Antonio Tiberti

Usefulness of the organ culture system when villous height/crypt depth ratio, intraepithelial lymphocyte count, or serum antibody tests are not diagnostic for celiac disease

The existence of mild forms of celiac disease (CD) can make the histology-based diagnosis difficult to reach. Since anti-endomysium (EMA) and anti-tissue transglutaminase (anti-tTG) are detectable in culture supernatants of duodenal biopsies from CD patients, our aim was to assess if this system can support the histology in the diagnostic work-up. A total of 559 suspected CD patients underwent serum EMA/anti-tTG detection, upper endoscopy with duodenal biopsy sampling, histologic analysis, and organ culture to detect EMA/anti-tTG in supernatants. A subgroup of 30 patients with organ culture positive results were put on a gluten-free diet (GFD). Their gluten-dependency was evaluated by the psychological general well-being and beck depression inventory indexes. Statistical analysis was performed by Cohen k inter-test, Friedman test, and Dunn multiple comparison. Two hundred forty-one out of 559 (43.1%) patients showed intestinal villous atrophy, whereas serum and organ culture EMA/anti-tTG were positive in 293/559 (52.4%) and 334/559 (59.7%) patients, respectively. The strength of agreement resulted good for serology vs histology (k = 0.730), good for organ culture vs histology (k = 0.662), and very good for serology vs organ culture (k = 0.852). After 12 months of GFD, psychological general well-being index significantly increased, and beck depression inventory index significantly decreased (P < 0.001 for each one). Data highlight the organ culture system as a useful tool to assist the histology in diagnosing CD, mainly in cases without villous atrophy or in seronegative patients. The marked improvement in quality of life after a GFD further supports the reliability of this system in diagnosing CD.

Weaknesses of histological analysis in celiac disease diagnosis: new possible scenarios

Abstract Introduction. Celiac disease (CD) is an autoimmune disorder triggered by the ingestion of gluten. Serology and organ culture system can support CD diagnosis, despite histology being the gold standard. Aim. We wanted to test the uniformity of application of Marsh–Oberhuber criteria by five different histologists. We also compared histological and serological data with cultural results to consider new possible strategies in CD diagnosis. Methods. We studied 114 patients, who were divided in two groups. Group A was composed of 66 patients on a gluten-containing diet, with gluten-related signs and symptoms, showing positive serological anti-endomysial antibodies (EMA) and anti-tissue transglutaminase (anti- tTG). Group B was composed of 48 disease-control patients, presenting serological EMA and anti-tTG negative results. All patients studied underwent esophagogastroduodenoscopy with duodenal biopsy and duodenal mucosa organ culture. All histological samples were evaluated by five different histologists according to an appropriate questionnaire following Marsh–Oberhuber classification. Cohen κ inter-test was used for evaluating the agreement between histologists regarding group A. Results. Strength of agreement was fair/moderate for villous:crypt ratio, moderate/good for villous height and crypt depth, and poor for intraepithelial lymphocytosis. Patients belonging to group A presented positive serological as well as cultural results in 100% of cases. None of the patients belonging to group B presented serological or cultural positive results. Discussion. Our study stresses the limits of histological interpretation due to the lack of uniformity in the use of Marsh–Oberhuber classification. These findings could cast doubt on the role of histology as CD gold standard and could open a debate on the most appropriate CD diagnostic procedure.

Intestinal, Systemic, and Oral Gluten-related Alterations in Patients With Nonceliac Gluten Sensitivity

Background: Nonceliac gluten sensitivity (NCGS) is an emergent condition, the framework of which is yet unclear, whereas the diagnosis is suggested only by gluten-dependent symptoms after excluding wheat allergy and celiac disease (CD). Our goal was to highlight intestinal, systemic, and oral alterations to clarify the NCGS pathogenesis and identify new diagnostic tools. Study: A total of 60 NCGS patients, 20 untreated CD, 20 treated CD, and 20 healthy volunteers were recruited. The differential diagnosis among gluten-related disorders was performed by serological, allergy, and histologic tools. NCGS patients were also subjected to antigliadin antibody (AGA) detection and HLA typing. All participants underwent an oral mucosa patch test for gluten (GOMPT), whereas an oral provocation test (OPT) for gluten was performed in 26 NCGS patients. Results: About 6/60 (10%) NCGS patients showed IgG AGA-positive results, whereas 45/60 (75%) patients carried HLA-DQ2 and/or HLA-DQ8 genes. GOMPT showed positive results in 45/60 (75%) NCGS patients, 3/20 (15%) untreated CD patients, 5/20 (25%) treated CD patients, and in no healthy volunteers. No significant difference was found between the severity of symptoms reported by NCGS patients subjected to OPT with gluten-containing croissants and those who underwent OPT with gluten-free croissants. Conclusions: GOMPT seems to be a specific tool for NCGS diagnosis, although further investigations are needed to overcome limits due to the small population studied and to contextualize GOMPT false-positive results.

