Carlo Tolone

Divergence of gut permeability and mucosal immune gene expression in two gluten-associated conditions: celiac disease and gluten sensitivity

BackgroundCeliac disease (CD) is an autoimmune enteropathy triggered by the ingestion of gluten. Gluten-sensitive individuals (GS) cannot tolerate gluten and may develop gastrointestinal symptoms similar to those in CD, but the overall clinical picture is generally less severe and is not accompanied by the concurrence of tissue transglutaminase autoantibodies or autoimmune comorbidities. By studying and comparing mucosal expression of genes associated with intestinal barrier function, as well as innate and adaptive immunity in CD compared with GS, we sought to better understand the similarities and differences between these two gluten-associated disorders.MethodsCD, GS and healthy, gluten-tolerant individuals were enrolled in this study. Intestinal permeability was evaluated using a lactulose and mannitol probe, and mucosal biopsy specimens were collected to study the expression of genes involved in barrier function and immunity.ResultsUnlike CD, GS is not associated with increased intestinal permeability. In fact, this was significantly reduced in GS compared with controls (P = 0.0308), paralleled by significantly increased expression of claudin (CLDN) 4 (P = 0.0286). Relative to controls, adaptive immunity markers interleukin (IL)-6 (P = 0.0124) and IL-21 (P = 0.0572) were expressed at higher levels in CD but not in GS, while expression of the innate immunity marker Toll-like receptor (TLR) 2 was increased in GS but not in CD (P = 0.0295). Finally, expression of the T-regulatory cell marker FOXP3 was significantly reduced in GS relative to controls (P = 0.0325) and CD patients (P = 0.0293).ConclusionsThis study shows that the two gluten-associated disorders, CD and GS, are different clinical entities, and it contributes to the characterization of GS as a condition associated with prevalent gluten-induced activation of innate, rather than adaptive, immune responses in the absence of detectable changes in mucosal barrier function.

The cannabinoid receptor type 2 Q63R variant increases the risk of celiac disease: implication for a novel molecular biomarker and future therapeutic intervention.

Celiac disease (CD) is a chronic inflammatory disease of the small bowel that occurs with the ingestion of gluten, found in several grains products. Although HLA-DQ2 variant is required for the gluten-derived peptide gliadin presentation by antigen-presenting cells to T-cells, non-HLA genetic factors account for the majority of heritable risk. Several genome-wide association studies have identified susceptibility loci for CD on chromosome 1. Cells of the immune system express the cannabinoid receptor type 2 (CB2), a plasma-membrane receptor activated by both endogenous and exogenous cannabinoids. Consistent data evidence that CB2 is linked to a variety of immune functional events and that, in the course of an inflammatory process, an increased number of receptors becomes available for activation. The cannabinoid receptor type 2 gene (CNR2; GeneID1269) maps on 1p36.11. In order to investigate the possible involvement of CB2 in CD establishment, immunohistochemistry toward CB2 receptor and CD4+ cells in small bowel biopsies from celiac children and association analysis, through TaqMan assay, of a CNR2 common missense variant, rs35761398 (CAA/CGG), resulting in the aminoacidic substitution of Glutamine at codon 63 with Arginine (Q63R), in a cohort of 327 South Italian children have been performed. We observed in this study that CB2 is up-regulated in CD small bowel biopsies and CNR2 rs35761398 is significantly associated with CD (χ(2) = 37.064; d.f. 1; p = 1.14 × 10(-9)). Our findings suggest a role of CB2 in CD. The Q63R variant, increasing more than six-fold the risk for CD susceptibility, might eventually represent a novel molecular biomarker for CD risk stratification. Indeed, we provide here further evidence that CB2 receptor plays a critical role in autoimmunity susceptibility and indicates that it represents a molecular target to pharmacologically modulate the immune components in CD.

Evaluation of Helicobacter Pylori eradication in pediatric patients by triple therapy plus lactoferrin and probiotics compared to triple therapy alone

BackgroundTo evaluate whether the addition of a probiotic could improve Helicobacter pylori (H.P.) eradication rates and reduce the side effects of treatment in children.MethodsBetween July 2008 and July 2011 all patients with a clinical, laboratory and endoscopic diagnosis of H.P. positive gastritis referred to our Unit were included in the study. Patients suffering from allergy to any of drugs used in the study, with previous attempts to eradicate H.P. and those who received antibiotics, PPIs or probiotics within 4 weeks were excluded from the present study. Patients were randomized into two therapy regimens (group A and B): both groups received standard triple treatment (omeprazole, amoxicillin and clarithromycin) while only group B patients were also given a probiotic (Probinul - Cadigroup). Patients compliance was evaluated at the end of the treatment. Successful eradication was defined as a negative 13 C-urea breath test (C13-ubt) result four weeks after therapy discontinuation.ResultsA total of 68 histopathologically proven H.P.-infection children (32 male and 36 females) were included in the study. All of the patients in both groups used more than 90% of the therapies and no patients were lost at follow up. All side effects were selflimiting and disappeared once the therapy was terminated. Epigastric pain was observed in 6 (17.6%) group A vs 2 (5.8%) group B patients (P<0.05), nausea in 3 (8.8%) group A vs 1 (2.9%) group B patients (P<0.05); vomiting and diarrhea were observed in 2(5.8%) and 8 (23.5%) group A patients, respectively and never in group B (P<0.05). There was no significant difference between the two groups in terms of constipation (5.8% in group A and B). Four weeks after the completion of therapy, 56/68 patients (82.3%) tested negative for H.P. on C13-ubt. H.P. was eradicated in 26 patients (76.4%) in group A and in 30 patients (88.2%) in group B. There was no significantly difference in the rate of H.P. eradication between group A and group B (p=0.1), although the success rate for H.P. eradication was higher in group B than in group A.ConclusionThe addition of a probiotic formula to triple therapy significantly decreased the frequency of epigastric pain, nausea, vomiting and diarrhea.

