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Background: Reduced activity of the sucrase-isomaltase (SI) enzyme can cause gastrointestinal symptoms. Biochemical measurement of SI activity in small intestinal biopsies is presently considered the gold standard for the diagnosis of SI deficiency, but this invasive test is not suitable as a routine diagnostic tool., Aim: To evaluate a 13 C-sucrose-breath test ( 13 CSBT) as a diagnostic tool for SI deficiency in an adult population., Methods: 13 CSBT results were compared to sucrase activity measured in duodenal biopsies., Results: Forty patients with gastrointestinal symptoms were included in the study, 4 of whom had celiac disease and the rest ( n  = 36) had normal histological findings. Nine patients (22.5%) had low sucrase activity measured using duodenal biopsies. No correlation was observed between enzymatic sucrase activity and the 13 CSBT results. The 13 CSBT-curves for the celiac patients versus patients with normal duodenal histology demonstrated that the patients with celiac disease were within the lower range of the distribution., Conclusion: We observed a mismatch between the 13 CSBT results and the biochemically measured sucrase activity, suggesting that SI activity is not uniformly distributed throughout the small intestines. This methodological discrepancy should be acknowledged when diagnosing SI deficiency.

We comment here on the article by Stefanolo et al entitled "Effect of Aspergillus niger prolyl endopeptidase in patients with celiac disease on a long-term gluten-free diet", published in the World Journal of Gastroenterology . Celiac disease is a well-recognized systemic autoimmune disorder. In genetically susceptible people, the most evident damage is located in the small intestine, and is caused and worsened by the ingestion of gluten. For that reason, celiac patients adopt a gluten-free diet (GFD), but it has some limitations, and it does not prevent re-exposure to gluten. Research aims to develop adjuvant therapies, and one of the most studied alternatives is supplementation with Aspergillus niger prolyl endopeptidase protease (AN-PEP), which is able to degrade gluten in the stomach, reducing its concentration in the small intestine. The study found a high adherence to the GFD, but did not address AN-PEP as a gluten immunogenic peptide reducer, as it was only tested in patients following a GFD and not in gluten-exposing conditions. This study opens up new research perspectives in this area and shows that further study is needed to clarify the points that are still in doubt., Competing Interests: Conflict-of-interest statement: The authors declare having no conflicts of interest., (©The Author(s) 2024. Published by Baishideng Publishing Group Inc. All rights reserved.)

Enzymatic modification of gliadin peptides by human transglutaminase 2 (TG2) is a key mechanism in the pathogenesis of celiac disease (CD) and represents a potential therapeutic target. Recently, we have identified the small oxidative molecule PX-12 as an effective inhibitor of TG2 in vitro. In this study, we further investigated the effect of PX-12 and the established active-site directed inhibitor ERW1041 on TG2 activity and epithelial transport of gliadin peptides. We analyzed TG2 activity using immobilized TG2, Caco-2 cell lysates, confluent Caco-2 cell monolayers and duodenal biopsies from CD patients. TG2-mediated cross-linking of pepsin-/trypsin-digested gliadin (PTG) and 5BP (5-biotinamidopentylamine) was quantified by colorimetry, fluorometry and confocal microscopy. Cell viability was tested with a resazurin-based fluorometric assay. Epithelial transport of promofluor-conjugated gliadin peptides P31-43 and P56-88 was analyzed by fluorometry and confocal microscopy. PX-12 reduced TG2-mediated cross-linking of PTG and was significantly more effective than ERW1041 (10 µM, 15 ± 3 vs. 48 ± 8%, p < 0.001). In addition, PX-12 inhibited TG2 in cell lysates obtained from Caco-2 cells more than ERW1041 (10 µM; 12 ± 7% vs. 45 ± 19%, p < 0.05). Both substances inhibited TG2 comparably in the intestinal lamina propria of duodenal biopsies (100 µM, 25 ± 13% vs. 22 ± 11%). However, PX-12 did not inhibit TG2 in confluent Caco-2 cells, whereas ERW1041 showed a dose-dependent effect. Similarly, epithelial transport of P56-88 was inhibited by ERW1041, but not by PX-12. Cell viability was not negatively affected by either substance at concentrations up to 100 µM. PX-12 did not reduce TG2 activity or gliadin peptide transport in confluent Caco-2 cells. This could be caused by rapid inactivation or degradation of the substance in the Caco-2 cell culture. Still, our in vitro data underline the potential of the oxidative inhibition of TG2. The fact that the TG2-specific inhibitor ERW1041 reduced the epithelial uptake of P56-88 in Caco-2 cells further strengthens the therapeutic potential of TG2 inhibitors in CD.

Coeliac disease (CD) is a chronic, immune-mediated small intestinal enteropathy, accompanied with gluten-triggered oxidative damage of duodenal mucosa. Previously, our research group reported an increased mucosal level of the antioxidant protein Parkinson's disease 7 (PARK7) in children with CD. In the present study, we investigated the role of increased PARK7 level on the epithelial cell and mucosal integrity of the small intestine. The presence of PARK7 was investigated using immunofluorescent staining on duodenal mucosa of children with CD and on FHs74Int duodenal epithelial cells. To investigate the role of oxidative stress, FHs74Int cells were treated with H 2 O 2 in the absence or presence of Comp23, a PARK7-binding compound. Intracellular accumulation of reactive oxygen species (ROS) was determined by DCFDA-based assay. Cell viability was measured by MTT, LDH, and Annexin V apoptosis assays. Disruption of cytoskeleton and cell adhesion was investigated by immunofluorescence staining and by real-time RT PCR. Effect of PARK7 on mucosal permeability was investigated ex vivo using intestinal sacs derived from control and Comp-23-pretreated mice. Comp23 treatment reduced the H 2 O 2 -induced intracellular accumulation of ROS, thus preserving the integrity of the cytoskeleton and also the viability of the FHs74Int cells. Accordingly, Comp23 treatment increased the expression of antioxidants ( NRF2 , TRX1 , GCLC , HMOX1 , NQO1 ), cell-cycle regulators ( TP53 , CDKN1A , PCNA , BCL2 , BAX ), and cell adhesion molecules ( ZO1 , CDH1 , VCL , ITGB5 ) of H 2 O 2 -treated cells. Pretreatment with Comp23 considerably decreased the small intestinal permeability. In this study, we demonstrate that PARK7-binding Comp23 reduces the oxidative damage of duodenal epithelial cells, via increased expression of NRF2- and P53-regulated genes. Our results suggest that PARK7 plays a significant role in the maintenance of mucosal integrity in CD., Competing Interests: The authors declare no conflict of interest., (Copyright © 2020 Apor Veres-Székely et al.)

Type 2 transglutaminase (TG2) is a ubiquitous enzyme able to modify gliadin peptides introduced into the organism through the diet. By means of its catalytic activity, TG2 seems to have an important pathogenetic role in celiac disease (CD), an inflammatory intestinal disease caused by the ingestion of gluten-containing cereals. A strong autoimmune response to TG2 characterizes CD development. Anti-TG2 antibodies specifically derange the uptake of the α-gliadin peptide 31-43 by control, but not by celiac dermal fibroblasts, underlying some different constitutive features regarding TG2 in healthy and celiac subjects. Our aim was to investigate whether these differences depended on a different TG2 subcellular distribution and whether peptide 31-43 differentially regulated TG2 expression and activity in cells of the two groups of subjects. We found that TG2 was more abundantly associated with membranes of celiac fibroblasts than of control cells, in particular with the early endosomal and autophagic compartments. We also found that peptide 31-43 differentially affected TG2 expression and activity in the two groups of cells, activating TG2 more in control than in celiac cells and inducing TG2 expression in celiac cells, but not in control ones. The different TG2 subcellular localization and the different way the peptide 31-43 modulates TG2 activity and availability into control and CD cells suggested that TG2 is involved in the definition of a constitutive CD cellular phenotype, thus having an important and still undefined role in CD pathogenesis.

