Secretion of celiac disease autoantibodies after in vitro gliadin challenge is dependent on small-bowel mucosal transglutaminase 2-specific IgA deposits

Background: In celiac disease gluten, the disease-inducing toxic component in wheat, induces the secretion of autoantibodies which are targeted against transglutaminase 2 (TG2). These autoantibodies are produced in the small-intestinal mucosa, where they can be found deposited extracellularly below the epithelial basement membrane and around mucosal blood vessels. In addition, during gluten consumption these autoantibodies can also be detected in patients' serum but disappear from the circulation on a gluten-free diet. Interestingly, after adoption of a gluten-free diet the serum autoantibodies disappear from the circulation more rapidly than the small-intestinal mucosal autoantibody deposits. The toxicity of gluten and the secretion of the disease-specific autoantibodies have been widely studied in organ culture of small-intestinal biopsy samples, but results hitherto have been contradictory. Since the mucosal autoantibodies disappear slowly after a gluten-free diet, our aim was to establish whether autoantibody secretion to organ culture supernatants in treated celiac disease patient biopsies is related to the duration of the diet and further to the preexistence of mucosal TG2-specific IgA deposits in the cultured biopsy samples. Results: In the organ culture system conducted with biopsies derived from treated celiac disease patients, gliadin induced secretion of autoantibodies to culture supernatants, reduced epithelial cell height and increased the density of lamina proprial CD25+ cells. However, these changes could be demonstrated only in biopsies from short-term treated celiac disease patients, where the small-intestinal mucosal TG2-specific IgA autoantibody deposits were still present. Furthermore, in these biopsies autoantibody secretion could be stimulated fully only after a 48-hour gliadin challenge. Conclusion: Our results show that studies focusing on the toxic effects of gliadin in the organ culture system should be carried out with biopsy samples from short-term treated celiac disease patients who are likely still to have mucosal IgA deposits present. In addition to providing an explanation for the discrepancies in previous publications, the present study also enables further validation of the organ culture method

The impact of human breast milk components on the infant metabolism

Background & aims Breastfeeding is beneficial for mothers and infants. Underlying mechanisms and biochemical mediators thus need to be investigated to develop and support improved infant nutrition practices promoting the child health. We analysed the relation between maternal breast milk composition and infant metabolism. Methods 196 pairs of mothers and infants from a European research project (PreventCD) were studied. Maternal milk samples collected at month 1 and month 4 after birth were analysed for macronutrient classes, hormone, and fatty acid (FA) content. Phospholipids, acylcarnitines, and amino acids were measured in serum samples of 4-month old infants. Associations between milk components and infant metabolites were analysed with spearman correlation and linear mixed effect models (LME). P-values were corrected for multiple testing (P-LME). Results Month 1 milk protein content was strongly associated with infant serum lyso-phosphatidylcholine (LPC) 14: 0 (P-LME = 0.009). Month 1 milk insulin was associated to infant acetylcarnitine (P-LME = 0.01). There were no associations between milk protein content and serum amino acids and milk total fat content and serum polar lipids. Middle- and odd-chain FA% in breast milk at both ages were significantly related to serum LPC and sphingomyelins (SM) species in infant serum (all P-LME < 0.05), while FA% 20: 5n(-3) and 22: 6n(-3) percentages were significantly associated to serum LPC 22:6 (P-LME = 1.91x10(-4)/7.93x10(-5)) in milk only at month 4. Other polyunsaturated fatty acids and hormones in milk showed only weak associations with infant serum metabolites. Conclusions Infant serum LPC are influenced by breast milk FA composition and, intriguingly, milk protein content in early but not late lactation. LPC 14:0, previously found positively associated with obesity risk, was the serum metabolite which was the most strongly associated to milk protein content. Thus, LPC 14:0 might be a key metabolite not only reflecting milk protein intake in infants, but also relating high protein content in milk or infant formula to childhood obesity risk

A gene-centric approach to biomarker discovery identifies transglutaminase 1 as an epidermal autoantigen

Publisher Copyright: © 2021 National Academy of Sciences. All rights reserved.Autoantigen discovery is a critical challenge for the understanding and diagnosis of autoimmune diseases. While autoantibody markers in current clinical use have been identified through studies focused on individual disorders, we postulated that a reverse approach starting with a putative autoantigen to explore multiple disorders might hold promise. We here targeted the epidermal protein transglutaminase 1 (TGM1) as a member of a protein family prone to autoimmune attack. By screening sera from patients with various acquired skin disorders, we identified seropositive subjects with the blistering mucocutaneous disease paraneoplastic pemphigus. Validation in further subjects confirmed TGM1 autoantibodies as a 55% sensitive and 100% specific marker for paraneoplastic pemphigus. This gene-centric approach leverages the wealth of data available for human genes and may prove generally applicable for biomarker discovery in autoimmune diseases.Peer reviewe

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

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

Mână de mână cu Boala Celiacă (BC)

Proiectul CD SKILLS PP13 a Universității de Stat de Medicină și Farmacie “Nicolae Testemițanu” din Republica Moldova și permisă spre traducere din limba engleză cu suportul tehnic al echipei de implementare: Tatiana Raba, Olesea Nicu, Anton Pivtora

Gluten-dependent Small Bowel Mucosal Transglutaminase 2–specific IgA Deposits in Overt and Mild Enteropathy Coeliac Disease

Objectives: In coeliac disease, immunoglobulin (Ig)A-class autoantibodies against transglutaminase-2 are produced in the small intestinal mucosa, where they are deposited extracellularly. It remains unclear whether positive intestinal transglutaminase-2-targeted IgA deposits in subjects having normal small bowel mucosal morphology are signs of early-stage coeliac disease. We evaluated the gluten dependency of these deposits in overt and mild enteropathy coeliac disease. Patients and Methods: All together 48 subjects suspected of coeliac disease but having normal small bowel mucosal villi were enrolled; 28 of them had latent coeliac disease. The remaining 20 having positive intestinal IgA deposits adopted a gluten-free diet before villous atrophy had developed. For comparison, 13 patients with overt coeliac disease and 42 noncoeliac controls were studied. Small bowel mucosal transglutaminase-2-specific autoantibodies were compared with villous morphology, intraepithelial lymphocyte densities, and serum coeliac autoantibodies. Results: Intestinal IgA deposits were seen in all but 1 of the patients with latent coeliac disease, when the morphology was still intact; the intensity of these deposits increased as villous atrophy developed and decreased again on a gluten-free diet. In 20 patients with intestinal IgA deposits in normal villi, the intensity of the deposits decreased with the diet similarly to that seen in patients with overt coeliac disease. Mucosal IgA deposits were seen initially only in 5% of noncoeliac controls and in 8% after extended gluten consumption. Conclusions: The response of small bowel mucosal transglutaminase-2-specific IgA deposits for dietary intervention was similar in overt and mild enteropathy coeliac disease. Detection of such IgA deposits thus offers a good diagnostic tool to uncover early-stage coeliac disease

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

<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 ( _**p<0.001 and _*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).

<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 ( _*p<0.01 and _**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.

<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 ( _*p<0.05 and _**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