Antibody Profile, Gene Expression and Serum Cytokines in At-Risk Infants before the Onset of Celiac Disease.

Immunological events that precede the development of villous atrophy in celiac disease (CeD) are still not completely understood. We aimed to explore CeD-associated antibody production (anti-native gliadin (AGA), anti-deamidated gliadin (DGP) and anti-tissue transglutaminase (anti-tTG)) in infants at genetic risk for CeD from the Italian cohorts of the PREVENT-CD and Neocel projects, as well as the relationship between antibody production and systemic inflammation. HLA DQ2 and/or DQ8 infants from families with a CeD case were followed from birth. Out of 220 at-risk children, 182 had not developed CeD by 6 years of age (CTRLs), and 38 developed celiac disease (CeD). The profiles of serum cytokines (INFγ, IL1ß, IL2, IL4, IL6, IL10, IL12p70, IL17A and TNFα) and the expression of selected genes (FoxP3, IL10, TGFß, INFγ, IL4 and IL2) were evaluated in 46 children (20 CeD and 26 CTRLs). Among the 182 healthy CTRLs, 28 (15.3%) produced high levels of AGA-IgA (AGA+CTRLs), and none developed anti-tTG-IgA or DGP-IgA, compared to 2/38 (5.3%) CeD infants (Chi Sq. 5.97, p = 0.0014). AGAs appeared earlier in CTRLs than in those who developed CeD (19 vs. 28 months). Additionally, the production of AGAs in CeD overlapped with the production of DGP and anti-tTG. In addition, gene expression as well as serum cytokine levels discriminated children who developed CeD from CTRLs. In conclusion, these findings suggest that the early and isolated production of AGA-IgA antibodies is a CeD-tolerogenic marker and that changes in gene expression and cytokine patterns precede the appearance of anti-tTG antibodies.

Progression of Celiac Disease in Children With Antibodies Against Tissue Transglutaminase and Normal Duodenal Architecture.

BACKGROUND & AIMS: Potential celiac disease is characterized by positive results from serologic tests for tissue transglutaminase antibodies (anti-TG2) but normal duodenal architecture (Marsh stages 0-1). There is controversy over the best way to manage these patients. We investigated risk factors associated with the development of villous atrophy in children with potential celiac disease. METHODS: We performed a prospective study of 280 children (ages 2-18 years) in Italy with suspected celiac disease, followed for up to 12 years (range, 18-150 months; median 60 months). The subjects had 2 consecutive positive results from tests for anti-TG2, tested positive for the endomysial antibody (anti-EMA), had total serum levels of immunoglobulin A in the normal range, normal duodenal architecture (Marsh stages 0-1) in 5 biopsies, and HLA DQ2- or DQ8-positive haplotypes. The children underwent serologic tests and clinical analyses every 6 months and a small bowel biopsy was taken every 2 years. A total of 210 patients of the original cohort were assessed at the 9-year follow-up evaluation. We performed multivariate analyses of clinical, genetic, and histologic data to identify factors associated with progression to villous atrophy. RESULTS: During the follow-up period, 42 (15%) of 280 children developed villous atrophy, whereas 89 (32%) children no longer tested positive for anti-TG2 or anti-EMA. The cumulative incidence of progression to villous atrophy was 43% at 12 years. In multivariate analysis, the baseline factors most strongly associated with development of villous atrophy were numbers of γδ intraepithelial lymphocyte cells followed by age and homozygosity for the HLA DQB1*02. In discriminant analysis, these baseline factors identified 80% of the children who developed baseline atrophy. CONCLUSIONS: In a long-term study of 280 children with suspected celiac disease (based on anti-TG2 and anti-EMA) on gluten-containing diets, the cumulative incidence of progression to villous atrophy was 43% over a 12-year period. We identified factors that can be used to identify children at highest risk for villous atrophy. This approach might be used to determine whether children with suspected celiac disease should immediately start a gluten-free diet or be monitored on their regular diet.

Presymptomatic Diagnosis of Celiac Disease in Predisposed Children: The Role of Gene Expression Profile.

