Design of a Quantitative LC-MS Method for Residual Toxins Adenylate Cyclase Toxin (ACT), Dermonecrotic Toxin (DNT) and Tracheal Cytotoxin (TCT) in Bordetella pertussis Vaccines.

The antigens for acellular pertussis vaccines are made up of protein components that are purified directly from Bordetella pertussis (B. pertussis) bacterial fermentation. As such, there are additional B. pertussis toxins that must be monitored as residuals during process optimization. This paper describes a liquid chromatography mass spectrometry (LC-MS) method for simultaneous analysis of residual protein toxins adenylate cyclase toxin (ACT) and dermonecrotic toxin (DNT), as well as a small molecule glycopeptide, tracheal cytotoxin (TCT) in a Pertussis toxin vaccine antigen. A targeted LC-MS technique called multiple reaction monitoring (MRM) is used for quantitation of ACT and TCT, which have established limits in drug product formulations. However, DNT is currently monitored in an animal test, which does not have an established quantitative threshold. New approaches for DNT testing are discussed, including a novel standard based on concatenated quantitation sequences for ACT and DNT. Collectively, the method represents a "3-in-1" analytical simplification for monitoring process-related residuals during development of B. pertussis vaccines.

Effects of microbial transglutaminase levels on donkey cheese production.

Microbial transglutaminase (MTGase) is an enzyme widely used in the dairy sector to improve the functional properties of protein-based products via the formation of a network between protein molecules. The aim of this study involving cheese from the milk of donkeys was to evaluate the effects of treatment with MTGase at the concentrations of 0 (control), 5, 8 and 10 U/g milk protein on the cheese-making process parameters, as well as the physical and chemical characteristics of the resulting cheese. MTGase influenced the time of gel formation from rennet addition (P < 0.05), with a delay at the two highest concentrations, accompanied by a lower (P < 0.01) pH of cheese and the lowest (P < 0.01) loss in cheese weight at 24 h of storage. The highest gel viscosity (P < 0.01) was observed at the highest concentration of the enzyme, reaching the value of 70 mPa⋅sec after 60 min. The chemical composition and color of the cheeses were not significantly affected by the inclusion of MTGase, regardless of the enzyme concentration. These findings may be of relevance in adapting the cheese-making process and might help in the design of new dairy products from donkey's milk.

Development of an 18F-Labeled Irreversible Inhibitor of Transglutaminase 2 as Radiometric Tool for Quantitative Expression Profiling in Cells and Tissues.

The transamidase activity of transglutaminase 2 (TGase 2) is considered to be important for several pathophysiological processes including fibrotic and neoplastic tissue growth, whereas in healthy cells this enzymatic function is predominantly latent. Methods that enable the highly sensitive detection of TGase 2, such as application of radiolabeled activity-based probes, will support the exploration of the enzyme's function in various diseases. In this context, the radiosynthesis and detailed in vitro radiopharmacological evaluation of an 18F-labeled Nε-acryloyllysine piperazide are reported. Robust and facile detection of the radiotracer-TGase 2 complex by autoradiography of thin layer plates and polyacrylamide gels after chromatographic and electrophoretic separation owing to irreversible covalent bond formation was demonstrated for the isolated protein, cell lysates, and living cells. By use of this radiotracer, quantitative data on the expression profile of activatable TGase 2 in mouse organs and selected tumors were obtained for the first time by autoradiography of tissue sections.

Optimization of soy protein isolate, microbial transglutaminase and glucono-δ-lactone in gluten-free rice noodles.

BACKGROUND: Rice flour does not contain gluten and lacks cohesion and extensibility, which is responsible for the poor texture of rice noodles. Different technologies have been used to mitigate this challenge, including hydrothermal treatments of rice flour, direct addition of protein in noodles, use of additives such as hydrocolloids and alginates, and microbial transglutaminase (MTG). Recently, the inclusion of soy protein isolate (SPI), MTG, and glucono-δ-lactone (GDL) in the rice noodles system yielded rice noodles with improved texture and more compact microstructure, hence the need to optimize the addition of SPI, MTG, and GDL to make quality rice noodles. RESULTS: Numerical optimization showed that rice noodles prepared with SPI, 68.32 (g kg-1 of rice flour), MTG, 5.06 (g kg-1 of rice flour) and GDL, 5.0 (g kg-1 of rice flour) gave the best response variables; hardness (53.19 N), springiness (0.76), chewiness (20.28 J), tensile strength (60.35 kPa), and cooking time (5.15 min). The pH, sensory, and microstructure results showed that the optimized rice noodles had a more compact microstructure with fewer hollows, optimum pH for MTG action, and overall sensory panelists also showed the highest preference for the optimized formulation, compared to other samples selected from the numerical optimization and desirability tests. CONCLUSION: Optimization of the levels of SPI, MTG, and GDL yielded quality noodles with improved textural, mechanical, sensory, and microstructural properties. This was partly due to the favourable pH value of the optimized noodles that provided the most suitable conditions for MTG crosslinking and balanced electrostatic interaction of proteins. © 2020 Society of Chemical Industry.