Background: Deamidated gliadin peptide (DGP) is a relatively new serologic assay used in diagnosis and monitoring of celiac disease. DGP IgG is recommended by some in pediatric patients <2 y. Use in other pediatric populations is not well established. The utility of the DGP screen (IgG + IgA) in patients with moderate increase of tissue transglutaminase (TTG) IgA has not been studied., Methods: Cases between January 2015 and October 2017 in which a patient had TTG IgA greater >19 and <100, DGP screen, and biopsy were collected. Indication for biopsy and diabetes diagnosis were recorded. Of 495 patients screened, 31 met criteria., Results: The sensitivity and specificity of DGP screen were calculated, and were 87.4% and 56%, respectively; though lower in patients with diabetes., Conclusions: The study suggests in patients with moderately increased TTG-IgA, DGP screen lacks specificity and does not provide additional information about whether or not to biopsy., (Copyright © 2019. Published by Elsevier B.V.)

Background and Purpose: Magnitude of gluten-specific T-cell responses in coeliac disease (CD) might be dependent on HLA-DQ2 gene dose. We aimed to investigate the effects of HLA-DQB1*02 allele dose on clinical outcomes., Methods: We reviewed the charts of all coeliac patients attending to three Hungarian university clinics after 1997 and included those patients, who (a) were diagnosed with CD, (b) underwent high-resolution HLA typing and (c) were ≥18 years at the time of data collection. HLA typing was performed to determine DQB1*02 allele dose. Patients were divided into risk groups by DQB1*02 allele dose, as follows: high-, intermediate- and low-risk groups corresponded to a double, single and zero doses, respectively. We used ANOVA and Pearson's chi-squared test to explore association between HLA risk and clinical variables., Results: A total of 727 coeliac patients attended the clinics but only 105 (14.4%) patients were eligible for inclusion. High, intermediate and low HLA risk patients comprised 35.3%, 52.3% and 12.3% of the study population, respectively. Double dose of HLA-DQB1*02 was more frequent in patient with high tTGA level (>10 times the upper limit of normal; p = 0.045). Gene dose was not associated with younger age at diagnosis (p = 0.549), gender (p = 0.739), more severe diagnostic histology (p = 0.318), more frequent classical presentation (p = 0.846), anaemia (p = 0.611), metabolic bone disease (p = 0.374), dermatitis herpetiformis (p = 0.381) and autoimmune diseases (p = 0.837)., Conclusions: Our study shows a significant gene dose effect in terms of tTGA level at diagnosis, but no significant association between HLA-DQB1*02 allele dose and the clinical outcomes in CD., (© 2019 The Authors. International Journal of Immunogenetics Published by John Wiley & Sons Ltd.)

Coeliac disease and dermatitis herpetiformis (DH) are characterized by autoantibodies targeting transglutaminase (TG)2 and TG3, respectively. Previous studies show that TG2 antibodies are produced in the gut and can be assessed in organ culture of small-intestinal biopsies from patients with coeliac disease. Thus far, no studies have investigated TG3 antibodies in organ culture of biopsies from patients with DH, or exploited the method in DH. The aim of this study was to investigate TG3 and TG2 antibody responses in serum and small-intestinal biopsies from patients with DH with active disease, and from those in remission. The majority of patients with DH were negative for both serum and organ culture medium TG2-targeting antibodies. Surprisingly, patients with active DH secreted TG3 antibodies into the culture medium despite seronegativity. In patients secreting high levels of TG3 antibodies into the culture medium, we also detected TG3-antibody-positive cells in the small-intestinal mucosa. These findings suggest that TG3 antibodies can be investigated in the organ culture system and that their secretion occurs in the small intestine, especially in active DH.

Objective: To do a serological screening for celiac disease in patients with unexplained liver cytolysis., Materials and Methods: Fifty-six patients with liver cytolysis without known aetiology were studied. Endomysial antibodies were determined by indirect immunofluorescence on human umbilical cord. Two thousand and five hundred blood donors served as control group. For statistical analysis, we used Chi-square or Fisher's exact test., Results: The frequency of IgA endomysial antibodies in our patients was significantly higher than in the control group (8.92% vs. 0.28%, p < .001). In female, endomysial antibodies were significantly more frequent in patients than in healthy subjects (12.12% vs. 0.4%; p < .001). In male, endomysial antibodies were significantly more frequent in patients than in healthy subjects (4.34% vs. 0.22%; p = .006). The frequency of positive EMA in female patients was higher than in male, but the difference was not statistically significant (12.12% vs. 4.43%; p = .6). Two patients were non-compliant with the gluten-free diet. One patient was out of touch. For the two other patients, transaminase levels reverted to normal level within six months of strict gluten withdrawal., Conclusions: A screening for celiac disease should be included within the diagnosis protocol of liver cytolysis.

It has been hypothesized that gluten-dependent production of anti-tissue-transglutaminase 2 (anti-TG2) antibodies may occur only at an intestinal level. We have investigated intestinal production of anti-TG2 antibodies in 136 patients with normal serum levels of anti-TG2 antibodies and normal duodenal mucosa. Intestinal deposits of anti-TG2 antibodies were evaluated by immunofluorescence and anti-TG2 antibodies released in organ culture supernatants measured by ELISA. Intestinal antibody libraries were obtained from 10 subjects. Immunohistochemistry for CD25⁺, CD3⁺, and TCR-γδ⁺ was assessed in subjects with positive ( n = 32) and negative ( n = 31) intestinal anti-TG2 antibodies. Globally 33/136 (24%) seronegative patients produced anti-TG2 autoantibodies at an intestinal level. Antibody libraries analysis confirmed the anti-TG2 antibodies mucosal production in all ( n = 8) positive subjects. Lamina propria CD25⁺ cell count was significantly ( p < 0.05) higher in patients with intestinal anti-TG2. Moreover, 13/32 (41%) of them showed high TCR-γδ⁺/CD3⁺ ratios. Intestinal anti-TG2 antibody production does not show absolute specificity for CD. It is seen more often in association with inflamed mucosa. Further investigations are necessary to prove the possible role of dietary gluten.

Objective: To find out correlation between serum anti-tissue transglutaminase immunoglobulin-A (tTGA) levels and Marsh grading on duodenal histopathology in Celiac disease (CD)., Methods: In a prospective cohort study, a total of 52 symptomatic patients between age group of 2-18 y were enroled. All enroled patients were subjected to upper GI endoscopy by an experienced endoscopist. Two biopsies each from the bulb (D1) and second part (D2) of the duodenum were taken and Marsh grading was performed by a single experienced pathologist. Serum tTGA levels were also performed to find out correlation between serum tTGA levels and Marsh grading., Results: The mean age of the patients was 8.21 ± 3.45 y (Range: 2-16 y). Anemia was the most common non-gastrointestinal (GI) sign and was present in 73% of the cases. However the authors could not find out any significant association between Marsh grading and hemoglobin levels (r = 0.32, p > 0.05). Serum tTGA levels were found to be positively correlated with Marsh grading (Spearmen correlation coefficient ρ = 0.74, p 0.000). Significant differences were found in tTGA levels between different Marsh gradings (ANOVA test) (p 0.000). Receiver-operator curve (ROC) analysis cut-off value of serum tTGA for predicting villous atrophy was 178.8 (nine times of cut-off value) with sensitivity of 100% and specificity of 85.7%., Conclusions: Serum tTGA levels can be used to predict villous atrophy and biopsy may be avoided in strongly suspected cases with more than 9 times of cut-offs.