Antibodies against Food Antigens in Patients with Autistic Spectrum Disorders

Purpose. Immune system of some autistic patients could be abnormally triggered by gluten/casein assumption. The prevalence of antibodies to gliadin and milk proteins in autistic children with paired/impaired intestinal permeability and under dietary regimen either regular or restricted is reported. Methods. 162 ASDs and 44 healthy children were investigated for intestinal permeability, tissue-transglutaminase (tTG), anti-endomysium antibodies (EMA)-IgA, and total mucosal IgA to exclude celiac disease; HLA-DQ2/-DQ8 haplotypes; total systemic antibodies (IgA, IgG, and IgE); specific systemic antibodies: α-gliadin (AGA-IgA and IgG), deamidated–gliadin-peptide (DGP-IgA and IgG), total specific gliadin IgG (all fractions: α, β, γ, and ω), β-lactoglobulin IgG, α-lactalbumin IgG, casein IgG; and milk IgE, casein IgE, gluten IgE, -lactoglobulin IgE, and α-lactalbumin IgE. Results. AGA-IgG and DPG-IgG titers resulted to be higher in ASDs compared to controls and are only partially influenced by diet regimen. Casein IgG titers resulted to be more frequently and significantly higher in ASDs than in controls. Intestinal permeability was increased in 25.6% of ASDs compared to 2.3% of healthy children. Systemic antibodies production was not influenced by paired/impaired intestinal permeability. Conclusions. Immune system of a subgroup of ASDs is triggered by gluten and casein; this could be related either to AGA, DPG, and Casein IgG elevated production or to impaired intestinal barrier function.

Low-protein diet and progression of retinal degeneration in gyrate atrophy of the choroid and retina: A twenty-six-year follow-up

Summary: Gyrate atrophy of the choroid and retina is an autosomal recessive chorioretinal dystrophy which leads to a slowly progressive loss of vision. The primary defect is due to a deficiency of the enzyme ornithine δ-aminotransferase, which is responsible for markedly elevated levels of ornithine in plasma and other body fluids. Although several therapeutic regimens have been proposed, the reduction in ornithine accumulation obtained by reducing the intake of its precursor arginine (semisynthetic low-arginine diet) is the one most practised. In this clinical and molecular study we report a patient with hyperornithinaemia and gyrate atrophy of the choroid and retina who had been diagnosed when she was 3 years 9 months old. She also presented mild mental retardation, delayed language development and speech defects. The patient has recently been found to be homozygous for the new Gly91Arg amino acid substitution of the enzyme ornithine δ-aminotransferase. This mutation lies in a region of the mature protein that is considered crucial for the mitochondrial targeting activity. In this patient, a 28-year treatment with a completely natural low-protein diet (0.8 g/kg per day of natural protein) has been able to significantly reduce ornithine plasma levels, and to greatly delay the natural progression of the chorioretinal changes. This study suggests that, in the long-term treatment of gyrate atrophy, the efficacy in slowing the progression of chorioretinal changes and the palatability of a completely natural low-protein diet make this treatment a potentially viable alternative in patients refusing the semisynthetic diet.

Concealed administration of frusemide simulating Bartter syndrome in a 4.5-year-old boy

A 4.5-year-old boy was admitted to three different hospitals because of a tendency towards dehydration and polyuria, along with normal blood pressure, hypochloraemia, hypokalaemia, metabolic alkalosis and an impaired urinary concentrating ability. A renal biopsy failed to reveal juxtaglomerular hyperplasia. The clinical and laboratory findings failed to improve despite supplementation with potassium chloride and treatment with indomethacin. The urine was found to contain frusemide. The parents denied any drug administration to the boy. The child is now doing well more than 1 year after separation from his mother. Since ingestion of diuretic cannot be differentiated from true Bartter syndrome by blood and urinary electrolyte measurements alone, a diuretic screen is warranted in children with findings consistent with Bartter syndrome.

A common CTLA4 polymorphism confers susceptibility to autoimmune thyroid disease in celiac children.