OBJECTIVE: The prevalence of celiac disease (CD) has increased significantly in recent years, and risk prediction and early diagnosis have become imperative especially in at-risk families. In a previous study, we identified individuals with CD based on the expression profile of a set of candidate genes in peripheral blood monocytes. Here we evaluated the expression of a panel of CD candidate genes in peripheral blood mononuclear cells from at-risk infants long time before any symptom or production of antibodies. METHODS: We analyzed the gene expression of a set of 9 candidate genes, associated with CD, in 22 human leukocyte antigen predisposed children from at-risk families for CD, studied from birth to 6 years of age. Nine of them developed CD (patients) and 13 did not (controls). We analyzed gene expression at 3 different time points (age matched in the 2 groups): 4-19 months before diagnosis, at the time of CD diagnosis, and after at least 1 year of a gluten-free diet. At similar age points, controls were also evaluated. RESULTS: Three genes (KIAA, TAGAP [T-cell Activation GTPase Activating Protein], and SH2B3 [SH2B Adaptor Protein 3]) were overexpressed in patients, compared with controls, at least 9 months before CD diagnosis. At a stepwise discriminant analysis, 4 genes (RGS1 [Regulator of G-protein signaling 1], TAGAP, TNFSF14 [Tumor Necrosis Factor (Ligand) Superfamily member 14], and SH2B3) differentiate patients from controls before serum antibodies production and clinical symptoms. Multivariate equation correctly classified CD from non-CD children in 95.5% of patients. CONCLUSIONS: The expression of a small set of candidate genes in peripheral blood mononuclear cells can predict CD at least 9 months before the appearance of any clinical and serological signs of the disease.

Epigenetics in Paediatric Gastroenterology, Hepatology, and Nutrition: Present Trends and Future Perspectives.

Epigenetics can be defined as stable, potentially heritable changes in the cellular phenotype caused by mechanisms other than alterations to the underlying DNA sequence. As such, any observed phenotypic changes including organ development, aging, and the occurrence of disease could be driven by epigenetic mechanisms in the presence of stable cellular DNA sequences. Indeed, with the exception of rare mutations, the human genome-sequence has remained remarkably stable over the past centuries. In contrast, substantial changes to our environment as part of our modern life style have not only led to a significant reduction of certain infectious diseases but also seen the exponential increase in complex traits including obesity and multifactorial diseases such as autoimmune disorders. It is becoming increasingly clear that epigenetic mechanisms operate at the interface between the genetic code and our environment, and a large body of existing evidence supports the importance of environmental factors such as diet and nutrition, infections, and exposure to toxins on human health. This seems to be particularly the case during vulnerable periods of human development such as pregnancy and early life. Importantly, as the first point of contact for many of such environmental factors including nutrition, the digestive system is being increasingly linked to a number of "modern" pathologies. In this review article, we aim to give a brief introduction to the basic molecular principals of epigenetics and provide a concise summary of the existing evidence for the role of epigenetic mechanisms in gastrointestinal health and disease, hepatology, and nutrition.

Potential celiac children: 9-year follow-up on a gluten-containing diet.

OBJECTIVES: Potential celiac disease (CD) is defined by the presence of serum anti-tissue-transglutaminase (anti-TG2) antibodies and normal duodenal mucosa. The major clinical problem is the management of asymptomatic patients and how to predict the development of villous atrophy. This prospective longitudinal cohort study describes the natural history of potential CD up to 9 years and explores risk factors associated with the development of mucosal damage. METHODS: Two hundred and ten potential CD children were eligible for the study; 175/210 asymptomatic children were left on a gluten-containing diet. Antibodies and clinical symptoms were checked every 6 months, and a small bowel biopsy was taken every 2 years to evaluate histological, immunohistochemical, and anti-TG2 deposits. Patients were genotyped for HLA and a set of non-HLA CD-associated genes. RESULTS: Forty-three percent of patients showed persistently elevated anti-TG2 level, 20% became negative during follow-up, and 37% showed a fluctuant anti-TG2 course with transiently negative values. At 3 years of follow-up, 86% of cases remained potential; 73 and 67% still had normal duodenal architecture at 6 and 9 years, respectively. Male sex, slight mucosal inflammation at time 0, and a peculiar genetic profile delineate a cohort of individuals who were prone to develop mucosal damage during time. CONCLUSIONS: A sizeable proportion of asymptomatic potential celiac patients showed fluctuation or negativization of antibody production, and many of these, with persistently positive anti-TG2, did not develop mucosal damage after 9 years of follow-up. Celiac population is a multivariate aggregate of individuals with different genetic and phenotypic profiles. Caution is required before prescribing a gluten-free diet for life to asymptomatic individuals with potential CD.