Objectives: Celiac disease is a chronic autoimmune disease in which gene-environment interactions cause the immune system to unfavorably react to naturally gluten-containing foods. PTPN22 plays a crucial role in regulating the function of various cells of the immune system, particularly T cells. Polymorphisms of the PTPN22 gene have been associated with many autoimmune diseases. The present genetic association study was conducted to investigate the possible associations between PTPNTT single nucleotide polymorphisms (SNPs) and celiac disease in an Iranian population., Materials and Methods: The study population consisted of 45 patients with celiac disease and 93 healthy controls. The study genotyped five SNPs of the PTPN22 gene: rs12760457, rs1310182, rs1217414, rs33996649, and rs2476601., Results and Conclusions: Control and patient groups did not differ on the genotype distribution of four of five investigated SNPs in the PTPN22 gene, for example, rs12760457, rs2476601, rs1217414, and rs33996649. The only investigated PTPN22 variant, which could be associated with CD, was rs1310182. A significant increase in the carriage of the T allele of rs1310182 in CD patients was observed (OR (95% CI) = 11.42 (5.41, 24.1), p value < 0.0001). The TT genotype of this SNP was significantly associated with celiac disease. Our study suggests that the rs1310182 SNP of PTPN22 gene may be a predisposing factor of celiac disease in the Iranian population. Further studies are required to investigate the issue in other racial and ethnic subgroups.

Celiac sprue, also known as gluten-sensitive enteropathy, is a chronic disease suffered by approximately 1% of the world's population. Engineered enzymes have been emerging to treat celiac disease by hydrolyzing the pathogenic peptides of gluten. For example, Kuma010 has been studied experimentally and proved to be a promising gluten hydrolase under gastric conditions. However, the detailed catalytic mechanism and the substrate specificity are still unclear. In this paper, quantum-mechanical/molecular-mechanical (QM/MM) molecular dynamics (MD) and free energy simulations were performed to determine the catalytic mechanism, the substrate specificity, and the role of the active-site residues during the reaction. The results given here demonstrate that the Kuma010 has a similar catalytic mechanism but different substrate specificity as wild-type kumamolisin-As. Binding properties of the enzyme (especially mutated residues) and substrate complex are discussed, and activation free energy barriers toward different substrates have also been examined. The computational free energy results are in reasonable agreement with the experimental data. The strategy for developing next-generation gluten hydrolases is discussed.

Initiation of celiac disease is triggered in the gastrointestinal tract by transglutaminase 2 (TG2) assisted deamidation of gluten peptides. Deamidation is a side-reaction to transamidation and occurs if primary amines are absent. In contrast to deamidation, transamidation does not trigger an immune response. The aim of the study was to identify a suitable food additive that interacts with TG2 binding motives in gluten-derived peptides to prevent deamidation/transamidation. Homology modelling of α2-gliadin and computational screening of compounds for their binding affinity to a common TG2 binding motive (P)QLP were done by using computational approaches followed by experimental testing of TG2 activity. A database containing 1174 potential food grade ligands was screened against the model of α2-gliadin (27 out of 33 aa). Out of the five best ligands, ascorbyl palmitate, was observed to decrease TG2 transamidation of gliadin by 82% ± 2%. To completely silence the transamidation, we added zinc chloride (ZnCl 2 ), and thereby reached a 99% ± 1% inhibition of TG2 activity. In addition, we conducted a pilot experiment in which ascorbyl palmitate was observed to decrease TG2 deamidation of gliadin completely. We propose ascorbyl palmitate in combination with ZnCl 2 with the future perspective to become an additive in celiac-safe foods.

Celiac disease is an autoimmune disorder that causes inflammation and destruction in the small intestine of genetically susceptible individuals following ingestion of gluten. Awareness of the disease has increased; however, it remains a challenge to diagnose. This review summarizes the intestinal and extraintestinal cross-sectional imaging findings of celiac disease. Small intestine fold abnormalities are the most specific imaging findings for celiac disease, whereas most other imaging findings reflect a more generalized pattern seen with malabsorptive processes. Familiarity with the imaging pattern may allow the radiologist to suggest the diagnosis in patients with atypical presentations in whom it is not clinically suspected. Earlier detection allows earlier treatment initiation and may prevent significant morbidity and mortality that can occur with delayed diagnosis. Refractory celiac disease carries the greatest risk of mortality due to associated complications, including cavitating mesenteric lymph node syndrome, ulcerative jejunoileitis, enteropathy-associated T cell lymphoma, and adenocarcinoma, all of which are described and illustrated. Radiologic and endoscopic investigations are complimentary modalities in the setting of complicated celiac disease.

Disaccharidases (DS) are brush border enzymes embedded in the microvillous membrane of small intestinal enterocytes. In untreated coeliac disease (CD), a general decrease of DS activities is seen. This manuscript reviews different aspects of DS activities in CD: their utility in the diagnosis and their application to in vitro toxicity testing. The latter has never been established in CD research. However, with the recent advances in small intestinal organoid techniques, DS might be employed as a biomarker for in vitro studies. This includes establishment of self-renewing epithelial cells raised from tissue, which express differentiation markers, including the brush border enzymes. Determining duodenal DS activities may provide additional information during the diagnostic workup of CD: (i) quantify the severity of the observed histological lesions, (ii) provide predictive values for the grade of mucosal villous atrophy, and (iii) aid diagnosing CD where minor histological changes are seen. DS can also provide additional information to assess the response to a gluten-free diet as marked increase of their activities occurs four weeks after commencing it. Various endogenous and exogenous factors affecting DS might also be relevant when considering investigating the role of DS in other conditions including noncoeliac gluten sensitivity and DS deficiencies.

Objectives: Persistent viral infections have been implicated in the etiology of autoimmune diseases in adulthood, but it is not known whether herpesviruses are associated with the development of celiac disease autoimmunity in childhood. We assessed whether herpesvirus infections are associated with transglutaminase type 2 antibody (TG2A) concentrations in children at 6 years of age., Methods: The present study was embedded within a population-based prospective cohort study. Serum immunoglobulin G levels against Epstein-Barr virus, cytomegalovirus (CMV), and herpes simplex virus type 1 were measured by enzyme-linked immunosorbent assay , and TG2A concentrations with fluorescence enzyme immunoassay in 4420 children at 6 years of age. Children were categorized based on TG2A concentrations into negative (<7 U/mL), positive (≥7-70 U/mL), and strongly positive (≥70 U/mL), that is, 10 times upper limit normal., Results: Fifty-nine children (1.3%) were TG2A positive, and of these 31 (53%) had concentrations 70 U/mL or more. Children with TG2A concentrations 70 U/mL or more were less often infected with CMV (adjusted odds ratio (aOR) 0.38, 95% CI 0.14-0.98, P = 0.04) and with any of the 3 viruses (aOR 0.38, 95% CI 0.18-0.78, P < 0.01) than children with TG2A negative concentrations. In addition, children with TG2A concentrations 70 U/mL or more were less often infected with 2 or more viruses than children with TG2A negative concentrations (aOR 0.15, 95% CI 0.03-0.65, P = 0.01)., Conclusions: Both CMV single infection and combined CMV, Epstein-Barr virus and/or herpes simplex virus type 1 infections are inversely associated with strongly TG2A positivity. This may indicate a protective effect of herpesvirus infections in the pathogenesis of celiac disease autoimmunity.