A New Formulation of Oral Viscous Budesonide in Treating of Paediatric Eosinophilic Oesophagitis: a Pilot Study

Objectives: Oral viscous budesonide is a recent therapeutic option for eosinophilic oesophagitis (EoE) compared with dietary restriction and inhaled steroids. This single-centre, open-label, not blinded study aims to evaluate the efficacy and safety of a new, preprepared oral viscous budesonide suspension (PVB) in children and adolescents with EoE. Methods: We treated 36 children with PVB (29 boys; median age 12 years) with EoE diagnosed according to European Society for Paediatric Gastroenterology Hepatology and Nutrition guidelines. Patients <150 and >150 cm height received 2 and 4 mg PVB daily, respectively, for 12 weeks. Upper gastrointestinal endoscopy was performed at baseline, after 12 weeks of therapy and 24 weeks after the end of therapy. Baseline and post-treatment scores were calculated for symptoms, endoscopy, and histology. Serum cortisol was performed at baseline, 12, and 36 weeks. Results: At the end of PVB trial, endoscopy showed macroscopic remission in 32 patients (88.9%), whereas at histology median pre- and post-treatment peak eosinophil count/high power field (HPF) markedly decreased from 42.2 (range: 15–100) to 2.9 (range: 0–30); moreover, mean symptom and histology scores impressively improved compared with baseline (P < 0.01). At 24 weeks after the end of PVB therapy, endoscopy showed oesophageal relapse in 21 patients (58.3%), whereas 15 (41.7%) were still in remission. Seven children (19.4%) with positive multichannel intraluminal impedance-pH were treated also with proton pump inhibitors. No significant difference between pre-/post-treatment morning cortisol levels occurred. Conclusions: The new PVB suspension presented in the present study is effective and safe for treating children with proven EoE. Larger placebo-controlled clinical trials would provide more information about dosing, efficacy, and long-term safety of this formulation, specifically designed for the oesophagus.

First Identification of Biallelic Inherited DUOX2 Inactivating Mutations as a Cause of Very Early Onset Inflammatory Bowel Disease

3 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116 117 118 119 120 Dear Editors: By catalyzing generation of hydrogen peroxide (H202) in enterocytes, DUOX2 participates in innate defense against intestinal microbes. One monoallelic exonic variant of DUOX2 was recently found in 15 out of 19 Crohn disease-affected patients in a large Ashkenazi family. Herein, we report the first identification of biallelic, inherited mutations of DUOX2 as a Mendelian cause of very early onset inflammatory bowel disease (VEO-IBD) in a boy born from unrelated parents of Italian origin. The patient developed pancolitis with bloody diarrhea at the age of 3. Histology showed dense lymphoplasmocytic and eosinophilic infiltrates, mucin depletion, architectural distortion and crypt abscesses without epitheloid granuloma (Figure 1A). No extragastrointestinal manifestation was reported. Mesalamine and systemic steroids induced symptom resolution, but clinical relapse occurred with steroid withdrawal. Thiopurines allowed complete remission but were discontinued after 1 month because of increased pancreatic enzymes. He is currently in remission under mesalamine. A known molecular defect was excluded using targeted sequencing of 68 VEO-IBD–associated genes. Whole exome sequencing performed on patient and parents identified compound heterozygous missense variants in DUOX2, both confirmed by Sanger sequencing (Figure 1B, C). The first variant substituted proline by serine (NM_014080.4:c.1825G>A; Minor Allele Frequency: 2.7 10 4 P609S,) within the first transmembrane domain (Figure 1D). The second missense variant replaced arginine with histidine (NM_014080.4:c.857G>A; R286H) in the peroxidase homology domain of DUOX2 (Figure 1D). R286H was absent from both public and Imagine databases. Multiple sequence alignment showed that mutations affected evolutionarily conserved residues (Supplementary Figure 1A), and both amino acid changes were predicted to be damaging. Membrane targeting and catalytic activity of the DUOX enzyme complexes requires heterodimerization of DUOX2 with DUOXA2. When expressing HA-tagged WT DUOX2 or mutants in H661-DUOXA2 cells, comparable amounts of WT DUOX2, R286H and P609S proteins were detected in total cell lysates (Supplementary Figure 1B). In contrast, flow cytometry revealed reduced surface expression of P609S compared to WT DUOX2, whereas R286H was undetectable (Figure 1E). Overall decreased surface expression of DUOX2 was observed when the mutants were expressed at 1:1 ratio (Figure 1E), suggesting that DUOX2