BACKGROUND Cytotoxic T-lymphocyte-associated protein 4 (CTLA4) is a strong candidate gene in autoimmunity susceptibility. In particular, the CTLA4 CT60 A/G dimorphism has been associated with celiac disease (CD) and was reported to be strongly associated with autoimmune thyroid disease (AITD). AIMS This study aimed to investigate the possible influences of the CTLA4 CT60 A/G polymorphism in the susceptibility of Italian children to CD and in the predisposition to develop AITD in children with CD. PATIENTS AND METHODS We genotyped 317 Italian celiac children, including 44 patients (13.9%) who developed AITD after CD diagnosis and 350 controls. RESULTS The CTLA4 CT60 GG genotype distribution did not show any significant difference between children with CD and control population (p=0.4). On the contrary, the frequency of the GG genotype was significantly higher in patients with CD complicated with AITD than in control subjects (p=0.002) and CD patients without AITD (p=0.02). CONCLUSION Our data show a significant effect of the CTLA4 CT60G allele at the homozygous state on the risk of developing AITD in children with CD and suggest that the reported association of the CTLA4 CT60 A/G polymorphism with CD is limited to the subgroup of patients who are or will be complicated with AITD.

Macroamylasemia in a 5-year-old Girl

In macroamylasemia, a macromolecular complex consisting of amylase linked to immunoglobulins circulates in the plasma and usually causes benign hyperamylasemia with low or normal amylasuria. Macroamylasemia is extremely rare in pediatric patients as it has been described in only four patients. We report herein the case of a 5-year-old girl with abdominal pain and macroamylasemia. To recognize macroamylase, we used agar gel electrophoresis, PEG precipitation, and fast protein liquid chromatography (FPLC). In our case, FPLC was found to be the most reliable method for the identification of the macromolecular complex. Macroamylasemia is merely a biochemical abnormality that is not associated with any kind of pathology. Its identification is therefore essential in order to avoid a wrong diagnosis, i.e., pancreatitis, with consequent inappropriate therapies.

Variable Response to the Diepoxybutane Test in two Dizygotic Twins with Fanconi's Anemia and Flow Cytometry for Diagnosis Confirmation

Fanconis anemia (FA) is a rare, genetically heterogeneous, autosomal recessive disorder characterized by bone marrow failure, congenital abnormalities, chromosome instability, and increased susceptibility to neoplasia. Congenital abnormalities vary in location and in severity and not all patients are affected. Although the primary defect of FA is unknown, hypersensitivity to the clastogenic effect of agents that introduce cross-links in the DNA, such as diepoxybutane (DEB), is a marker of the FA phenotype in patients suffering from aplastic anemia without the physical characteristics of the syndrome and, conversely, in cases with abnormalities in the preanemic phase. We report the case of two dizygotic twins suffering from FA with discordant hematologic data. The DEB test repeated several times in various laboratories yielded conflicting results, whereas cell cycle studies by flow cytometry revealed a pattern typical of FA patients. Moreover, the flow cytometric pattern was correlated with the clinical severity of the disease.

Incidence and distribution of coeliac disease in Campania (Italy): 2011-2013.

Background There exists a wide variation in the reported incidence of coeliac disease in recent decades. We aimed to evaluate the incidence rate of coeliac diagnoses performed in an Italian region, Campania, between 2011 and 2013 and its variation therein. Methods All coeliac diagnoses made from 2011 to 2013 and registered within the Campania coeliac disease register (CeliacDB) were identified. Incidence rates were analysed by sex, age and province of residence, with a Poisson model fitted to determine incidence rate ratios. Results We found 2049 coeliac disease diagnoses registered in the CeliacDB between 2011 and 2013; 1441 of these patients were female (70.4%) and 1059 were aged less than 19 years (51.7%). The overall incidence of coeliac disease in Campania was 11.8 per 100,000 person-years (95% CI 11.3–12.3) during the study period, with marked variation by age [27.4 per 100,000 person-years (95% CI 25.8–29.1) in children under 19 years of age and 7.3 per 100,000 (95% CI 6.8–7.8) in adults] and sex [16.1 per 100,000 person-years in females (95% CI 15.3–16.9) and 7.2 per 100,000 person-years in males (95% CI 6.6–7.8)]. Coeliac disease incidence was roughly similar in Naples, Salerno, Caserta and Avellino, but about half in Benevento. More than 80% of our study population was diagnosed by the combination of positive antitransglutaminase IgA and Marsh 3. More than half of the patients were symptomatic at the time of coeliac disease diagnosis (39.7% had a classical presentation and 21.1% a non-classical one according to the Oslo definition). Conclusions Coeliac disease incidence was roughly similar among Campania provinces, except in Benevento where it was about half, probably due to less awareness of coeliac disease in this area. The incidence of coeliac disease in Campania appears to be lower than that reported by most of the previous literature, suggesting the necessity of new coeliac awareness programmes.