Enrico Valletta

In Celiac Disease, a Subset of Autoantibodies against Transglutaminase Binds Toll-Like Receptor 4 and Induces Activation of Monocytes

Background Celiac disease is a small intestine inflammatory disorder with multiple organ involvement, sustained by an inappropriate immune response to dietary gluten. Anti-transglutaminase antibodies are a typical serological marker in patients with active disease, and may disappear during a gluten-free diet treatment. Involvement of infectious agents and innate immunity has been suggested but never proven. Molecular mimicry is one of the mechanisms that links infection and autoimmunity. Methods and Findings In our attempt to clarify the pathogenesis of celiac disease, we screened a random peptide library with pooled sera of patients affected by active disease after a pre-screening with the sera of the same patients on a gluten-free diet. We identified a peptide recognized by serum immunoglobulins of patients with active disease, but not by those of patients on a gluten-free diet. This peptide shares homology with the rotavirus major neutralizing protein VP-7 and with the self-antigens tissue transglutaminase, human heat shock protein 60, desmoglein 1, and Toll-like receptor 4. We show that antibodies against the peptide affinity-purified from the sera of patients with active disease recognize the viral product and self-antigens in ELISA and Western blot. These antibodies were able to induce increased epithelial cell permeability evaluated by transepithelial flux of [3H] mannitol in the T84 human intestinal epithelial cell line. Finally, the purified antibodies induced monocyte activation upon binding Toll-like receptor 4, evaluated both by surface expression of activation markers and by production of pro-inflammatory cytokines. Conclusions Our findings show that in active celiac disease, a subset of anti-transglutaminase IgA antibodies recognize the viral protein VP-7, suggesting a possible involvement of rotavirus infection in the pathogenesis of the disease, through a mechanism of molecular mimicry. Moreover, such antibodies recognize self-antigens and are functionally active, able to increase intestinal permeability and induce monocyte activation. We therefore provide evidence for the involvement of innate immunity in the pathogenesis of celiac disease through a previously unknown mechanism of engagement of Toll-like receptor 4.

Peak expiratory flow variation and bronchial hyperresponsiveness in asthmatic children during periods of antigen avoidance and reexposure

Changes of diurnal variation of peak expiratory flow rate (%PEF variation) and their relationship with bronchial hyperresponsiveness (BHR) to methacholine (PC20) were evaluated in 12 children with mild‐to‐moderate asthma and house‐dust mite allergy, during successive periods of stay in a mite‐free environment at high altitude (1756 m) and at their home at sea level. The children remained at the high altitude from October until the end of December; then they spent a 3‐week period at home and returned to high altitude residence in January. PEF was measured daily, in the morning and in the evening, during the 3 months’ stay at high altitude and then for 10 days after the return in January. PC20 was assessed in 8/12 children, once a month from October to December, and at the return in January. Mean absolute PEF values did not change significantly throughout the study. From October to December, patients showed a significant decrease of mean %PEF variation (P= 0.04), while PC20 showed an increase (P= 0.05). After the 3 weeks at home, both %PEF variation (P= 0.03) and PC20 (P= 0.05) significantly worsened. The correlation between PC20 values and mean %PEF variation in the 2 days before and after each methacholine test was r=−0.63 (P= 0.001). Our data suggest that there is a beneficial effect of a prolonged stay in a mite‐free environment, on both PEF variability and BHR, also in asthmatic children with good pulmonary function. PEF variability and bronchial responsiveness to methacholine were significantly correlated also for small changes of the two variables.

FEF25–75 as a Marker of Airway Obstruction in Asthmatic Children During Reduced Mite Exposure at High Altitude

Sensitivity of forced expiratory flow between 25% and 75% of the vital capacity (FEF25-75) in detecting airway obstruction was investigated in 14 children with mild-moderate asthma, allergic to house dust mites, while at high altitude (1756 m). Forced vital capacity (FVC), forced expiratory volume in 1 sec (FEV1), FEF25-75, and peak expiratory flow (PEF) were measured every 2 weeks for 12 weeks (total, 84 measurements). The presence or absence of wheezing at the chest auscultation was ascertained before each test. During the study period, a significant improvement of both mean (SD) FEF25-75 [61 (12)% vs. 68 (11)% of the predicted value, p = 0.005] and PEF [95 (16)% vs. 103 (13)%, p = 0.002] was observed. FEV1 changed only marginally [82 (7)% vs. 86 (6)%, p = 0.05]. Wheezing was present on 12/84 occasions. Wheezing was associated with abnormal FEF25-75 values on most occasions but not with abnormal FEV1 or PEF. FEF25-75 was decreased on 51% of days in which wheezing was absent. FEV1 and PEF were, respectively, normal in 69% (p < 0.0001) and 92% (p < 0.0001) of measurements in which FEF25-75 was abnormal. These results suggest that FEF25-75 may be considered a good indicator of airflow obstruction and a sensitive marker of respiratory improvement in asthmatic children during reduced antigen exposure.