Celiac disease is triggered by partially digested gluten proteins. Enzyme therapies that complete protein digestion in vivo could support a gluten-free diet, but the barrier to completeness is high. Current options require enzyme amounts on the same order as the protein meal itself. In this study, we evaluated proteolytic components of the carnivorous pitcher plant (Nepenthes spp.) for use in this context. Remarkably low doses enhance gliadin solubilization rates, and degrade gliadin slurries within the pH and temporal constraints of human gastric digestion. Potencies in excess of 1200:1 (substrate-to-enzyme) are achieved. Digestion generates small peptides through nepenthesin and neprosin, the latter a novel enzyme defining a previously-unknown class of prolyl endoprotease. The digests also exhibit reduced TG2 conversion rates in the immunogenic regions of gliadin, providing a twin mechanism for evading T-cell recognition. When sensitized and dosed with enzyme-treated gliadin, NOD/DQ8 mice did not show intestinal inflammation, when compared to mice challenged with only pepsin-treated gliadin. The low enzyme load needed for effective digestion suggests that gluten detoxification can be achieved in a meal setting, using metered dosing based on meal size. We demonstrate this by showing efficient antigen processing at total substrate-to-enzyme ratios exceeding 12,000:1.

Introduction: Transglutaminases (TGases) are a class of enzymes that play multifunctional roles. Their protein-crosslinking activity has been linked to fibrosis and Huntington's disease, their glutamine deamidation activity has been related to celiac disease and their GTP-binding activity has been implicated in cancer. All of these physiological disorders have prompted the development of inhibitors, which has accelerated dramatically over the past decade., Areas Covered: This review presents an overview of TGase inhibitors published in the patent literature, from the first compounds developed in the late 1980's, to the current date. This article is focussed on the chemical structure of new inhibitors and their probable mechanism of action., Expert Opinion: Comparison of effective TGase inhibitors reveals common structural features that may guide future design. Many of these elements are embodied in the first TGase inhibitor to recently enter into clinical trials.

Background: Celiac disease is an autoimmune systemic disorder in genetically predisposed individuals precipitated by gluten ingestion., Objective: In this study, we aimed to determine asymptomatic spike-and-wave findings on electroencephalography in children with celiac disease., Methods: A total of 175 children with the diagnosis of celiac disease (study group) and 99 age- and sex-matched healthy children as controls (control group) were included in the study. In order to determine the effects of gluten free diet on laboratory and electroencephalography findings, the celiac group is further subdivided into two as newly-diagnosed and formerly-diagnosed patients. Medical histories of all children and laboratory findings were all recorded and neurologic statuses were evaluated. All patients underwent a sleep and awake electroencephalography., Results: Among 175 celiac disease patients included in the study, 43 were newly diagnosed while 132 were formerly-diagnosed patients. In electroencephalography evaluation of patients the epileptiform activity was determined in 4 (9.3%) of newly diagnosed and in 2 (1.5%) of formerly diagnosed patients; on the other hand the epileptiform activity was present in only 1 (1.0%) of control cases. There was a statistically significant difference between groups in regards to the presence of epileptiform activity in electroencephalography. Pearson correlation analysis revealed that epileptiform activity in both sleep and awake electroencephalography were positively correlated with tissue transglutaminase levels (P=0.014 and P=0.019, respectively)., Conclusion: We have determined an increased epileptiform activity frequency among newly-diagnosed celiac disease patients compared with formerly-diagnosed celiac disease patients and control cases. Moreover the tissue transglutaminase levels were also correlated with the presence of epileptiform activity in electroencephalography. Among newly diagnosed celiac disease patients, clinicians should be aware of this association and be alert about any neurological symptoms.

Antibodies to the autoantigen transglutaminase 2 (TG2) are a hallmark of celiac disease. We have studied the interaction between TG2 and an anti-TG2 antibody (679-14-E06) derived from a single gut IgA plasma cell of a celiac disease patient. The antibody recognizes one of four identified epitopes targeted by antibodies of plasma cells of the disease lesion. The binding interface was identified by small angle x-ray scattering, ab initio and rigid body modeling using the known crystal structure of TG2 and the crystal structure of the antibody Fab fragment, which was solved at 2.4 Å resolution. The result was confirmed by testing binding of the antibody to TG2 mutants by ELISA and surface plasmon resonance. TG2 residues Arg-116 and His-134 were identified to be critical for binding of 679-14-E06 as well as other epitope 1 antibodies. In contrast, antibodies directed toward the two other main epitopes (epitopes 2 and 3) were not affected by these mutations. Molecular dynamics simulations suggest interactions of 679-14-E06 with the N-terminal domain of TG2 via the CDR2 and CDR3 loops of the heavy chain and the CDR2 loop of the light chain. In addition there were contacts of the framework 3 region of the heavy chain with the catalytic domain of TG2. The results provide an explanation for the biased usage of certain heavy and light chain gene segments by epitope 1-specific antibodies in celiac disease., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Objectives: Guidelines recommend routine screening of liver function tests (LFTs) in patients diagnosed with celiac disease (CD). However, little is known about the prevalence of liver disorders in CD outside of Europe. Our aims were to estimate the prevalence of LFT abnormalities in CD and to evaluate the effect of a gluten-free diet (GFD) on LFTs., Methods: Adult patients with biopsy-proven CD were identified from a prospectively maintained database and matched with healthy controls. LFT levels for women and men were defined as abnormal based on the Third National Health and Nutrition Examination Survey (NHANES III) criteria. Data on demographics, coexisting liver diseases, and laboratory work-ups including aspartate transaminase (AST) and alanine transaminase (ALT) values at the time of diagnosis and on a GFD were recorded. Subsequently, data from this cohort were compared with data from 7,789 individuals participating in the National Health and Nutrition Examination Survey, 2009-2010. Univariate logistic regression, Wilcoxon signed-ranks, Student's t-test, χ(2), and Fischer's exact test were used for statistical analysis., Results: In 463 CD patients with ALT or AST levels at the time of CD diagnosis, 40.6% had elevated LFTs compared with 24.2% of treated CD patients (P<0.001) and 16.6% of matched controls (P<0.001). Similarly, 36.7% of CD patients on the NHANES database had abnormal ALT values compared with 19.3% of non-celiac patients (P=0.03). Approximately, 78.6% of CD patients with elevated LFTs at diagnosis normalized LFTs on a GFD after a mean duration of 1.5±1.5 years., Conclusions: Forty percent of individuals will have elevated LFTs at CD diagnosis; however, the majority will normalize with standard CD therapy. LFTs should be checked in all patients with CD and coexisting liver disorder should be considered in patients whose LFTs have not improved within a year on a GFD.

Anti-transglutaminase antibodies are the diagnostic marker of celiac disease, and are considered to be synthesized only by intestinal B-lymphocytes. During an infectious disease, these antibodies are transiently detected in serum. We show that these infection-triggered antibodies may not originate in the intestinal mucosa and are not an indication of celiac disease., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Introduction: The only current treatment for celiac disease is a strict gluten-free diet. The ubiquitous presence of gluten in groceries, however, makes the diet burdensome and difficult to maintain, and alternative treatment options are thus needed. Here, the important role of transglutaminase 2 (TG2) in the pathogenesis of celiac disease makes it an attractive target for drug development., Areas Covered: The present paper gives an overview of TG2 and addresses its significance in the pathogenesis of celiac disease. Moreover, the article summarizes preclinical studies performed with TG2 inhibitors and scrutinizes issues related to this therapeutic approach., Expert Opinion: Activation of TG2 in the intestinal mucosa is central in celiac disease pathogenesis and researchers have therefore suggested TG2 inhibitors as a potential therapeutic approach. However, a prerequisite for such a drug is that it should be specific for TG2 and not affect the activity of other members of the transglutaminase family. Such compounds have already been introduced and tested in vitro, but a major obstacle to further development is the lack of a well-defined animal model for celiac disease. Nonetheless, with encouraging results in preclinical studies clinical trials with TG2 inhibitors are eagerly awaited.

The results of the examination of patients with type I children,which was diagnosed with celiac disease and pancreatic exocrine insufficiency. To correct the PEN used pancreatin preparations and studied its effectiveness.

