Antibodies against neo-epitope of microbial and human transglutaminase complexes as biomarkers of childhood celiac disease.

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.

Different DRB1*03:01-DQB1*02:01 haplotypes confer different risk for celiac disease.

Celiac disease is associated with the HLA-DR3-DQA1*05:01-DQB1*02:01 and DR4-DQA1*03:01-DQB1*03:02 haplotypes. In addition, there are currently over 40 non-HLA loci associated with celiac disease. This study extends previous analyses on different HLA haplotypes in celiac disease using next generation targeted sequencing. Included were 143 patients with celiac disease and 135 non-celiac disease controls investigated at median 9.8 years (1.4-18.3 years). PCR-based amplification of HLA and sequencing with Illumina MiSeq technology were used for extended sequencing of the HLA class II haplotypes HLA-DRB1, DRB3, DRB4, DRB5, DQA1 and DQB1, respectively. Odds ratios were computed marginally for every allele and haplotype as the ratio of allelic frequency in patients and controls as ratio of exposure rates (RR), when comparing a null reference with equal exposure rates in cases and controls. Among the extended HLA haplotypes, the strongest risk haplotype for celiac disease was shown for DRB3*01:01:02 in linkage with DQA1*05:01-DQB1*02:01 (RR = 6.34; P-value < .0001). In a subpopulation analysis, DRB3*01:01:02-DQA1*05:01-DQB1*02:01 remained the most significant in patients with Scandinavian ethnicity (RR = 4.63; P < .0001) whereas DRB1*07:01:01-DRB4*01:03:01-DQA1*02:01-DQB1*02:02:01 presented the highest risk of celiac disease among non-Scandinavians (RR = 7.94; P = .011). The data also revealed 2 distinct celiac disease risk DR3-DQA1*05:01-DQB*02:01 haplotypes distinguished by either the DRB3*01:01:02 or DRB3*02:02:01 alleles, indicating that different DRB1*03:01-DQB1*02:01 haplotypes confer different risk for celiac disease. The associated risk of celiac disease for DR3-DRB3*01:01:02-DQA1*05:01-DQB1*02:01 is predominant among patients of Scandinavian ethnicity.

Role of proneurotensin as marker of paediatric coeliac disease.

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.

Serum cytokine pattern in young children with screening detected coeliac disease.

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)-1ß, 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-1ß, 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.

A gluten-free diet lowers NKG2D and ligand expression in BALB/c and non-obese diabetic (NOD) mice.

The interplay between diet and immune parameters which could affect type 1 diabetes (T1D) pathogenesis is not sufficiently clarified. Intestinal up-regulation of the activating receptor natural killer group 2D (NKG2D) (CD314) and its ligands is a hallmark of coeliac disease. However, the direct effect of gluten on NKG2D 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 NKG2D expression on DX5(+) (CD49b) natural killer (NK) cells, CD8(+) T cells and in intestinal and islet levels of NKG2D and ligands in BALB/c and non-obese diabetic (NOD) mice. Gluten-free NOD mice had lower insulitis (P < 0·0001); reduced expression of NKG2D 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 NKG2D and DX5 mRNA in intestine (P < 0·05). Differences in intestinal mRNA expression were found in mice at 8, 13 and 20 weeks. Intestinal expression of NKG2D ligands was reduced in SGF mice with lower expression of all ligands. In isolated islets, a SGF diet induced a higher expression of specific NKG2D ligands. Our data show that a gluten-free diet reduces the level of NKG2D and the expression of NKG2D ligands. These immunological changes may contribute to the lower T1D incidence associated with a gluten-free diet.

Reduction of tissue transglutaminase autoantibody levels by gluten-free diet is associated with changes in subsets of peripheral blood lymphocytes in children with newly diagnosed coeliac disease.

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+CD25(high)CCR4+ 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.

Using radioligand-binding assays to measure tissue transglutaminase autoantibodies in young children.