Screening of coeliac disease in north Italian children with type 1 diabetes: limited usefulness of HLA-DQ typing

Aim: To determine the contribution of HLA‐DQA1* and HLA‐DQB1* genes to the risk of coeliac disease (CD) in a cohort of children with type 1 diabetes mellitus (T1DM) from northern Italy. Methods: Three hundred and fifty‐seven children with T1DM, attending the Childhood Diabetes Unit of the University of Verona, have been regularly tested for serum IgA endomysial antibodies (EMA). All patients with positive EMA underwent small bowel biopsy to confirm the diagnosis of CD. HLA typing was performed in subjects with T1DM and CD, and in a control group of 79 EMA‐negative patients with T1DM. Results: Of the 357 patients tested, 25 (7%) had CD. The frequency of HLA‐ DQA1*0501‐DQB1*0201 (T1DM + CD 68% vs T1DM 62%) and of DQA1*0301‐DQB1*0302 (T1DM + CD 40% vs T1DM 35%) haplotypes, between T1DM patients with and without CD, was statistically comparable. A trend towards a reduction of the risk of CD (p= 0.055, OR: 0.22, CI 0.05: 1.04) was observed in patients with T1DM (28% vs T1DM + CD 2%) who did not carry either the HLA‐DQA1*0501‐DQB1*0201 or the DQA1*0301‐DQB1*0302 haplotype.

Effects of allergen exposure-avoidance on inflammation in asthmatic children

Inflammation of the airways has long been known to be the classic pathologic feature of asthmatics who have died in status asthmaticus (1, 2). However, it has recently been appreciated, from studies of bronchial lavage and bronchial biopsies, that inflammatory changes are present in even mildly symptomatic adult patients (3). Experimental evidence has linked the induction of bronchial hyperresponsiveness (BHR) to the presence of inflammation (4-7). The recognition of allergic airways inflammation, as a cause of both transient and persistent airway hyperresponsiveness, has led to increased appreciation of importance of atopy in the pathogenesis of asthma (4-8). Recently, it has been shown that eosinophil counts in lavage fluid are correlated with histamine reactivity in children (9) as well as in adults (3). Furthermore, ultrastructural examination of the airways of two asthmatic children undergoing open lung biopsy during clinical remission showed features similar to lung tissue from two children who had died in status asthmaticus with the exception of a larger number of submucosal eosinophils and more extensivedenudation ofthe epithelium in fatal asthma (10). Evidence from these findings suggest that airways-inflammation may play a role also in asthmatic children. This hypothesis can be further supported by studies which have shown that allergen avoidance can improve BHR and asthma symptoms both in adults (11-12) and children (13-15) with asthma. Because of the importance of better understanding the correlation of allergen exposure with BHR and inflammation we evaluated changes in BHR and serum markers of inflammation when allergic asthmatic children sensitive to house dust mites moved from an environment rich in allergens (sea level) to an environment free from mites at a high altitude (Istituto Pi0 XII, Misurina 1756111) (16-17) and back again to sea level. The migration of asthmatic children from sea level to the alpine environment and back again, offers a natural allergen exposureavoidance-exposure challenge model.

Oral ondansetron versus domperidone for symptomatic treatment of vomiting during acute gastroenteritis in children: multicentre randomized controlled trial

BackgroundVomiting in children with acute gastroenteritis (AG) is not only a direct cause of fluid loss but it is also a major factor of failure of oral rehydration therapy (ORT). Physicians who provide care to paediatric patients in the emergency department (ED) usually prescribe intravenous fluid therapy (IVT) for mild or moderate dehydration when vomiting is the major symptom. Thus, effective symptomatic treatment of vomiting would lead to an important reduction in the use of IVT and, consequently, of the duration of hospital stay and of frequency of hospital admission. Available evidence on symptomatic treatment of vomiting shows the efficacy of the most recently registered molecule (ondansetron) but a proper evaluation of antiemetics drugs largely used in clinical practice, such as domperidone, is lacking.ObjectivesTo compare the efficacy of ondansetron and domperidone for the symptomatic treatment of vomiting in children with AG who have failed ORT.Methods/DesignMulticentre, double-blind randomized controlled trial conducted in paediatric EDs. Children aged from 1 to 6 years who vomiting, with a presumptive clinical diagnosis of AG, and without severe dehydration will be included. After the failure of a initial ORS administration in ED, eligible children will be randomized to receive: 1) ondansetron syrup (0,15 mg/Kg of body weight); 2) domperidone syrup (0,5 mg/Kg of body weight); 3) placebo. The main study outcome will be the percentage of patients needing nasogastric or IVT after symptomatic oral treatment failure, defined as vomiting or fluid refusal after a second attempt of ORT. Data relative to study outcomes will be collected at 30 minute intervals for a minimum of 6 hours. A telephone follow up call will be made 48 hours after discharge. A total number of 540 children (i.e. 180 patients in each arm) will be enrolled.DiscussionThe trial results would provide evidence on the efficacy of domperidone, which is largely used in clinical practice despite the lack of proper evaluation and a controversial safety profile, as compared to ondansetron, which is not yet authorized in Italy despite evidence supporting its efficacy in treating vomiting. The trial results would contribute to a reduction in the use of IVT and, consequently, in hospital admissions in children with AG. The design of this RCT, which closely reflect current clinical practice in EDs, will allow immediate transferability of results.Trial RegistrationClinicalTrials.gov: NCT01257672

