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Summary: Tissue transglutaminase (tTG) and microbial transglutaminase (mTG) cross‐link gliadins to form complexes that expose immunogenic neo‐epitopes to produce tTG and mTG‐neo‐epitope antibodies. The aim of this study was to test the diagnostic performance of antibodies against non‐complexed and complexed forms of transglutaminases, to correlate their activities to the intestinal damage and to explore age group dependency in celiac disease (CD). A total of 296 children with untreated CD and 215 non‐celiac disease controls were checked by in‐house enzyme‐linked immunosorbent assays detecting immunoglobulin (Ig)A, IgG or combined detection of IgA and IgG (check) against tTG, AESKULISA® tTG New Generation (tTG‐neo) and mTG‐neo (RUO), IgA and IgG antibodies against deamidated gliadin peptide (DGP) and human IgA anti‐endomysium antibodies (EMA) using AESKUSLIDES® EMA. Intestinal pathology was graded according the revised Marsh criteria, and age dependencies of the antibody activities were analysed. Using cut‐offs estimated from receiver operating characteristic (ROC) curves, the highest area under curve (AUC) of the TG assays was 0·963 for tTG‐neo check, followed by tTG check (0·962) when the diagnosis was based on enteric mucosal histology. tTG‐neo check was the most effective to reflect the intestinal abnormalities in CD (r = 0·795, P < 0·0001). High levels of anti‐mTG‐neo IgG and anti‐tTG‐neo IgG appeared in the earlier age groups, as compared to anti‐tTG IgG (P < 0·001). Considering antibody diagnostic performance based on AUC, enteric damage reflection and predictability at an early age, the anti‐neo tTG check was the most effective diagnostic biomarker for pediatric CD. The mTG neo check might represent a new marker for CD screening, diagnosis and predictability. [ABSTRACT FROM AUTHOR]

Neurotensin (NT) is a gut hormone functioning proinflammatory through nuclear factor kappa B (NF-κB) and interleukin (IL)−8 secretion or anti-inflammatory through epidermal growth factor receptors. NT mRNA is down-regulated in duodenal biopsies of children with untreated coeliac disease. The aim of this study was to investigate if plasma pro-NT levels correlated with the degree of intestinal mucosal damage and tissue transglutaminase autoantibody (tTGA) levels in children with coeliac disease. Fasting plasma samples from 96 children with coeliac disease and 89 non-coeliac disease controls were analysed for NT precursor fragment pro-NT 1-117 by a chemiluminometric immunoassay. Pro-NT levels were compared with NT mRNA from duodenal biopsies, assessed previously with quantitative polymerase chain reaction (PCR). Illumina core exome arrays were used for human leucocyte antigen (HLA) typing and the Marsh criteria applied to score mucosal damage. Tissue TGA was measured by radio binding assay. A general linear model compared pro-NT levels with diagnosis of coeliac disease, Marsh score and HLA DQ haplotype. Spearman's rank test was used to compare pro-NT levels with tTGA, age and duodenal NT mRNA levels, respectively. Plasma pro-NT levels were elevated in children with coeliac disease (median 23 pmol/l higher, P = 0·003) and in those with severe intestinal mucosal damage (median 24 pmol/l higher for ≥ Marsh 3b versus not, P = 0·0004). Pro-NT levels correlated further with tTGA ( r2 = 0·22, P = 0·002), but not with duodenal NTS mRNA levels ( r2 = −0·12, P = 0·14). Pro-NT was not associated with any of the HLA risk-haplotypes. Elevated peripheral pro-NT levels reflect more severe forms of active coeliac disease, indicating a potential role of NT in intestinal inflammation. [ABSTRACT FROM AUTHOR]