AIM: To measure autoantibodies against tissue transglutaminase (tTG) in young children prospectively screened for coeliac disease (CD). METHODS: In total, 652 children aged 2.9 (2.5-4.2) y were analysed for IgA-tTG and IgG-tTG with radioligand-binding assays and IgA endomysial antibodies (EMA) by indirect immunofluorescence. Antibody-positive children were retested after 1.2 (range 0.2-1.9) y. Intestinal biopsy was performed on children with persistently high antibody levels. RESULTS: In total, 3.2% (95% CI: 1.9-4.6%) of the 652 children were positive for at least one antibody at baseline: 2.5% (95% CI: 1.3-3.7%) for IgA-tTG, 1.7% (95% CI: 0.7-2.7%) for IgG-tTG and 2.9% (95% CI: 1.6-4.2%) for IgA-EMA, respectively. Ten children were positive for all three antibodies, five for both IgA-tTG and EMA, four for EMA only, one for IgA-tTG and another for IgG-tTG. IgA-EMA titres correlated with IgA-tTG levels (r = 0.73, p = 0.0003). At follow-up, seven of 20 children remained positive for all three antibodies, three for IgA-tTG only, one for both IgA-tTG and EMA, one for IgA-tTG and IgG-tTG, and the remaining child refused further participation. Three biopsies showed villous atrophy, two increased intraepithelial lymphocytes and two normal findings. Biopsy was not performed in four children with low or declining tTG antibody levels at follow-up and in one child who declined. CD was evident in 0.5% (95% CI: 0.0-1.0%) (3/652). CONCLUSION: This study revealed a high number of young children positive for tTG antibodies as well as EMA, but the majority showed declining levels in both antibodies over time. We suggest using radioligand-binding assays for quantitative measurement of tTG antibodies when change in antibody levels is studied in young children.

Tissue transglutaminase autoantibodies and human leucocyte antigen in Down's syndrome patients with coeliac disease.

UNLABELLED: The association between autoantibodies against tissue transglutaminase (tTG) and human leucocyte antigen (HLA)-DQB1 alleles was tested in Down's syndrome (DS) patients with and without coeliac disease (CD). Immunoglobulin A (IgA) and G (IgG) anti-tTG were measured in radioligand binding assays and compared with conventionally analysed IgA antibodies against gliadin (AGA) and IgA autoantibodies against endomysium (EMA) in 48 DS patients. HLA-DQB1 typing was carried out by polymerase chain reaction and hybridization with allele-specific probes in 41/48 patients. Both IgA-tTG and IgG-tTG, as well as EMA, were detected in 7/48 and AGA in 15/48 patients. Intestinal biopsy showed histopathological changes consistent with CD in 9/16 patients. HLA-DQB1 typing, available for 8/9 patients with and for 33/39 without CD, demonstrated that 5/8 with CD had DQB1*02 compared with 7/33 of those without (p = 0.0345). In patients with anti-tTG, 5/6 had the DQB1*02 allele compared with 7/35 of those without (p = 0.0053). CONCLUSIONS: Anti-tTG are HLA-DQB1*02-associated autoantibodies which together could be useful screening tests for silent CD in DS patients. In patients with gastrointestinal symptoms or clinical signs of malabsorption, anti-tTG should be combined with AGA to detect other forms of enteropathies and CD.

Prediction of silent celiac disease at diagnosis of childhood type 1 diabetes by tissue transglutaminase autoantibodies and HLA.