Aim. To clinically evaluate the activity of glucoamylase, maltase, saccharase, and lactase in the small intestinal mucosa (SIM) of patients with celiac disease. Subjects and methods. Twenty-nine patents with celiac disease were examined. The disease was first detected in 8 patients; in the remaining patients, it had been diagnosed 6 months to 35 years before. The diagnosis was verified by histological examinations of duodenal biopsy specimens and by determination of immunoglobulin (Ig) A and G antibodies to tissue transglutaminase (atTG) and gliadin (AGA) by an enzyme-linked immunosorbent assay (ELISA). Carbohydrase activities were estimated in the duodenal biopsy specimens, by applying the method of A. Dahlquist. Results. In the control group, the activities of glucoamylase, maltase, saccharase, and lactase averaged 598.8+184.2, 825.3+239.3, 180.2-68.1, and 53.4+16.3 ng/glucose/mg tissue min, respectively. In the patients with celiac disease, the average activities of all the examined enteric enzymes were significantly below the normal value even they had been on a gluten-free diet (GFD) for 10 years or longer. Complete SIM structural recovery (Marsh stage 0) occurred in only 7 of 18 patients who had been on a strictly GFD. Serological (atTG and AGA) tests got also negative in all the 7 patients with completely recovered SIM. Six of the latter patients continued to have abdominal bloating and borborygmus, unstable stool with a propensity for diarrhea and weakness. Each was detected to have a lower activity of one or a few enzymes. The activity of all the carbohydrases reached its normal value in only 1 patient and she felt healthy, without perceiving any food intolerance. Conclusion. The activity of membrane enzymes may serve as a marker for the degree of SIM recovery in patients with celiac disease.

A new amperometric immunosensor based on the covalent immobilization of tissue transglutaminase enzyme in its open conformation (open-tTG) was developed and optimized for determination of anti-tissue transglutaminase antibodies (anti-tTG) in human serum. Experimental design allowed us to find the optimal conditions for quantification of both IgA and IgG isotypes of anti-tTG in order to assess suitability of the device for diagnostic purposes. The glassy carbon electrodic substrate was electrochemically functionalized with gold nanoparticles and subsequently derivatized with a self-assembled monolayer of 11-mercaptoundecanoic acid for the covalent anchoring of the enzyme. This step was performed under carefully controlled conditions in order to keep the open conformation of the tTG. The immunosensor showed good analytical performance with limit of detection levels (1.7 AU mL(-1) for IgA and 2.7 AU mL(-1) for IgG) below the diagnostic threshold value (3.0 AU mL(-1)) and inter-sensor reproducibility giving RSD lower than 10%. The developed sensor was validated in serum samples from pediatric patients for clinical applications, using two ELISA kits specific for the determination of anti-tTG IgA and IgG antibodies as reference methods; good recovery rates ranging from 74% to 117% were calculated., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Rothia mucilaginosa, a natural microbial inhabitant of the oral cavity, cleaves gluten (gliadin) proteins at regions that are resistant to degradation by mammalian enzymes. The aim of this study was to investigate to what extent the R. mucilaginosa cell-associated enzymes abolish gliadin immunogenic properties. Degradation of total gliadins and highly immunogenic gliadin 33-mer or 26-mer peptides was monitored by SDS-PAGE and RP-HPLC, and fragments were sequenced by liquid chromatography and electrospray ionization tandem mass spectrometer (LC-ESI-MS/MS). Peptide deamidation by tissue transglutaminase (TG2), a critical step in rendering the fragments more immunogenic, was assessed by TG2-mediated cross-linking to monodansyl cadaverine (MDC), and by a +1-Da mass difference by LC-ESI-MS. Survival of potential immunogenic gliadin epitopes was determined by use of the R5 antibody-based ELISA. R. mucilaginosa-associated enzymes cleaved gliadins, 33-mer and 26-mer peptides into smaller fragments. TG2-mediated cross-linking showed a perfect inverse relationship with intact 33-mer and 26-mer peptide levels, and major degradation fragments showed a slow rate of MDC cross-linking of 6.18 ± 2.20 AU/min compared with 97.75 ± 10.72 and 84.17 ± 3.25 AU/min for the intact 33-mer and 26-mer, respectively, which was confirmed by reduced TG2-mediated deamidation of the fragments in mass spectrometry. Incubation of gliadins with Rothia cells reduced R5 antibody binding by 20, 82, and 97% after 30 min, 2 h, and 5 h, respectively, which was paralleled by reduced reactivity of enzyme-treated 33-mer and 26-mer peptides in the R5 competitive ELISA. Our broad complementary approach to validate gluten degrading activities qualifies R. mucilaginosa-associated enzymes as promising tools to neutralize T cell immunogenic properties for treatment of celiac disease., (Copyright © 2014 the American Physiological Society.)

Dermatitis herpetiformis (DH) is an extraintestinal manifestation of coeliac disease. Untreated coeliac disease patients are known to have transglutaminase 2 (TG2)-targeted IgA deposits in the small bowel mucosa. To evaluate whether similar intestinal IgA deposits are also present in DH and whether the deposits disappear with gluten-free diet, 47 untreated and 27 treated DH patients were studied. Seventy-nine percent of untreated and 41% of the treated DH patients had TG2-specific IgA deposits in the small bowel, and the presence of the deposits showed a significant association with the degree of small bowel villous atrophy (p < 0.001). Other coeliac-disease related inflammatory markers were also investigated, and the density of small bowel mucosal intraepithelial γδ(+) T cells was increased in 91% of untreated and 73% of treated DH patients. The results show that the majority of untreated DH patients have similar gluten-dependent TG2-specific IgA deposits the small bowel mucosa as coeliac disease patients.

Background & Aims: Gluten ingestion leads to small intestinal mucosal injury in patients with celiac disease, necessitating strict life-long exclusion of dietary gluten. Despite adherence to a gluten-free diet, many patients remain symptomatic and still have small intestinal inflammation. In this case, nondietary therapies are needed. We investigated the ability of ALV003, a mixture of 2 recombinant gluten-specific proteases given orally, to protect patients with celiac disease from gluten-induced mucosal injury in a phase 2 trial., Methods: We established the optimal daily dose of gluten to be used in a 6-week challenge study. Then, in the intervention study, adults with biopsy-proven celiac disease were randomly assigned to groups given ALV003 (n = 20) or placebo (n = 21) together with the daily gluten challenge. Duodenal biopsies were collected at baseline and after gluten challenge. The ratio of villus height to crypt depth and densities of intraepithelial lymphocytes were the primary end points., Results: A daily dose of 2 g gluten was selected for the intervention study. Sixteen patients given ALV003 and 18 given placebo were eligible for efficacy evaluation. Biopsies from subjects in the placebo group showed evidence of mucosal injury after gluten challenge (mean villus height to crypt depth ratio changed from 2.8 before challenge to 2.0 afterward; P = .0007; density of CD3(+) intraepithelial lymphocytes changed from 61 to 91 cells/mm after challenge; P = .0003). However, no significant mucosal deterioration was observed in biopsies from the ALV003 group. Between groups, morphologic changes and CD3(+) intraepithelial lymphocyte counts differed significantly from baseline to week 6 (P = .0133 and P = .0123, respectively). There were no statistically significant differences in symptoms between groups., Conclusions: Based on a phase 2 trial, the glutenase ALV003 appears to attenuate gluten-induced small intestinal mucosal injury in patients with celiac disease in the context of an everyday gluten-free diet containing daily up to 2 g gluten. Clinicaltrial.gov,, Numbers: NCT00959114 and NCT01255696., (Copyright © 2014 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background: Scalloping of duodenal folds noted on esophagogastroduodenoscopy (EGD) has been associated with various illnesses including celiac disease (CD). The aim of the present study was to examine the frequency of scalloping in pediatric patients undergoing EGD and to assess its significance in the diagnosis of CD. We also evaluated the association of scalloping with the histopathology and celiac serology in the subgroup of celiac patients., Patients and Methods: All children (0-18 years) who underwent EGD at Shaare Zedek Medical Center for any reason during a 2.5-year period were retrospectively included, yielding a consecutive cohort without selection bias. Relevant data were obtained from the patient files., Results: During the study period, 623 children underwent EGD of whom 149 (24%) were eventually diagnosed with CD. In 74/623children (12%), scalloping was seen and had a sensitivity of 48% (95% CI 0.40-0.57), specificity of 99% (0.98-0.99) and positive predictive value of 97% (0.9-0.99) to diagnose CD. The prevalence of scalloping increased with advancing stage of the Marsh classification from 33% (7/21) in Marsh 1 to 63% (34/54) in Marsh 3c (P < 0.001). Scalloping was associated with a significantly higher median tissue transglutaminase level (153 [IQR 98-168] versus 49 [IQR 11-143]; P = 0.011)., Conclusion: The results suggest that the diagnosis of CD is almost certain if isolated scalloping is observed during EGD done to rule out CD. Thus, attention to this finding may serve as an additional tool in the diagnosis of CD., (© 2013 The Authors. Digestive Endoscopy © 2013 Japan Gastroenterological Endoscopy Society.)

