14 research outputs found

    Thioredoxin is involved in endothelial cell extracellular transglutaminase 2 activation mediated by celiac disease patient IgA

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    Purpose: To investigate the role of thioredoxin (TRX), a novel regulator of extracellular transglutaminase 2 (TG2), in celiac patients IgA (CD IgA) mediated TG2 enzymatic activation. Methods: TG2 enzymatic activity was evaluated in endothelial cells (HUVECs) under different experimental conditions by ELISA and Western blotting. Extracellular TG2 expression was studied by ELISA and immunofluorescence. TRX was analysed by Western blotting and ELISA. Serum immunoglobulins class A from healthy subjects (H IgA) were used as controls. Extracellular TG2 enzymatic activity was inhibited by R281. PX12, a TRX inhibitor, was also employed in the present study. Results: We have found that in HUVECs CD IgA is able to induce the activation of extracellular TG2 in a dose-dependent manner. Particularly, we noted that the extracellular modulation of TG2 activity mediated by CD IgA occurred only under reducing conditions, also needed to maintain antibody binding. Furthermore, CD IgA-treated HUVECs were characterized by a slightly augmented TG2 surface expression which was independent from extracellular TG2 activation. We also observed that HUVECs cultured in the presence of CD IgA evinced decreased TRX surface expression, coupled with increased secretion of the protein into the culture medium. Intriguingly, inhibition of TRX after CD IgA treatment was able to overcome most of the CD IgA-mediated effects including the TG2 extracellular transamidase activity. Conclusions: Altogether our findings suggest that in endothelial cells CD IgA mediate the constitutive activation of extracellular TG2 by a mechanism involving the redox sensor protein TRX

    Endothelial extracellular transglutaminase 2 (TG2) activity analyses by monodansylcadaverine (MDC) incorporation.

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    <p>The relative extracellular TG2 activity (<b>A</b>) at different time points and with (<b>B</b>) increasing concentrations of class A immunoglobulins are shown. The dashed line indicates TG2 activity in untreated endothelial cells (HUVECs). Bars represent mean MDC incorporation as fold of control and error bars indicate standard error of the mean. P-value < 0.05 was considered significant ( <sub>**</sub>p<0.001 and <sub>*</sub>p<0.01). Data derived from three independent experiments, repeated in quadruplicate. HUVECs were treated with celiac disease patient (CD IgA) and non-celiac subject’s immunoglobulin-A (H IgA). Extracellular TG2 activity was inhibited by administering a non-permeable, site directed TG2 inhibitor, R281.</p

    Celiac IgA CD IgA) mediated activation of extracellular transglutaminase 2 (TG2) by thioredoxin (TRX).

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    <p>(<b>A</b>) The relative surface expression of TG2 as well as (<b>B</b>) the relative TG2 activity on HUVECs treated with CD IgA autoantibodies was investigated by ELISA prior preincubation of endothelial cells with TRX inhibitor, PX12. The dashed line indicates TG2 expression and activity in untreated HUVECs. Bars represent mean values as fold of control and error bars indicate standard error of the mean. P-value < 0.05 was considered significant ( <sub>*</sub>p<0.01 and <sub>**</sub>p<0.001). (<b>C</b>) The secretion of TRX in endothelial cell (HUVECs) culture media was analysed by Western blotting in the presence of a specific TRX inhibitor, PX12 (1-methylpropyl 2-imidazolyl disulfide). Representative Western blots of HUVECs culture supernatants show amounts of TRX and ƴ-tubulin used as loading control for quality sample. Data derived from at least three independent experiments, repeated in quadruplicate are shown. Endothelial cells were treated with CD IgA and non-celiac subject’s immunoglobulin-A (H IgA).</p

    Celiac IgA (CD IgA) promotes secretion of thioredoxin.