Beta catenin and cytokine pathway dysregulation in patients with manifestations of the "PTEN hamartoma tumor syndrome".

BACKGROUND: The "PTEN hamartoma tumor syndrome" (PHTS) includes a group of syndromes caused by germline mutations within the tumor suppressor gene "phosphatase and tensin homolog deleted on chromosome ten" (PTEN), characterized by multiple polyps in the gastrointestinal tract and by a highly increased risk of developing malignant tumours in many tissues. The current work clarifies the molecular basis of PHTS in three unrelated Italian patients, and sheds light on molecular pathway disregulation constitutively associated to PTEN alteration. METHODS: We performed a combination of RT-PCR, PCR, sequencing of the amplified fragments, Real Time PCR and western blot techniques. RESULTS: Our data provide the first evidence of ß-catenin accumulation in blood cells of patients with hereditary cancer syndrome caused by germ-line PTEN alteration. In addition, for the first time we show, in all PHTS patients analysed, alterations in the expression of TNFα, its receptors and IL-10. Importantly, the isoform of TNFRI that lacks the DEATH domain (TNFRSF1ß) was found to be overexpressed. CONCLUSION: In light of our findings, we suggest that the PTEN pathway disregulation could determine, in non-neoplastic cells of PHTS patients, cell survival and pro-inflammatory stimulation, mediated by the expression of molecules such as ß-catenin, TNFα and TNFα receptors, which could predispose these patients to the development of multiple cancers.

Gluten consumption and inflammation affect the development of celiac disease in at-risk children.

Gene expression, lipidomic and growth impairment findings suggest that the natural history of celiac disease (CD) starts before the gluten-induced immune response. Gluten intake in the first years of life is a controversial risk factor. We aimed to estimate the risk of developing CD associated with the amount of gluten intake and the serum inflammatory profile in genetically predisposed infants. From an Italian cohort of children at risk for CD, we enrolled 27 children who developed CD (cases) and 56 controls matched by sex and age. A dietary interview at 9, 12, 18, 24 and 36 months was performed. Serum cytokines (INFγ, IL1ß, IL2, IL4, IL6, IL10 IL12p70, IL17, and TNFα) were analysed at 4 and 36 months. Infants who developed CD by 6 years showed an increase in serum cytokines (INFγ, IL1ß, IL2, IL6, IL10, IL12p70 and TNFα) at 4 months of age before gluten introduction. CD cases ate significantly more gluten in the second year of life than controls, and gluten intake in the second year of life was strongly correlated with serum cytokines (INFγ, IL2, IL4, IL12p70, IL17) at 36 months only in CD cases. The dietary pattern of infants who developed CD was characterized by high consumption of biscuits and fruit juices and low intake of milk products, legumes, vegetables and fruits. Genetically predisposed infants who developed CD showed a unique serum cytokine profile at 4 months before gluten consumption. The amount of gluten was strongly correlated with an inflammatory profile in serum cytokines at 36 months only in infants who developed CD.

In Celiac Disease Patients the In Vivo Challenge with the Diploid Triticum monococcum Elicits a Reduced Immune Response Compared to Hexaploid Wheat.

SCOPE: Gluten from the diploid wheat Triticum monococcum (TM) has low content of immunostimulatory sequences and a high gastro-intestinal digestibility. Gluten-reactive T cells elicited by diploid and hexaploid (Triticum aestivum-TA) wheat in celiac disease (CD) patients upon a brief oral challenge are analyzed. METHODS AND RESULTS: Seventeen patients with CD (median age 13 years) consumed for 3 days sandwiches made with TM (cultivar Norberto-ID331, N=11), or TA (cultivar Sagittario, N=11) flours, corresponding to 12 gr of gluten/die. Immunostimulatory properties are assessed in blood by measuring the IFN-γ-secreting T cells by EliSpot and the expression of inflammatory cytokines/receptors (IL-12A, IL-15, IL-18RAP, IFN-γ) by qPCR. TA mobilizes a remarkable number of gliadin-specific, IFN-γ-secreting T cells (p<0.05), while no significant cell mobilization is induced by TM (p=ns). Similar results are obtained in response to five immunogenic peptides from α-, ω-, and γ-gliadins, although with a large individual variability. An increased mRNA expression for IL-12A and IFN-γ is detected in the group eating TA compared to those consuming TM (p<0.05). CONCLUSIONS: Although T. monococcum is a cereal not suitable for the diet of celiacs, this diploid wheat elicits a reduced in vivo T-cell response compared to T. aestivum in celiac patients.