Introduction: Celiac disease (CD) is an autoimmunological gluten sensitive enteropathy occuring to genetically predisposed individuals. Active CD is accompanied by presence of multiple antibodies. Anti alpha enolase antibodies were reported in several autoimmunological disorders like rheumatoid arthritis, primary sclerosing cholangitis. Data about its presence and role in CD is avaricious., Aim: The aim of this study was to determine presence of anti alpha enolase antibodies in CD, correlation with gluten exposure, presence of anti tranglutaminase antibodies and Marsh scale., Methods: Sera from 31 patients with CD (21 females, 10 males) and 6 healthy subjects were collected. Evaluation of CD activity and adherence to gluten free diet were obtained by serology tests (presence of endomyslum antibodies and/or anti transglutamineses in IgA or IgG classes) and histological hallmarks. Anti alpha enolase antibodies were identified in sera using ELISA kit. Titres of anti alpha enolase antibodies were identified among patients with newly diagnosed CD, CD patients non adhering to gluten free diet (GFD), adhering to GFD and among healthy subjects., Results: Mean titre of anti alpha enolase antibodies was higher in CD patients (both treated and non treated) in comparison to control group, respectively 1.1 ng/mL, and 0.795 ngl mL. Among CD patients non adhering to gluten free diet mean titre was 1.4 ng/mL., Conclusions: Higher anti alpha enolase antibodies titres in non treated CD suggest usefulness of its measurement. These antibodies might be a novel marker of chronic inflammation among CD patients non adhering to GFD.

Background: Celiac disease is an inflammatory condition of the small intestine that affects genetically predisposed individuals after dietary wheat gliadin ingestion. Type 2-transglutaminase (TG2) activity seems to be responsible for a strong autoimmune response in celiac disease, TG2 being the main autoantigen. Several studies support the concept that celiac anti-TG2 antibodies may contribute to disease pathogenesis. Our recent findings on the ability of anti-TG2 antibodies to induce a rapid intracellular mobilization of calcium ions, as well as extracellular signal-regulated kinase phosphorylation, suggest that they potentially act as signaling molecules. In line with this concept, we have investigated whether anti-TG2 antibodies can induce phosphoproteome modification in an intestinal epithelial cell line., Methods and Principal Findings: We studied phosphoproteome modification in Caco-2 cells treated with recombinant celiac anti-TG2 antibodies. We performed a two-dimensional electrophoresis followed by specific staining of phosphoproteins and mass spectrometry analysis of differentially phosphorylated proteins. Of 14 identified proteins (excluding two uncharacterized proteins), three were hypophosphorylated and nine were hyperphosphorylated. Bioinformatics analyses confirmed the presence of phosphorylation sites in all the identified proteins and highlighted their involvement in several fundamental biological processes, such as cell cycle progression, cell stress response, cytoskeletal organization and apoptosis., Conclusions: Identification of differentially phosphorylated proteins downstream of TG2-antibody stimulation suggests that in Caco-2 cells these antibodies perturb cell homeostasis by behaving as signaling molecules. We hypothesize that anti-TG2 autoantibodies may destabilize the integrity of the intestinal mucosa in celiac individuals, thus contributing to celiac disease establishment and progression. Since several proteins here identified in this study were already known as TG2 substrates, we can also suppose that transamidating activity and differential phosphorylation of the same targets may represent a novel regulatory mechanism whose relevance in celiac disease pathogenesis is still unexplored.

Background: Common marmosets (Callithrix jacchus) are susceptible to gastrointestinal diseases. Sensitivity to nutritional elements, for example gluten, has been suggested, but a serological screening has not been performed yet., Methods: A gluten-containing diet was offered to 24 animals, followed by a gluten-free diet. During these diets, serum IgA antibodies to gliadin (AGA), tissue transglutaminase (tTG), deamidated gliadin (ADGA), and glycoprotein 2 (AGP2A) were determined. Body weight, diarrhea, and other clinical symptoms were recorded., Results: Gluten increased AGA, tTG, and AGP2A concentrations in 13 of 24 animals. A significant decline of AGA and AGP2A was seen on gluten withdrawal. Positive (AGA, tTG) animals presented diarrhea more frequently on gluten-containing diet and showed significantly increased body weight on gluten-free diet compared to negative animals., Conclusion: Gluten ingestion caused gastrointestinal symptoms in common marmosets, which disappeared on gluten withdrawal. Considering the immunological response to both diets, gluten sensitivity seems to be most likely., (© 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

An electrochemical magneto immunosensor for the detection of anti-transglutaminase antibodies (ATG2) in celiac disease was developed. The immunological reaction is performed on magnetic beads (MBs) as a solid support in which the transglutaminase enzyme (TG2) is covalently immobilized (TG2-MB) and then ATG2 were revealed by an antibody labeled with peroxidase. The electrochemical response of the enzymatic reaction with o-phenilendiamine and H₂O₂ as substrates by square wave voltammetry was correlated with the ATG2. Graphite-epoxi composite cylindrical electrodes and screen printed electrodes were used as transducers in the immunosensor. A total number of 29 sera from clinically confirmed cases of celiac disease and 19 negative control sera were tested by the electrochemical magneto immunosensor. The data were submitted to the receiver-operating characteristic plot (ROC) analysis which indicated that 16.95 units was the most effective cut-off value (COV) to discriminate correctly between celiac and non-celiac patients. Using this point for prediction, sensitivity was found to be 100%, while specificity was 84%., (Copyright © 2013 Elsevier B.V. All rights reserved.)

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.

