In order to identify the children who produced significant amounts of AGA antibodies, we estimated the threshold of the 95% percentile of the distribution of AGAs in controls and calculated a value of 10 units/mL. IL1, IL2, IL4, IL6, IL10, IL12p70, IL17A and TNF) and the expression of selected genes (FoxP3, IL10, TGF, INF, 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, = 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 usually a CeD-tolerogenic marker and that changes in gene expression and cytokine patterns XY1 precede the appearance of anti-tTG antibodies. Keywords: celiac disease, prospective cohorts, infants at risk for celiac disease, anti-gliadin antibodies, anti-tissue transglutaminase antibodies, serum cytokines and gene expression, tolerance 1. Introduction Celiac disease (CeD) is usually characterized by chronic intestinal inflammation caused by an abnormal immune response to prolamins found in wheat and other cereals [1]. Prolamins not tolerated by CeD patients contain specific sequences rich in proline and glutamine amino acids that are resistant to human gastrointestinal proteases and therefore remain partially digested in the intestinal tract [2,3,4,5]. At the site of the intestinal mucosa, the human tissue transglutaminase enzyme (tTG) deamidates specific glutamine (Q) residues, which facilitates the presentation of gluten peptides to intestinal pro-inflammatory T cells [6]. At the same time, antibodies against tTG are produced, representing the diagnostic hallmark of the disease [7]. The genetic profile XY1 [8,9,10] and a set of environmental factors contribute to the multiplicative risk of developing the disease [9,10,11,12,13]. In fact, Rabbit Polyclonal to BTLA prenatal events, early feeding patterns [11,12,13,14], viral infections [15,16] and other unknown factors may contribute to an increased risk of CeD. Recently, in the German and Hungarian cohorts of the PREVENT-CD project, the profile of antibody development was explored [17]. In their recently published study, Dis et al. evaluated the progression from the early recognition of the native gliadin peptide, resulting in the production of anti-gliadin antibodies (AGAs), to the production of the anti-deamidated gliadin antibody (DGP), and finally to the development of anti-tTG autoantibodies. The authors suggested that the simple recognition of the antigen and the exclusive production of AGAs are not predictive of disease development [17]. However, the description of what happens between the loss of tolerance to gluten and the beginning of intestinal damage is still unclear. The humoral response to gliadin peptides, XY1 the deamidation of QXP-sequence-containing sequences [18] and the resulting formation of the complex with tTG [19], a prerequisite for the production of anti-tTG autoantibodies [20], are likely to be key to the development of the full-blown disease. The early production of AGAs could allow for distinguishing children who develop tolerance from those who progress to an abnormal immune response to gluten. In order to investigate the role of AGA production in the development of tolerance after early dietary gluten exposure, here, we explored the relationship between the early production of antibodies with the development of villous atrophy in a longitudinal cohort of children at risk for CeD. In addition, we evaluated the levels of serum cytokines and the expression of a set of pro-inflammatory genes in relation to the production of antibodies. 2. Results 2.1. Antibody Production in At-Risk Children Who Develop CeD Thirty-eight genetically at-risk infants who developed CeD by 6 years of age frequently XY1 started to produce anti-tTG antibodies after the second year of life, with a peak XY1 at a median age of 42 months (Supplementary Table S1). The peaks of AGA and anti-tTG antibodies in CeD, which were estimated by the longitudinal profile of each child, are shown in Table 1. The peak of AGAs occurred slightly before the production of anti-tTG: the first peak was observed around 28 months and the highest peak occurred around 36C42 months of age, when anti-tTG antibodies were also detected. Similarly, in the few cases where DGP antibodies were estimated, their profile overlapped with that of AGAs (Supplementary Physique S1). Indeed, in CeD (8 children, 50 samples), both DGP-IgA and DGP-IgG correlated strongly with anti-tTG (Pearsons r = 0.669 and r = 0.807, both < 0.0001). DGP-IgA also.