Growth rate of coeliac children is compromised before the onset of the disease.

INTRODUCTION: Growth impairment has often been described in children who develop coeliac disease (CD). Based on data from the multicentre, longitudinal PreventCD study, we analysed the growth patterns of infants at genetic risk of CD, comparing those who developed CD by 6 years of age (CD 'cases', 113 infants) versus those who did not develop CD by 6 years (no CD 'controls', 831 infants). METHODS: Weight and length/height were measured using a longitudinal protocol. Raw measurements were standardised, computing z-scores for length/height and weight; a linear mixed model was fitted to the data in order to compare the rate of growth in the two cohorts. RESULTS: Neither cases nor controls had significant growth failure. However, when the mean z-scores for weight and height were analysed, there was a difference between the two groups starting at fourth month of life. When the growth pattern in the first year was analysed longitudinally using mixed models, it emerged that children who develop CD had a significantly lower growth rate in weight z-score (-0.028/month; 95% CI -0.038 to -0.017; p<0.001) and in length/height z-score (-0.018/month; 95% CI -0.031 to -0.005; p=0.008) than those who do not develop CD. When the whole follow-up period was analysed (0-6 years), differences between groups in both weight and length/height z-scores were confirmed. CONCLUSION: The growth of children at risk of CD rarely fell below 'clinical standards'. However, growth rate was significantly lower in cases than in controls. Our data suggest that peculiar pathways of growth are present in children who develop CD, long before any clinical or serological signs of the disease appear.

Implication of adenomatous polyposis coli and MUTYH mutations in familial colorectal polyposis.

PURPOSE: Familial adenomatous polyposis is an autosomal dominantly inherited syndrome characterized by hundreds or thousands of colorectal polyps and a high risk of colorectal cancer at a young age. Truncating germline mutations in the adenomatous polyposis coli gene are detected in approximately 80 percent of patients with classical familial adenomatous polyposis and in approximately 10 percent of the attenuated familial adenomatous polyposis patients. METHODS: We investigated the adenomatous polyposis coli and MUTYH genes mutations in a well-characterized series of 25 unrelated Italian patients with familial adenomatous polyposis. RESULTS: We characterized the specific adenomatous polyposis coli gene mutation in 10 probands, and identified eight truncating mutations (4 novel and 4 known mutations) and two splicing mutations. One of these, a novel missense mutation in exon 15, activates an exonic splicing enhancer control sequence. Moreover, 11 MUTYH gene mutations have been identified in 7 patients without a dominant family history of polyposis. CONCLUSIONS: This study enlarges the genotype-phenotype correlations of familial adenomatous polyposis and suggests that messenger alterations could be responsible for a subset of familial adenomatous polyposis cases without germ-line adenomatous polyposis coli or MUTYH gene mutations. It also confirms that genotype-phenotype correlations in MUTYH-associated polyposis are very complex.

Respiratory Infections and the Risk of Celiac Disease.