Aim: To assesses the safety and efficacy of Aspergillus niger prolyl endoprotease (AN-PEP) to mitigate the immunogenic effects of gluten in celiac patients., Methods: Patients with initial diagnosis of celiac disease as confirmed by positive serology with subtotal or total villous atrophy on duodenal biopsies who adhere to a strict gluten-free diet (GFD) resulting in normalised antibodies and mucosal healing classified as Marsh 0 or I were included. In a randomised double-blind placebo-controlled pilot study, patients consumed toast (approximately 7 g/d gluten) with AN-PEP for 2 wk (safety phase). After a 2-wk washout period with adherence of the usual GFD, 14 patients were randomised to gluten intake with either AN-PEP or placebo for 2 wk (efficacy phase). Measurements at baseline included complaints, quality-of-life, serum antibodies, immunophenotyping of T-cells and duodenal mucosa immunohistology. Furthermore, serum and quality of life questionnaires were collected during and after the safety, washout and efficacy phase. Duodenal biopsies were collected after the safety phase and after the efficacy phase. A change in histological evaluation according to the modified Marsh classification was the primary endpoint., Results: In total, 16 adults were enrolled in the study. No serious adverse events occurred during the trial and no patients withdrew during the trial. The mean score for the gastrointestinal subcategory of the celiac disease quality (CDQ) was relatively high throughout the study, indicating that AN-PEP was well tolerated. In the efficacy phase, the CDQ scores of patients consuming gluten with placebo or gluten with AN-PEP did not significantly deteriorate and moreover no differences between the groups were observed. During the efficacy phase, neither the placebo nor the AN-PEP group developed significant antibody titers. The IgA-EM concentrations remained negative in both groups. Two patients were excluded from entering the efficacy phase as their mucosa showed an increase of two Marsh steps after the safety phase, yet with undetectable serum antibodies, while 14 patients were considered histologically stable on gluten with AN-PEP. Also after the efficacy phase, no significant deterioration was observed regarding immunohistological and flow cytometric evaluation in the group consuming placebo compared to the group receiving AN-PEP. Furthermore, IgA-tTG deposit staining increased after 2 wk of gluten compared to baseline in four out of seven patients on placebo. In the seven patients receiving AN-PEP, one patient showed increased and one showed decreased IgA-tTG deposits., Conclusion: AN-PEP appears to be well tolerated. However, the primary endpoint was not met due to lack of clinical deterioration upon placebo, impeding an effect of AN-PEP.

Coeliac disease is characterized by intestinal inflammation caused by gluten, proteins which are widely contained in the Western diet. Mammalian digestive enzymes are only partly capable of cleaving gluten, and fragments remain that induce toxic responses in patients with coeliac disease. We found that the oral microbiome is a novel and rich source of gluten-degrading organisms. Here we report on the isolation and characterization of the cultivable resident oral microbes that are capable of cleaving gluten, with special emphasis on the immunogenic domains. Bacteria were obtained by a selective culturing approach and enzyme activities were characterized by: (i) hydrolysis of paranitroanilide-derivatized gliadin-derived tripeptide substrates; (ii) gliadin degradation in-gel (gliadin zymography); (iii) gliadin degradation in solution; (iv) proteolysis of the highly immunogenic α-gliadin-derived 33-mer peptide. For selected strains pH activity profiles were determined. The culturing strategy yielded 87 aerobic and 63 anaerobic strains. Species with activity in at least two of the four assays were typed as: Rothia mucilaginosa HOT-681, Rothia aeria HOT-188, Actinomyces odontolyticus HOT-701, Streptococcus mitis HOT-677, Streptococcus sp. HOT-071, Neisseria mucosa HOT-682 and Capnocytophaga sputigena HOT-775, with Rothia species being active in all four assays. Cleavage specificities and substrate preferences differed among the strains identified. The approximate molecular weights of the enzymes were ~75 kD (Rothia spp.), ~60 kD (A. odontolyticus) and ~150 kD (Streptococcus spp.). In conclusion, this study identified new gluten-degrading microorganisms in the upper gastrointestinal tract. A cocktail of the most active oral bacteria, or their isolated enzymes, may offer promising new treatment modalities for coeliac disease., (© 2013 The Authors Clinical Microbiology and Infection © 2013 European Society of Clinical Microbiology and Infectious Diseases.)

Background: Celiac disease (CD) is an autoimmune disorder that occurs in genetically predisposed people and is caused by a reaction to the gluten protein found in wheat, which leads to intestinal villous atrophy. Currently there is no drug for treatment of CD. The only known treatment is lifelong gluten-free diet. The main aim of this work is to develop a mathematical model of the immune response in CD patients and to predict the efficacy of a transglutaminase-2 (TG-2) inhibitor as a potential drug for treatment of CD., Results: A thorough analysis of the developed model provided the following results:1. TG-2 inhibitor treatment leads to insignificant decrease in antibody levels, and hence remains higher than in healthy individuals.2. TG-2 inhibitor treatment does not lead to any significant increase in villous area.3. The model predicts that the most effective treatment of CD would be the use of gluten peptide analogs that antagonize the binding of immunogenic gluten peptides to APC. The model predicts that the treatment of CD by such gluten peptide analogs can lead to a decrease in antibody levels to those of normal healthy people, and to a significant increase in villous area., Conclusions: The developed mathematical model of immune response in CD allows prediction of the efficacy of TG-2 inhibitors and other possible drugs for the treatment of CD: their influence on the intestinal villous area and on the antibody levels. The model also allows to understand what processes in the immune response have the strongest influence on the efficacy of different drugs. This model could be applied in the pharmaceutical R&D arena for the design of drugs against autoimmune small intestine disorders and on the design of their corresponding clinical trials.

Coeliac disease (CD) is characterized by several markers, including anti-transglutaminase auto-antibodies (tTGAb) directed against multiple epitopes of the gliadin protein. We aimed to investigate the correlation among CD duodenal lesions, tTGAb titres and the immunoreactivity against tTG constructs. A total of 345 CD patients (209 females, 136 males, overall median age: 7.3 years) were tested for full-length (fl) tTGAb with a fluid-phase radioimmunoassay. Out of the total, 231 patients were also tested for immunoreactivity against tTG fragments (F1: a.a. 227-687 and F2: a.a. 473-687). Patients were classified according to diffuse (D), patchy (P) or bulb (B) histological lesions. All sera were found fltTGAb positive. Patients with D, P and B lesions had a mean Ab index of 0.84±0.39, 0.57±0.39 and 0.45±0.24, respectively. Mean tTGAb titre varied between D and localized (P+B) patients (0.84±0.39 versus 0.52±0.34, P < 0.0001). Overall, 86.1% of patients were F1 auto-antibody (F1Ab) positive (D: 89%, P: 75%, B: 40%; D versus P+B: P = 0.004) and 49% of patients were F2 auto-antibody (F2Ab) positive (D: 53%, P: 19%, B: 10%; D versus P+B: P = 0.0006). Of the D patients 50.7% showed combined F1Ab-F2Ab (D versus P+B: P = 0.001), whereas 60% of B patients were negative for both F1Ab and F2Ab (B versus D: P < 0.0001). Coeliac-specific tTGAb immunoreactivity correlates with the grading and extension of histological duodenal lesions in CD patients at diagnosis. The immunoreactivity against single and combined tTG fragments is significantly higher in patients with D lesions. This is the first evidence of a distinct coeliac-specific immunoreactivity in patients with different duodenal involvement.

Objectives: The previous finding of an immunologic response primarily directed against transglutaminase (TG)6 in patients with gluten ataxia (GA) led us to investigate the role of TG6 antibodies in diagnosing GA., Methods: This was a prospective cohort study. We recruited patients from the ataxia, gluten/neurology, celiac disease (CD), and movement disorder clinics based at Royal Hallamshire Hospital (Sheffield, UK) and the CD clinic, Tampere University Hospital (Tampere, Finland). The groups included patients with idiopathic sporadic ataxia, GA, and CD, and neurology and healthy controls. All were tested for TG6 antibodies. Duodenal biopsies were performed in patients with positive serology. In addition, biopsies from 15 consecutive patients with idiopathic sporadic ataxia and negative serology for gluten-related disorders were analyzed for immunoglobulin A deposits against TG., Results: The prevalence of TG6 antibodies was 21 of 65 (32%) in idiopathic sporadic ataxia, 35 of 48 (73%) in GA, 16 of 50 (32%) in CD, 4 of 82 (5%) in neurology controls, and 2 of 57 (4%) in healthy controls. Forty-two percent of patients with GA had enteropathy as did 51% of patients with ataxia and TG6 antibodies. Five of 15 consecutive patients with idiopathic sporadic ataxia had immunoglobulin A deposits against TG2, 4 of which subsequently tested positive for TG6 antibodies. After 1 year of gluten-free diet, TG6 antibody titers were significantly reduced or undetectable., Conclusions: Antibodies against TG6 are gluten-dependent and appear to be a sensitive and specific marker of GA.