Coeliac disease is an autoimmune disease characterized by inflammation localized to the small bowel, but less is known about systemic signs of inflammation. The aim was to measure cytokines of the T helper 1 ( Th1) and T helper 2 ( Th2) cell patterns in children with screening-detected coeliac disease before and after treatment with a gluten-free diet. Serum samples selected before and after the start of a gluten-free diet from 26 3-year-old children diagnosed with biopsy-proven coeliac disease and from 52 matched controls were assayed in an multiplex enzyme-linked immunosorbent assay ( ELISA) for the 10 cytokines: interferon ( IFN)-γ, interleukin ( IL)-1β, IL-2, IL-4, IL-5, IL-8, IL-10, IL-12p70, IL-13 and tumour necrosis factor ( TNF)-α. Among Th1 cytokines, IFN-γ and IL-12p70 were elevated significantly in children with coeliac disease compared to controls ( P < 0·001 and P = 0·001, respectively). Similar findings were demonstrated for the Th2 cytokines IL-5 ( P < 0·001), IL-10 ( P = 0·001) and IL-13 ( P = 0·002). No difference in cytokine levels between the two groups was found for TNF-α, IL-1β, IL-2, IL-4 and IL-8. After gluten-free diet, levels of IL-5, IL-12 and IL-10 decreased significantly ( P < 0·001, P = 0·002 and P = 0·007) and IFN-γ levels were reduced ( P = 0·059). Young children with coeliac disease detected by screening demonstrate elevated levels of serum cytokines at time of diagnosis. A prolonged systemic inflammation may, in turn, contribute to long-term complications known to be associated with untreated coeliac disease. [ABSTRACT FROM AUTHOR]

The interplay between diet and immune parameters which could affect type 1 diabetes ( T1 D) pathogenesis is not sufficiently clarified. Intestinal up-regulation of the activating receptor natural killer group 2D ( NKG2 D) ( CD314) and its ligands is a hallmark of coeliac disease. However, the direct effect of gluten on NKG2 D expression is not known. We studied, by fluorescence activated cell sorter (lymphoid tissues) and reverse transcription-quantitative polymerase chain reaction (intestine and pancreatic islets), if a gluten-free diet ( GF diet) from 4 weeks of age or a gluten-free diet introduced in breeding pairs ( SGF diet), induced changes in NKG2 D expression on DX5+(CD49b) natural killer ( NK) cells, CD8+ T cells and in intestinal and islet levels of NKG2 D and ligands in BALB/c and non-obese diabetic ( NOD) mice. Gluten-free NOD mice had lower insulitis ( P < 0·0001); reduced expression of NKG2 D on DX5+ NK cells in spleen and auricular lymph nodes ( P < 0·05); and on CD8+ T cells in pancreas-associated lymph nodes ( P = 0·04). Moreover, the level of CD71 on DX5+ NK cells and CD8+ T cells ( P < 0·005) was markedly reduced. GF and SGF mice had reduced expression of NKG2 D and DX5 m RNA in intestine ( P < 0·05). Differences in intestinal m RNA expression were found in mice at 8, 13 and 20 weeks. Intestinal expression of NKG2 D ligands was reduced in SGF mice with lower expression of all ligands. In isolated islets, a SGF diet induced a higher expression of specific NKG2 D ligands. Our data show that a gluten-free diet reduces the level of NKG2 D and the expression of NKG2 D ligands. These immunological changes may contribute to the lower T1 D incidence associated with a gluten-free diet. [ABSTRACT FROM AUTHOR]

Tissue transglutaminase (tTG) autoantibodies decline after gluten-free diet in patients with coeliac disease. We tested the hypothesis that gluten-free diet-induced change in tTG autoantibody levels affects subsets of peripheral blood lymphocytes. Peripheral blood was obtained from 20 children with biopsy-proven active coeliac disease. Gluten-free diet was initiated and the children examined again after three and six months. tTG autoantibodies were measured in radioligand binding assays and lymphocyte subsets by flow cytometry. IgA-tTG levels at diagnosis, 2204 U/ml (median, range 113–24990), were reduced over six months to 76 U/ml (median, range 1–1261) ( P < 0·001). At six months, 12/20 (60%) children had reduced their IgA-tTG levels to < 100 U/ml and these children showed a decrease in B cells (mean change −3·8%, P = 0·014), CD4+ T cells (mean −4·32%, P = 0·011) and CD4+ T cells expressing CD25high (mean change −0·62%, P = 0·036). In contrast, the CD4+CD25highCCR4+ T cell population increased during the same period (mean change 11·5%, P = 0·0036). The decline in IgA-tTG levels correlated to the decrease in B cells ( r = 0·56, P = 0·01), CD4+ T cells ( r = 0·66, P = 0·004) as well as CD4+CD25high T cells ( r = 0·59, P = 0·01). A negative correlation was found between the decline in IgA-tTG and CD4+CD25high T cells expressing CD45RO ( r = –0·49, P = 0·03) and CCR4 ( r = –0·54, P = 0·01). This is the first observational study on the effect of gluten-free diet on concurrent changes of tTG autoantibodies and specific peripheral blood lymphocyte subsets. Our data suggest that flow cytometry may be a useful complement to tTG autoantibodies when studying the effects of gluten-free diet in children with coeliac disease. [ABSTRACT FROM AUTHOR]