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    <p><b>(TRX) and decreases its endothelial surface expression</b>. (<b>A</b>) Representative Western blots from total cell lysates and supernatants show amounts of TRX and ƴ-tubulin used as loading control for protein extracts. (<b>B</b>) Relative surface expression of TRX was studied by ELISA. The dashed line indicates TRX surface expression in untreated HUVECs. Bars represent mean TRX expression as fold of control and error bars indicate standard error of the mean. P-value <0.05 was considered significant ( <sub>*</sub>p<0.05 and <sub>**</sub>p<0.001). Data derived from three independent experiments, repeated in quadruplicate are shown. Endothelial cells were treated with CD IgA and non-celiac subject’s immunoglobulin-A (H IgA). Extracellular TG2 activity was inhibited by administering a site directed, non-permeable inhibitor, R281.</p

    Celiac patient immunoglobulins A (CD IgA) increase transglutaminase 2 (TG2) surface deposition on endothelial cells.

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    <p>(<b>A</b>) The relative surface expression of TG2 on non-permeabilized endothelial cells (HUVECs) was studied by ELISA. The dashed line indicates TG2 surface expression in untreated HUVECs. Bars represent mean TG2 expression as fold of control and error bars indicate standard error of the mean. P-value <0.05 was considered significant ( <sub>*</sub>p<0.001). Data derived from three independent experiments, repeated in quadruplicate are shown. (<b>B</b>) Surface expression of TG2 on non-permeabilized HUVECs was also investigated by immunofluorescence (IF) with the monoclonal TG2 antibody, 4G3. Representative IF stainings are shown. Control non-celiac subject’s immunoglobulin-A (H IgA). Extracellular TG2 activity was inhibited by administering a site directed, non-permeable inhibitor, R281.</p

    Celiac disease IgA (CD IgA) can activate fibronectin-bound transglutaminase 2 (FN-TG2) under reducing conditions.

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    <p>The effect of CD IgA on TG2 enzymatic activity was assessed by EZ-Link Pentylamine-Biotin (5-BP) incorporation on FN. In the absence of both the reducing agent, dithiothreitol (DTT) and Ca<sup>2+</sup>, TG2, alone or with any IgA, was enzymatically inactive. Maximal TG2 activity was obtained in the presence of DTT and Ca<sup>2+</sup> even in the presence of class A immunoglobulins. On the contrary, in the presence of a reducing agent, DTT, but without exogenous Ca<sup>2+</sup>, CD IgA was able to induce the activation of TG2 in contrast to non-celiac IgA (H IgA). Bars represent mean 5-BP incorporation on FN as fold of TG2 in the absence of DTT and Ca<sup>2+</sup>. Error bars indicate standard error of the mean; P-value < 0.05 was considered significant ( <sub>***</sub>p<0.001, <sub>**</sub>p<0.01 and <sub>*</sub>p<0.05). Data derived from three independent experiments, repeated in quadruplicate are shown.</p

    Anti-microbial antibodies in celiac disease: Trick or treat?

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    AIM: To determine the prevalence of a new set of anti-glycan and anti-outer membrane protein (anti-OMP) antibodies in a Hungarian cohort of adult Celiac disease (CD) patients

    Uncovering the gap: Coeliac disease knowledge among healthcare professionals in the Danube region

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    Abstract Background Several studies have shown that the knowledge about coeliac disease (CD) is not satisfactory among healthcare professionals (HCP). The aim of our study was to assess the knowledge of HCPs about CD in the Danube region. Methods HCPs from 8 countries in the Danube region were asked to complete the web-based questionnaire about CD. Scores of HCPs were compared according to their speciality, work experience and country of residence. The results were compared with the results of a similar study conducted in Central Europe within the Focus IN CD project in 2016. Results Questionnaire was completed by 799 HCPs from Austria, Croatia, Czech Republic, Hungary, Moldova, Romania, Serbia, and Slovenia. Mean score achieved by HCPs was 52.2%. Paediatric gastroenterologists scored the highest (75.3%). Comparing the data with the study conducted in Central Europe in 2016, we found a significant rise (p < 0.001) in the knowledge of paediatric gastroenterologists. Also, HCPs who previously took part in the Focus IN CD project, achieved higher score (61.1% vs. 50.8%; p < 0.001). Conclusion The knowledge about CD among HCPs in Danube region is not satisfactory. There has been a significant increase in the knowledge of paediatric gastroenterologists, showing the benefit of various awareness raising activities that were carried out recently
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