BACKGROUND AND OBJECTIVES: The increasing incidence of celiac disease (CD) suggests that common infections before the onset of autoimmune diseases could be an important factor in switching the immune response. We aimed to explore the relationship between early clinical events and the development of CD in genetically predisposed infants. METHODS: In this study, 373 newborns from families with at least 1 relative with CD were recruited, and human leukocyte antigen DQ2- or DQ8-positive infants were followed up with clinical and serological evaluations. Cross tabulation and odds ratios were used to explore the risk associated with single variables, and logistic regression analysis was performed to determine the variables that contributed to the risk of developing CD. Stepwise discriminant analysis was used to determine which variables could distinguish case patients from controls before diagnosis. RESULTS: The cumulative incidence of CD in this cohort was 6% at 3 years and 13.5% at 5 years of age, and l34 children (14%) developed CD before the sixth year of life. An analysis of adverse events showed a higher frequency of respiratory tract infections among CD patients during the first 24 months of life. In a stepwise discriminant analysis, which included sex and human leukocyte antigen risk class, only respiratory infections in the second and first years of life significantly contributed to discrimination of case patients versus controls. CONCLUSIONS: A multivariate model of discriminant analysis showed that the frequency of respiratory infections in the first 2 years of life could distinguish children who developed CD from those who did not.

Gene Expression Profiling of Celiac Biopsies and Peripheral Blood Monocytes Using Taqman Assays.

Quantitative real-time PCR (qPCR) allows for highly sensitive, rapid, and reproducible quantification of mRNA: it has become an established technology for the quantification of gene expression with the 5' nuclease assay using TaqMan(®) probes. It is used for a broad range of applications, including quantification of gene expression, measuring RNA interference, biomarker discovery, pathogen detection, and drug target validation. When studying gene expression with qPCR, scientists usually investigate changes-increases or decreases-in the quantity of particular gene products or a set of gene products. Investigations typically evaluate gene response to biological conditions such as disease states, exposure to pathogens or chemical compounds, organ or tissue location, and cell cycle or differentiation status. Here we describe this technique applied to molecular profiling of candidate genes in celiac biopsies and peripheral blood monocytes. Using data obtained by gene expression experiments, a discriminant equation has been developed that allows the correct classification of Celiac Disease (CD) patients compared to healthy controls, CD patients on a Gluten Free Diet (GFD), and other disease controls.

High Frequency of Haplotype HLA-DQ7 in Celiac Disease Patients from South Italy: Retrospective Evaluation of 5,535 Subjects at Risk of Celiac Disease.

BACKGROUND: Celiac disease (CD) has a strong genetic component mainly due to HLA DQ2/DQ8 encoding genes. However, a minority of CD patients are DQ2/DQ8-negative. To address this issue, we retrospectively characterized HLA haplotypes in 5,535 subjects at risk of CD (either relatives of CD patients or subjects with CD-like symptoms) referred to our center during a 10-year period. METHODS: We identified loci DQA1/DQB1/DRB1 by sequence-specific oligonucleotide-PCR and sequence-specific primer-PCR; anti-transglutaminase IgA/IgG and anti-endomysium IgA by ELISA and indirect immunofluorescence, respectively. RESULTS: We diagnosed CD in 666/5,535 individuals, 4.2% of whom were DQ2/DQ8-negative. Interestingly, DQ7 was one of the most abundant haplotypes in all CD patients and significantly more frequent in DQ2/DQ8-negative (38%) than in DQ2/DQ8-positive CD patients (24%) (p<0.05). CONCLUSION: Our data lend support to the concept that DQ7 represents an additive or independent CD risk haplotype with respect to DQ2/DQ8 haplotypes but this finding should be verified in other large CD populations.

Gene expression profile of peripheral blood monocytes: a step towards the molecular diagnosis of celiac disease?

AIM: Celiac disease (CD) is a multifactorial autoimmune disease induced by ingestion of gluten in genetically predisposed individuals. Despite technological progress, the diagnosis of CD is still based on duodenal biopsy as it was 50 years ago. In this study we analysed the expression of CD-associated genes in small bowel biopsies of patients and controls in order to explore the multivariate pathway of the expression profile of CD patients. Then, using multivariant discriminant analysis, we evaluated whether the expression profiles of these genes in peripheral blood monocytes (PBMs) differed between patients and controls. PARTICIPANTS: Thirty-seven patients with active and 11 with treated CD, 40 healthy controls and 9 disease controls (Crohn's disease patients) were enrolled. RESULTS: Several genes were differentially expressed in CD patients versus controls, but the analysis of each single gene did not provided a comprehensive picture. A multivariate discriminant analysis showed that the expression of 5 genes in intestinal mucosa accounted for 93% of the difference between CD patients and controls. We then applied the same approach to PBMs, on a training set of 20 samples. The discriminant equation obtained was validated on a testing cohort of 10 additional cases and controls, and we obtained a correct classification of all CD cases and of 91% of the control samples. We applied this equation to treated CD patients and to disease controls and obtained a discrimination of 100%. CONCLUSIONS: The combined expression of 4 genes allows one to discriminate between CD patients and controls, and between CD patients on a gluten-free diet and disease controls. Our results contribute to the understanding of the complex interactions among CD-associated genes, and they may represent a starting point for the development of a molecular diagnosis of celiac disease.

