and 99% and 95%, respectively, and both had 88% diagnostic accuracy. EmA and anti-tTG assayed in the culture medium had 98% sensitivity, 100% specificity, and 98% diagnostic accuracy (vs serum assays; P <0.0001). Twenty-nine CD patient specimens (16%) were negative for serum anti-tTG and EmA; for 24 of these patients, anti-tTG assay of the culture medium was positive. The CD patients whose biopsy specimens were positive for serum antibodies showed the following intestinal histologies: total villous atrophy, 35%; severe villous atrophy, 25%; mild atrophy, 25%; villi with no atrophy but with increased intraepithelial lymphocytes, 15%. None of the CD patients whose specimens were negative for serum antibodies showed total or severe villous atrophy; 77% had mild villous atrophy, and 23% had no villous atrophy but had increased intraepithelial lymphocyte counts. Mild villous atrophy was also seen in specimens from approximately 15% of patients without CD. CONCLUSION: Anti-tTG assay of the culture medium of biopsy specimens can improve the accuracy of CD diagnosis in patients negative for serum antibodies.

Gliadin, zonulin and gut permeability: Effects on celiac and non-celiac intestinal mucosa and intestinal cell lines.

OBJECTIVE: Little is known about the interaction of gliadin with intestinal epithelial cells and the mechanism(s) through which gliadin crosses the intestinal epithelial barrier. We investigated whether gliadin has any immediate effect on zonulin release and signaling. MATERIAL AND METHODS: Both ex vivo human small intestines and intestinal cell monolayers were exposed to gliadin, and zonulin release and changes in paracellular permeability were monitored in the presence and absence of zonulin antagonism. Zonulin binding, cytoskeletal rearrangement, and zonula occludens-1 (ZO-1) redistribution were evaluated by immunofluorescence microscopy. Tight junction occludin and ZO-1 gene expression was evaluated by real-time polymerase chain reaction (PCR). RESULTS: When exposed to gliadin, zonulin receptor-positive IEC6 and Caco2 cells released zonulin in the cell medium with subsequent zonulin binding to the cell surface, rearrangement of the cell cytoskeleton, loss of occludin-ZO1 protein-protein interaction, and increased monolayer permeability. Pretreatment with the zonulin antagonist FZI/0 blocked these changes without affecting zonulin release. When exposed to luminal gliadin, intestinal biopsies from celiac patients in remission expressed a sustained luminal zonulin release and increase in intestinal permeability that was blocked by FZI/0 pretreatment. Conversely, biopsies from non-celiac patients demonstrated a limited, transient zonulin release which was paralleled by an increase in intestinal permeability that never reached the level of permeability seen in celiac disease (CD) tissues. Chronic gliadin exposure caused down-regulation of both ZO-1 and occludin gene expression. CONCLUSIONS: Based on our results, we concluded that gliadin activates zonulin signaling irrespective of the genetic expression of autoimmunity, leading to increased intestinal permeability to macromolecules.

Multiple food hypersensitivity as a cause of refractory chronic constipation in adults.

Chronic constipation that is unresponsive to laxative treatment is a severe illness, but children unresponsive to laxatives have been successfully treated with an elimination diet. We report the first cases of refractory chronic constipation caused by food hypersensitivity in adults. Four patients with refractory constipation who were unresponsive to high doses of laxatives were put on an oligo-antigenic diet and underwent successive double-blind, placebo-controlled, food challenges (DBPFC). Routine laboratory tests, immunological assays, colonoscopy, esophago-gastroduodenoscopy and rectal and duodenal histology were performed. While on an elimination diet, bowel habits normalized in all patients and a DBPFC challenge triggered the reappearance of constipation. In comparison with another 13 patients with refractory constipation unresponsive to the elimination diet, observed over the same period, the patients with food-hypersensitivity-related constipation had the following characteristics: longer duration of illness (p < 0.03), lower body mass index (p < 0.03), higher frequency of self-reported food intolerance (p < 0.01), higher frequency of nocturnal abdominal pain and anal itching (p < 0.01). In patients with food hypersensitivity, hemoglobin concentrations and peripheral leukocytes were lower than those in controls (p < 0.03). The duodenal and rectal mucosa histology showed lymphocyte and eosinophil infiltration, and the duodenal villi were flattened in two cases. In adult patients, refractory chronic constipation may be caused by food hypersensitivity and an elimination diet is effective in these subjects.

Food intolerance and chronic constipation: manometry and histology study.

BACKGROUND: Chronic constipation in children can be caused by cows' milk intolerance (CMI), but its pathogenesis is unknown. AIMS: To evaluate the histology and manometry pattern in patients with food intolerance-related constipation. PATIENTS AND METHODS: Thirty-six consecutive children with chronic constipation were enrolled. All underwent an elimination diet and successive double-blind food challenge. All underwent rectal biopsy and anorectal manometry. RESULTS: A total of 14 patients were found to be suffering from CMI and three from multiple food intolerance. They had a normal stool frequency on elimination diet, whereas constipation recurred on food challenge. The patients with food intolerance showed a significantly higher frequency of erosions of the mucosa, and the number of intra-epithelial lymphocytes and eosinophils. The rectal mucous gel layer showed that the food-intolerant patients had a significantly lower thickness of mucus than the other subjects studied. Manometry showed a higher anal sphincter resting pressure and a lower critical volume in food intolerance patients than in the others suffering from constipation unrelated to food intolerance. Both histology and manometry abnormalities disappeared on the elimination diet. CONCLUSIONS: Food intolerance-related constipation is characterized by proctitis. Increased anal resting pressure and a reduced mucous gel layer can be considered to be contributory factors in the pathogenesis of constipation.

Chronic constipation and food intolerance: a model of proctitis causing constipation.

OBJECTIVE: Chronic constipation in children can be linked to cow's milk intolerance (CMI) but the existence of a food intolerance-dependent proctitis is still debated. The aim of this study was to evaluate the histologic data in patients with food intolerance-related constipation. MATERIAL AND METHODS: Fifty-two consecutive patients (22 M, median age 4 years) with chronic constipation unresponsive to common treatment were enrolled. All patients were put on a cow's milk-free diet for 4 weeks and those uncured on this diet underwent a subsequent 4-week period of oligoantigenic diet. In the patients cured on elimination diet, a subsequent double-blind food challenge was performed to confirm the diagnosis of food intolerance. At entry to the study, routine hemato-chemical and immunologic assays, rectoscopy, and histologic study of the rectal mucosa were performed. In the patients cured on elimination diet, rectal histology was repeated when they were cured. RESULTS: Twenty-four patients were found to be suffering from CMI and 6 from multiple food intolerance. These patients had a normal stool frequency on elimination diet, while constipation reappeared on food challenge. The condition of the remaining 22 patients did not improve on elimination diet. The patients with food intolerance showed a significantly higher frequency of erosions of the mucosa, number of intraepithelial lymphocytes and eosinophils, and number of eosinophils in the lamina propria. Study of the rectal mucus gel layer showed that the food-intolerant patients had a significantly lower thickness than the other subjects studied. In the food intolerant patients, histologic abnormalities disappeared on elimination diet, when the patients were well. CONCLUSIONS: Food intolerance-related constipation is characterized by proctitis with eosinophil infiltrate of the rectal mucosa. A reduced mucus gel layer can be considered a contributory factor in the pathogenesis of the constipation.