Gastroesophageal reflux in young children and adolescents: Is there a relation between symptom severity and esophageal histological grade?

Background: The pediatric literature about the correlation between symptoms and histological lesions in patients investigated for gastroesophageal reflux disease is scarce and inconclusive. The primary aim of the present study was to assess the relation between the complained symptom severity and the esophageal histological grade, through the use of validated and reliable scores. Methods: All children ages between 2 and 17 years referred to perform upper gastrointestinal endoscopy because of gastroesophageal reflux disease symptoms were asked to complete the Pediatric Gastroesophageal Symptom and Quality of Life validated questionnaire, investigating the main symptoms complained and their impact on daily life and school activities. Esophageal mucosal samples taken during the procedure were analyzed and scored according to the Yerian‐Fiocca classification. Results: A total of 164 children were included in the study. No significant association was found between symptomatic score and histological score (rs: 0.05, P: 0.49). Even when focusing only on adolescents with heartburn or chest pain, no correlation between symptom severity and esophageal lesions was found (rs: −0.18, P: 0.264). Intercellular space diameter values did not mirror symptom severity. Conclusions: The main finding of this study on children with reflux symptoms is the lack of correlation between symptom severity and esophageal histological grade. The magnitude of intercellular spaces was found not to be related with the clinical score as well.

One-step immunochromatographic visual assay for anti-transglutaminase detection in organ culture system: An easy and prompt method to simplify the in vitro diagnosis of celiac disease

Anti‐tissue transglutaminase (anti‐tTG) and endomysium antibodies (EMA) are detectable in duodenal culture media of celiac disease (CD) patients. To improve the management of this organ culture system, we evaluated the anti‐tTG occurrence by immunochromatographic assay (ICA).

RIP3 AND pMLKL promote necroptosis-induced inflammation and alter membrane permeability in intestinal epithelial cells

BACKGROUND Necroptosis is an inflammatory form of programmed cell death requiring receptor-interacting protein kinase 3 (RIP3) and mixed lineage kinase domain-like protein (MLKL). AIMS The aim of this study is to examine in depth in vitro and ex vivo the contribution of necroptosis to intestinal inflammation. METHODS In vitro: we used an intestinal cell line, HCT116RIP3, produced in our laboratory and overexpressing RIP3. Ex vivo: intestinal mucosal biopsies were taken from patients with inflammatory bowel disease (IBD) (20 with Crohns disease; 20 with ulcerative colitis) and from 20 controls. RESULTS RIP3-induced necroptosis triggers MLKL activation, increases cytokine/alarmin expression (IL-8, IL-1β, IL-33, HMGB1), NF-kBp65 translocation and NALP3 inflammasome assembly. It also affects membrane permeability by altering cell-cell junctional proteins (E-cadherin, Occludin, Zonulin-1). Targeting necroptosis through Necrostatin-1 significantly reduces intestinal inflammation in vitro and in cultured intestinal explants from IBD. CONCLUSION We show for the first time in vitro and ex vivo that RIP3-driven necroptosis seriously affects intestinal inflammation by increasing pMLKL, activating different cytokines and alarmins, and altering epithelial permeability. The inhibition of necroptosis causes a significant decrease of all these effects. These data strongly support the view that targeting necroptosis may represent a promising new option for the treatment of inflammatory enteropathies.