Celiac disease (CD) is an immune-mediated, inflammatory disorder of the small intestines with a defined genetic etiological component associated with the expression of HLA-DQ2 and/or HLA-DQ8 haplotypes. The dietary consumption of gluten-rich cereals triggers a gluten-specific immune response in genetically susceptible individuals leading to a spectrum of clinical manifestations ranging from an inapparent subclinical disease, to overt enteropathy that can in some individuals progress to enteropathy-associated T cell lymphoma (EATL). The tissue-destructive pathologic process of CD is driven by activated NK-like intraepithelial CD8(+) lymphocytes and the proinflammatory cytokine IL-15 has emerged to be pivotal in orchestrating this perpetual tissue destruction and inflammation. Moreover, transgenic mice that over-express human IL-15 from an enterocyte-specific promoter (T3(b)-hIL-15 Tg) recapitulate many of the disease-defining T and B cell-mediated pathologic features of CD, further supporting the evolving consensus that IL-15 represents a valuable target in devising therapeutic interventions against the form of the disease that is especially refractory to gluten-free diet. In the present study, we evaluated the potential efficacy of tofacitinib, a pan-JAK inhibitor that abrogates IL-15 signaling, as a therapeutic modality against CD using T3(b)-hIL-15 Tg mice. We demonstrate that tofacitinib therapy leads to a lasting reversal of pathologic manifestations in the treated mice, thereby highlighting the potential value of tofacitininb as a therapeutic modality against refractory CD for which no effective therapy exists currently. Additionally, the visceral adiposity observed in the tofacitinib-treated mice underscores the importance of continued evaluation of the drug's impact on the lipid metabolism.

Anti-tissue transglutaminase (tTG) antibodies are specifically produced in the small-intestinal mucosa of celiac disease (CD) patients. It is now recognized that these antibodies, acting on cell-surface tTG, may play an active role in CD pathogenesis triggering an intracellular response via the activation of different signal transduction pathways. In this study, we report that anti-tTG antibodies, both commercial and from a CD patient, induce a rapid Ca(2+) mobilization from intracellular stores in Caco-2 cells. We characterized the mechanism of Ca(2+) release using thapsigargin and carbonylcyanide-p-trifluoromethoxyphenylhydrazone, which are able to deplete specifically endoplasmic reticulum and mitochondria of Ca(2+), respectively. Our data highlight that both pathways of calcium release were involved, thus indicating that the spectrum of cellular responses downstream can be very wide. In addition, we demonstrate that the increased Ca(2+) level in the cells evoked by anti-tTG antibodies was sufficient to activate tTG, which is normally present as a latent protein due to the presence of low Ca(2+) and to the inhibitory effect of GTP/GDP. Herein, we discuss the importance of intracellular tTG activation as central in the context of CD pathogenesis.

Purpose: Celiac disease is an autoimmune-mediated enteropathy characterized by adaptive and innate immune responses to dietary gluten in wheat, rye and barley in genetically susceptible individuals. Gluten-derived gliadin peptides are deamidated by transglutaminase 2 (TG2), leading to an immune response in the small-intestinal mucosa. TG2 inhibitors have therefore been suggested as putative drugs for celiac disease. In this proof-of-concept study we investigated whether two TG2 inhibitors, cell-impermeable R281 and cell-permeable R283, can prevent the toxic effects of gliadin in vitro and ex vivo., Methods: Intestinal epithelial Caco-2 cells were treated with peptic-tryptic-digested gliadin (PT-gliadin) with or without TG2 inhibitors and thereafter direct toxic effects (transepithelial resistance, cytoskeletal rearrangement, junction protein expression and phoshorylation of extracellular-signal-regulated kinase 1/2) were determined. In an organ culture of celiac-patient-derived small-intestinal biopsies we measured secretion of TG2-autoantibodies into the culture medium and the densities of CD25- and interleukin (IL) 15-positive cells, forkhead box P3 (FOXP3)-positive regulatory T cells (Tregs) and Ki-67-positive proliferating crypt cells., Results: Both TG2 inhibitors evinced protective effects against gliadin-induced detrimental effects in Caco-2 cells but the cell-impermeable R281 seemed slightly more potent. In addition, TG2 inhibitor R281 modified the gluten-induced increase in CD25- and IL15-positive cells, Tregs and crypt cell proliferation, but had no effect on antibody secretion in celiac-patient-derived biopsies., Conclusions: Our results suggest that TG2 inhibitors are able to reduce certain gliadin-induced effects related to responses in vitro and ex vivo.

Homozygosity for a nonsense mutation in the fucosyltransferase 2 (FUT2) gene (rs601338G>A) leads to the absence of ABH blood groups (FUT2 non-secretor status) in body fluids. As the secretor status has been shown to be a major determinant for the gut microbial spectrum, assumed to be important in the gut immune homeostasis, we studied the association of rs601338-FUT2 with celiac disease (CelD) and inflammatory bowel disease (IBD) in the Finnish population. Rs601338 was genotyped in CelD (n = 909), dermatitis herpetiformis (DH) (n = 116), ulcerative colitis (UC) (n = 496) and Crohn's disease (CD) (n = 280) patients and healthy controls (n = 2738). CelD showed significant genotypic [P = 0.0074, odds ratio (OR): 1.28] and recessive (P = 0.015, OR: 1.28) association with the rs601338-AA genotype. This was also found in the combined CelD+DH dataset (genotype association: P = 0.0060, OR: 1.28; recessive association: P < 0.011, OR: 1.28). The A allele of rs601338 showed nominal association with dominant protection from UC (P = 0.044, OR: 0.82) and UC+CD (P = 0.035, OR: 0.84). The frequency of non-secretors (rs601338-GG) in controls, CelD, DH, UC and CD datasets was 14.7%, 18%, 18.1%, 14.3% and 16.1%, respectively. No association was evident in the DH or CD datasets alone. In conclusion, FUT2 non-secretor status is associated with CelD susceptibility and FUT2 secretor status may also play a role in IBD in the Finnish population., (© 2012 John Wiley & Sons A/S.)

Objectives: Production of autoantibodies to the enzyme tissue transglutaminase (tTG) is a hallmark of coeliac disease (CD). We have previously demonstrated that the immumoglobulin (Ig) A response to tTG in adult CD specifically targets its catalytic core region, containing the active-site triad of amino acids. The aim of the present study was to investigate this phenomenon in paediatric patients with CD, and to elucidate the contribution of each active-site residue to epitopes recognised. The specificity of the IgG anti-tTG response was also investigated and compared with that of the IgA anti-tTG response, in both paediatric and adult patients with CD., Methods: Wild-type and novel variants of tTG were generated via site-directed mutagenesis and expressed as glutathione-S-transferase-fusion proteins in Escherichia coli BL-21. The mutagenic variants of tTG had substitutions of 1, 1, or all of the 3 of the catalytic triad amino acids. All of the recombinant tTGs were tested for their antigenicity in IgA and IgG enzyme-linked immunosorbent assays with cohorts of paediatric (n=63) and adult (n=30) CD sera., Results: Substitution of even 1 amino acid in the catalytic triad resulted in a significant reduction of CD IgA and IgG anti-tTG binding, with all of the mutant proteins displaying diminished antigenicity compared with the wild-type protein., Conclusions: The core region of tTG is specifically targeted from early on in disease course by CD patient autoantibodies of both the IgA and IgG class.