39 research outputs found

    Loss and Gain of Tolerance to Pancreatic Glycoprotein 2 in Celiac Disease

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    <div><p>Background</p><p>Autoantibodies against pancreatic secretory-granule membrane glycoprotein 2 (GP2) have been demonstrated in patients with Crohn’s disease but recently also with celiac disease (CD). Both entities are characterized by intestinal barrier impairment with increased gut permeability. Pathophysiological hallmark of CD is a permanent loss of tolerance to alimentary gliadin and a transient loss of tolerance to the autoantigen human tissue transglutaminase (tTG). Therefore, we explored the behavior of loss of tolerance to GP2 reported in CD.</p><p>Methods</p><p>We assessed prevalences and levels of autoantibodies against GP2, CD-specific antibodies to endomysial antigens and tTG as well as Crohn’s disease-specific anti-<i>Saccharomyces cerevisiae</i> antibodies in sera of 174 patients with active CD, 84 patients under gluten-free diet (GFD) and 129 controls. Furthermore, we looked for an association between anti-GP2 antibody positivity and degree of mucosal damage in CD.</p><p>Results</p><p>We found significantly elevated anti-GP2 IgA positivity in active CD patients (19.5%) compared to CD patients under GFD (0.0%) and controls (5.4%, p < 0.001, respectively). Anti-GP2 IgA levels correlated significantly with CD-specific antibodies (p < 0.001). Anti-GP2 autoantibody positivity disappeared under GFD similarly to CD-specific autoantibodies against tTG and endomysial antigens. For the first time, IgA antibody levels to GP2 are demonstrated to be associated with degree of villous atrophy according to Marsh classification.</p><p>Conclusions</p><p>Anti-GP2 IgA seems to be associated with disease activity in a distinct subgroup of patients with CD. The observed loss of tolerance to GP2 in a subset of patients with CD is transient and disappears under GFD.</p></div

    Clinical characteristics of the study groups.

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    <p><sup>a</sup> In this group, 51 patients of group 1 with active CD and follow-up serology under gluten-free diet (GFD) are included.</p><p><sup>b</sup> For the other 21 patients, the original histology was not available. In one patient diagnosis was done according to the new ESPGHAN guidelines without biopsy [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128104#pone.0128104.ref018" target="_blank">18</a>].</p><p><sup>c</sup> The 36 patients with family history of CD were from 23 different families.</p><p>celiac disease (CD), gluten-free diet (GFD).</p><p>Clinical characteristics of the study groups.</p

    Association between anti-GP2 IgA level and degree of villous atrophy.

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    <p>IgA antibody levels to glycoprotein 2 (GP2) determined in 153 patients with active celiac disease are associated with the grade of villous atrophy according to Marsh classification. The dotted line indicates cut-off value of 20 U/mL for anti-GP2 IgA. Data are displayed as U/mL in Box-and-Whisker plots with far out values, defined as values that are smaller than the lower quartile minus 3 times the interquartile range, or larger than the upper quartile plus 3 times the interquartile range, displayed as solid triangles. * p = 0.001, ** p = 0.002.</p

    Antibody kinetics in 4 celiac disease patients.

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    <p>Antibody kinetics in 4 celiac disease patients (A-D) with at least two consecutive samples before and after the onset of a gluten free diet. In fact, in all patients anti- glycoprotein 2 (GP2) IgA levels were reduced to values below the cut-off. Interestingly, in one patient (patient B) with co-existing type 1 diabetes who became positive for anti-GP2 IgA in parallel with anti- tissue transglutaminase (tTG) IgA, anti-GP2 IgA turned also negative under gluten free diet.</p

    Demonstration of autoantibody specificity by inhibition in ELISA.

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    <p>A: Inhibition of serum IgA reactivity to glycoprotein 2 (GP2). A serum with high anti-GP2 IgA level at a dilution giving an optical density (OD) of 0.8 was pre-incubated with recombinant GP2 (Isoform alpha) and tissue transglutaminase (TG) at decreasing concentrations from 0–10 μg/mL. B: Inhibition of serum IgA reactivity to tTG. A serum with high anti-tTG IgA level at a dilution giving an optical density (OD) of 1.5 was pre-incubated with recombinant GP2 (Isoform alpha) and tTG at decreasing concentrations from 0–10 μg/mL. Data are presented as means of triplicate measurements with the corresponding standard deviation.</p

    Correlation of anti-GP2 IgA with celiac disease (CD) specific antibodies.