Early bronchopulmonary involvement in Crohn disease: a case report

BackgroundBronchopulmonary manifestations of Crohn disease have been rarely described in children, including both subclinical pulmonary involvement and severe lung disease.Case presentationA 6.5-year-old girl is described with early recurrent bronchopulmonary symptoms both at presentation and in the quiescent phase of Crohn disease. Pulmonary function tests (lung volumes and flows, bronchial reactivity and carbon monoxide diffusing capacity) were normal. Bronchoalveolar cytology showed increased (30%) lymphocyte counts and bronchial biopsy revealed thickening of basal membrane and active chronic inflammation.ConclusionsClinical and histological findings in our young patient suggest involvement of both distal and central airways in an early phase of lung disease. The pathogenesis of Crohn disease-associated lung disorders is discussed with reference to the available literature. A low threshold for pulmonary evaluation seems to be advisable in all children with CD.

Measurement of NADPH oxidase activity in detergent lysates of human and mouse macrophage monolayers

An assay to measure NADPH oxidase activity in detergent lysates of macrophage monolayers is described. The addition of a reaction mixture containing appropriate concentrations of disrupting detergents, NADPH as oxidase substrate and cytochrome c as electron acceptor, to macrophages monolayers permits the reliable detection of a superoxide dismutase-sensitive NADPH-dependent cytochrome c reductive activity. This activity is strictly substrate dependent and NADH could not substitute for NADPH. The NADPH-dependent superoxide anion-forming activity (NADPH oxidase) was investigated in different populations of human and mouse macrophages. NADPH oxidase was activated by stimulation of macrophages with phorbol-myristate acetate and activity levels correlated with ability of intact cells to produce superoxide anion. The optimal conditions for assay of NADPH oxidase were investigated and the assay was used to measure the kinetic properties of the NADPH oxidase. The assay permits investigations of the enzymatic basis of oxidative metabolism in macrophages cultivated as adherent cells without any requirements for recovery of the cells in suspension and subcellular fractionation.

Normal or defective immune response to Hepatitis B vaccine in patients with diabetes and celiac disease

A defective production of protective levels of antibodies to Hepatitis B (HB) vaccine is reported to occur in 4–10% of healthy subjects and a correlation with the presence of specific human leukocyte antigen (HLA) molecules, including DQ2, which also confers genetic predisposition to celiac disease (CD) and type I diabetes mellitus (T1DM), has been suggested. The aim of this study was to analyze the serological response to HB vaccine and measles-containing vaccines in 69 diabetic patients (T1DM), 42 patients with celiac disease (CD) and 79 healthy control subjects (CT). The median interval between the third dose of HB vaccine and serum collection was 6.8, 3.5, and 4.7 years for T1DM, CD and CT groups, respectively. 50/69 (72%) T1DM patients, 32/42 (76%) CD patients and 61/79 (77%) CT subjects showed protective anti-HBs antibodies after vaccination, with no statistically significant difference. On the contrary, a lower statistically significant difference was found in the mean HBsAb level of T1DM subjects when compared with the other two groups. No correlation between HLA DQ2 expression in T1DM and vaccine response was detected. The comparison of serological response to measles after vaccination also showed no statistically significant differences in the three groups. Contrasting results between these data and those reported in the literature might be due to differences in the time intervals between vaccination and testing. Prospective studies in pathological and healthy groups with the same age at HBV vaccination and with the same time interval for blood sample collection to determine antibody titers are necessary in order to provide more conclusive data.

Incomplete gastric metaplasia in children with insulin-dependent diabetes mellitus and celiac disease. An ultrastructural study

BackgroundThe association of insulin-dependent diabetes mellitus (IDDM) and celiac disease (CD) has been widely reported in children but the relationship between the two conditions is incompletely understood. Moreover, specific studies on intestinal biopsies of patients with the association of the two diseases are still lacking.MethodsWe studied the ultrastructure of the duodenal mucosa in 12 patients with both IDDM and CD.ResultsAll patients had either total or partial atrophy of duodenal mucosa. In seven subjects, an accumulation of electrondense granules in the apical cytoplasm of groups of enterocytes was found. In four of them, a double population of granules existed (mean diameter: 400-800 nm and 100-200 nm respectively) showing a biphasic pattern. In the other three patients, only smaller granules (100- 200 nm) were found in the enterocytes.ConclusionsThe present work suggests that patients with IDDM/CD may represent a subgroup in the context of the CD population. Intestinal biopsies of such individuals often show accumulation of electrondense granules in the apical cytoplasm of enterocytes that can be interpreted as incomplete gastric metaplasia.