AIMS: The aims were to estimate the diagnostic sensitivity and specificity of autoantibodies to tissue transglutaminase (IgA- and IgG-tTG), gliadin (AGA) and endomysium (EMA) in relation to human leukocyte antigen (HLA)-DQB1 alleles to identify silent celiac disease at diagnosis of type 1 diabetes. METHODS: IgA- and IgG-tTG were measured in radioligand binding assays in 165 type 1 diabetic patients. Data on HLA-DQB1 were available for 148 patients and on both AGA and EMA for 164 patients. For patients considered positive for AGA or EMA, or both, an intestinal biopsy was suggested. HLA-DQB1 typing was carried out by polymerase chain reaction and hybridization with allele specific probes. RESULTS: Three patients, left out from further study of antibodies, but not from HLA-DQB1 analysis, had treated celiac disease at diagnosis. Out of the other 162 type 1 diabetic patients tested, nine had IgA-tTG, six IgG-tTG, eight EMA, and 11 AGA. Biopsy was suggested for nine patients, of whom six showed villous atrophy, one did not and two refused to participate. Thus, silent celiac disease was probable in 8/162 and biopsy-verified in 6/162, where five patients were AGA-positive and six either EMA-, IgA-tTG- or IgG-tTG-positive. Of the 11 patients with celiac disease (three with treated and eight with silent celiac disease), 10 were HLA-DQB1-typed, of whom 65% (13/20) had the DQB1*02 allele, compared with 36% (100/276; p = 0.011) of those without celiac disease. IgA-tTG levels were higher in patients having either *02 or *0302 (0.6; -1.3-112.4 RU) compared with those not having these alleles (0.4; -0.7-3.4 RU; p = 0.023). CONCLUSION: IgA-tTG are HLA-DQB1*02-associated autoantibodies with high sensitivity and specificity for silent celiac disease at diagnosis of type 1 diabetes.

Inverse relationship between GAD65 antibody levels and severe retinopathy in younger type 1 diabetic patients.

Several risk factors for severe non-proliferative and proliferative retinopathy in type 1 diabetes mellitus have been proposed without explaining the rapid progression of retinopathy in some patients. Since GAD65 autoantibodies (GAD65Abs) are detected against glutamic acid decarboxylase (GAD), which is mainly expressed in islets and nervous tissue in type 1 diabetic patients, the aim of the present investigation was to test the hypothesis whether GAD65Abs are associated with rapidly progressing severe retinopathy. Patients with severe non-proliferative or proliferative retinopathy (n = 27) were compared with another group, which in spite of long diabetes duration had no or only mild signs of retinopathy (n = 28). GAD65Abs were analysed in a radioimmunoassay using in vitro translated human GAD65, and the levels were expressed as an index in relation to positive and negative reference samples. Using a cut-off level representing the 99th percentile of normals, 6/27 (22%) with and 9/28 (32%) without severe retinopathy were considered GAD65Ab positive. Although there was no difference in the number of GAD65Ab positive patients, the GAD65Ab levels were lower in patients with (0.30; 0.11-0.64) than without (0.68; 0.34-1.12) severe retinopathy (P = 0.03). The patients were also subjected to HLA-DR and DQ typing by PCR and hybridization with oligospecific probes. DQ2/8 was more common in patients with (56%) than without (29%) severe retinopathy (P = 0.05), but DQ2/8 could not account for the lower GAD65Ab levels in patients with severe retinopathy. It is concluded that GAD65Ab levels are inversely correlated with severe retinopathy in young type 1 diabetic patients.

HLA-DQB1*0201/0302 is associated with severe retinopathy in patients with IDDM.

Some insulin-dependent diabetic (IDDM) patients develop severe forms of retinopathy. Putative risk factors such as hypertension, poor metabolic control, nephropathy and growth hormone levels do not fully explain the progress of retinopathy in these patients. It has been discussed whether there is a genetic marker, since some diabetic patients without any known predisposing risk factors develop severe retinopathy and others do not. In the present study, HLA-DR and DQ were compared in two patient groups with IDDM. One group consisted of patients with early-onset diabetes, with severe non-proliferative or proliferative retinopathy; the other group had no or only mild signs of retinopathy. High resolution HLA typing was carried out by polymerase chain reaction (PCR) and hybridization with allele specific probes. Alleles on the DR3-DQ2 haplotype, DRB1*0301, DQA1*0501 and DQB1*0201, were more frequent in patients with severe retinopathy. A difference was seen when combining certain alleles in the genotypes of DQA1*03/0501 (p > 0.05) and DQB1*0201/0302 (p < 0.01). The findings of the present study suggest that DQB1*0201/0302 is the strongest genetic marker for severe retinopathy and DRB1*0301/0401 only has a secondary influence when combined with this genotype. It seems as if IDDM patients who are positive for the genotype DR3-DQ2/DR4-DQ8 (DRB1*0301-DQA1*0501-DQB1*0201/DRB1*0401 -DQA1*03-DQB1*0302) are at greater risk of developing severe retinopathy.