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    <p>Association of anti-GP2 IgA with endomysial antibodies (EmA) (A) and anti- tissue transglutaminase (tTG) IgA (B) in 174 patients with active CD. (Spearman's coefficient of rank correlation for (A) 0.466 and (B) 0.494 (p<0.001, respectively).</p

    Flow cytometry analysis of γH2AX-stained DNA double-strand breaks.

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    <p>Mean γH2AX intensity was assessed in PBMCs immediately, 1 h and 20 h after indicated exposure conditions. (A) Representative overlay histogram of γH2AX-intensity 1 h after indicated exposure (black line) and of corresponding control (gray line). (B) Difference of mean fluorescence intensity (MFI) of γH2AX and IgG-isotype control staining from 16 independent experiments at three different time points after exposure as mean ± SEM (***: P ≤ 0.001; ns: P > 0.05).</p

    Analysis of DNA Double-Strand Breaks and Cytotoxicity after 7 Tesla Magnetic Resonance Imaging of Isolated Human Lymphocytes

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    <div><p>The global use of magnetic resonance imaging (MRI) is constantly growing and the field strengths increasing. Yet, only little data about harmful biological effects caused by MRI exposure are available and published research analyzing the impact of MRI on DNA integrity reported controversial results. This in vitro study aimed to investigate the genotoxic and cytotoxic potential of 7 T ultra-high-field MRI on isolated human peripheral blood mononuclear cells. Hence, unstimulated mononuclear blood cells were exposed to 7 T static magnetic field alone or in combination with maximum permissible imaging gradients and radiofrequency pulses as well as to ionizing radiation during computed tomography and γ-ray exposure. DNA double-strand breaks were quantified by flow cytometry and automated microscopy analysis of immunofluorescence stained γH2AX. Cytotoxicity was studied by CellTiter-Blue viability assay and [<sup>3</sup>H]-thymidine proliferation assay. Exposure of unstimulated mononuclear blood cells to 7 T static magnetic field alone or combined with varying gradient magnetic fields and pulsed radiofrequency fields did not induce DNA double-strand breaks, whereas irradiation with X- and γ-rays led to a dose-dependent induction of γH2AX foci. The viability assay revealed a time- and dose-dependent decrease in metabolic activity only among samples exposed to γ-radiation. Further, there was no evidence for altered proliferation response after cells were exposed to 7 T MRI or low doses of ionizing radiation (≤ 0.2 Gy). These findings confirm the acceptance of MRI as a safe non-invasive diagnostic imaging tool, but whether MRI can induce other types of DNA lesions or DNA double-strand breaks during altered conditions still needs to be investigated.</p></div

    Proliferation assay of subsequently PHA-stimulated PBMCs.

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    <p>[<sup>3</sup>H]-thymidine incorporation was determined 84 h after indicated exposure conditions. Diagram displays mean ± SEM of 16 independent experiments (***: P ≤ 0.001; ns: P > 0.05).</p

    ESM increases MuSK expression in HEp-2 M4 cells.

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    <p>A. Exponentially growing HEp-2 M4 cells were treated (right chart) or not treated (left chart) with the epigenetic supplement mixture (ESM) as indicated. Cell cycle phases were analyzed by FACS. Although there is an increase of cells in G2/M phase with ESM, this treatment apparently does not induce cell cycle arrest. B. Cell extracts from exponentially growing transfected HEp-2 M4 or untransfected parental HEp-2 cells were processed for Western Blotting. Cells had been treated or not with the epigenetic supplement mixture (ESM) as indicated. Detection of the MuSK-V5 fusion protein was performed as described in the materials section. Expected band of expressed MuSK is indicated by arrow, other bands are probably degradation products. C. Clone HEp-2 M4 or parental HEp-2 cells were treated or not with ESM as indicated, and MuSK expression was analyzed by immunofluorescence using MuSK autoantibody-positive patient serum (1). A serum pretested by radioimmunoassay to be negative for anti-MuSK autoantibodies was used as control (2). Immunofluorescence images were evaluated by AKLIDES system (10× objective, TRANSFECT modus).</p
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