A celiac cellular phenotype, with altered LPP sub-cellular distribution, is inducible in controls by the toxic gliadin peptide P31-43.

Celiac disease (CD) is a frequent inflammatory intestinal disease, with a genetic background, caused by gliadin-containing food. Undigested gliadin peptides P31-43 and P57-68 induce innate and adaptive T cell-mediated immune responses, respectively. Alterations in the cell shape and actin cytoskeleton are present in celiac enterocytes, and gliadin peptides induce actin rearrangements in both the CD mucosa and cell lines. Cell shape is maintained by the actin cytoskeleton and focal adhesions, sites of membrane attachment to the extracellular matrix. The locus of the human Lipoma Preferred Partner (LPP) gene was identified as strongly associated with CD using genome-wide association studies (GWAS). The LPP protein plays an important role in focal adhesion architecture and acts as a transcription factor in the nucleus. In this study, we examined the hypothesis that a constitutive alteration of the cell shape and the cytoskeleton, involving LPP, occurs in a cell compartment far from the main inflammation site in CD fibroblasts from skin explants. We analyzed the cell shape, actin organization, focal adhesion number, focal adhesion proteins, LPP sub-cellular distribution and adhesion to fibronectin of fibroblasts obtained from CD patients on a Gluten-Free Diet (GFD) and controls, without and with treatment with A-gliadin peptide P31-43. We observed a "CD cellular phenotype" in these fibroblasts, characterized by an altered cell shape and actin organization, increased number of focal adhesions, and altered intracellular LPP protein distribution. The treatment of controls fibroblasts with gliadin peptide P31-43 mimics the CD cellular phenotype regarding the cell shape, adhesion capacity, focal adhesion number and LPP sub-cellular distribution, suggesting a close association between these alterations and CD pathogenesis.

Synergistic effect of interleukin-10-receptor variants in a case of early-onset ulcerative colitis.

AIM: To investigated the molecular cause of very early-onset ulcerative colitis (UC) in an 18-mo-old affected child. METHODS: We analysed the interleukin-10 (IL10) receptor genes at the DNA and RNA level in the proband and his relatives. Beta catenin and tumor necrosis factor-α (TNFα) receptors were analysed in the proteins extracted from peripheral blood cells of the proband, his relatives and familial adenomatous polyposis (FAP) and PTEN hamartoma tumor syndrome (PHTS) patients. Samples were also collected from the proband's inflamed colorectal mucosa and compared to healthy and tumour mucosa collected from a FAP patient and patients affected by sporadic colorectal cancer (CRC). Finally, we examined mesalazine and azathioprine effects on primary fibroblasts stabilised from UC and FAP patients. RESULTS: Our patient was a compound heterozygote for the IL10RB E47K polymorphism, inherited from his father, and for a novel point mutation within the IL10RA promoter (the -413G->T), inherited from his mother. Beta catenin and tumour necrosis factor α receptors-I (TNFRI) protein were both over-expressed in peripheral blood cells of the proband's relatives more than the proband. However, TNFRII was over-expressed only in the proband. Finally, both TNFα-receptors were shown to be under-expressed in the inflamed colon mucosa and colorectal cancer tissue compared to healthy colon mucosa. Consistent with this observation, mesalazine and azathioprine induced, in primary fibroblasts, IL10RB and TNFRII over-expression and TNFRI and TNFα under-expression. We suggest that ß-catenin and TNFRI protein expression in peripheral blood cells could represent molecular markers of sub-clinical disease in apparently healthy relatives of patients with early-onset UC. CONCLUSION: A synergistic effect of several variant alleles of the IL10 receptor genes, inherited in a Mendelian manner, is involved in UC onset in this young child.