Nuevos biomarcadores en la enfermedad celiaca / No disponible

Los avances en el conocimiento de la enfermedad celiaca han permitido disponer de marcadores para el diagnóstico como los anticuerpos antitransglutaminasa tisular y antigliadina deaminada. La amplia disponibilidad de estos anticuerpos junto con los estudios genéticos del HLA-DQ y la biopsia duodenal constituyen los pilares para el diagnóstico definitivo. Sin embargo, en ocasiones surgen dificultades tanto en el diagnóstico como en el seguimiento del paciente celiaco que no se resuelven con estas herramientas. En este artículo se revisa la evidencia científica junto con la posible utilidad clínica de diferentes biomarcadores. Se ha estructurado la revisión en aquellos que han sido evaluados desde el punto de vista fisiopatológico en relación con el daño intestinal o la respuesta inmunológica, junto con la utilidad clínica que pudieran tener en el diagnóstico y seguimiento de la enfermedad celiaca

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In situ detection of intracellular tissue transglutaminase based on aggregation-induced emission.

Herein, a novel strategy for in situ imaging and real-time monitoring of intracellular tissue transglutaminase (TG2) is presented based on aggregation-induced emission (AIE). It has high sensitivity and specificity, minimal background signal and can also effectively distinguish different cell types (drug-resistant cancer cells, cancer cells and normal cells).

Transglutaminase 1 and 2 are localized in different blood cells in the freshwater crayfish Pacifastacus leniusculus.

In the present study we show that hemocytes in the freshwater crayfish Pacifastacus leniusculus express two different transglutaminases. We describe the sequence of a previously unknown TGase (Pl_TGase1) and named this as Pl_TGase2 and compared this sequence with similar sequences from other crustaceans. The catalytic core domain is similar to the previously described TGase in P. leniusculus, but Pl_TGase2 has significant differences in the N-terminal and C-terminal domains. Further, we show conclusive evidences that these different transglutaminases are specific for different hemocyte types so that Pl_TGase1 is expressed in the hematopoietic tissue and in the cytoplasm of semigranular hemocytes, while Pl_TGase2 is expressed in vesicles in the granular hemocytes. By in situ hybridization we show that both Pl_TGase1 and Pl_TGase2 mRNA are present only in a subset of the respective hemocyte population. This observation indicates that there may be different subtypes of semigranular as well as granular hemocytes which may have different specific functions.

Low Prevalence of Celiac Disease among Patients with Functional Gastrointestinal Disorders in Latvia.

BACKGROUND AND AIMS: Studies suggest that the prevalence of celiac disease (CD) is increased in individuals with functional gastrointestinal disorders (FGIDs), in particular, irritable bowel syndrome (IBS); however, the evidence is conflicting. We aimed to analyze the prevalence of CD in patients with FGIDs in Latvia. METHODS: This retrospective study included patients with FGIDs, referred for a gastroenterologist consultation in a secondary gastroenterology practice unit. Patients were divided into three groups - patients only with IBS (IBS group), patients only with functional dyspepsia (FD) (FD group), patients with mixed symptoms IBS and FD (Mixed group). Patient levels of tissue transglutaminase IgA (tTG-IgA) and/or antiendomysial IgA group antibodies (EMA-IgA) were evaluated. Four duodenal biopsies were obtained and reported according to Marsh classification. Patients diagnosed or being referred for confirmation of CD were excluded from the study. RESULTS: Overall, 1,833 FGIDs patients were enrolled. Celiac serology was available for 1,570 patients, duodenal histology for 582 patients, both histology and serology for 319 patients. In total, celiac seropositivity was present in 1.78% (28/1570) (3.18% in IBS group, 0.90% in FD group and 1.11% of cases in the mixed group). Fifteen patients had histopathological changes (2.58%; 15/582). Three IBS patients (2.36%) were both serology and biopsy positive. None of the FD patients had CD. CONCLUSION: Prevalence of biopsy-proven CD in patients from Latvia with FGIDs was low. Routine screening for CD could be considered only among patients with IBS.

Imaging of the ex vivo transglutaminase activity in liver macrophages of sepsis mice.

Sepsis is the leading cause of death in hospitalized patients and is characterized by a dysregulated inflammatory response to infection and multiple organ failure, including the liver. Transglutaminase 2 (TG2) is a multifunctional enzyme that exhibits transamidase, GTPase, and integrin-binding activities and has opposing roles in the regulation of cell growth, differentiation, and apoptosis. TG2 plays both pathogenic and protective roles in liver diseases, revealing the need to examine the activities of TG2. Here, we introduced an ex vivo imaging approach to examine the in vivo transamidase activity of TG2 based on the combination of intraperitoneal injection of 5-biotinamidopentylamine (5BAPA), a biotinylated substrate for TG2, and fluorescent streptavidin staining in frozen liver sections. Increased 5BAPA signals was observed in the livers of lipopolysaccharide (LPS) and cecal ligation and puncture (CLP)-induced sepsis mice. Pharmacological inhibition of TG2 activity ameliorated LPS-induced liver injury. 5BAPA signals were observed in TG2-expressing and F4/80-positive midzonal macrophages, providing direct evidence that activated macrophages are the major cellular source of active TG2 in the livers of sepsis mice. Further studies focusing on the activation of 5BAPA-stained midzonal macrophages may improve understanding of the molecular pathophysiology and the development of therapeutic strategies for sepsis.

Physiochemical and rheological properties of oxidized Japanese seerfish (Scomberomorus niphonius) myofibrillar protein.

Myofibrillar protein (MFP) of Japanese seerfish (JS) was oxidized by Fenton system (1, 4, 8, 16 mM H2 O2 for 0, 2, 4, 6 hr). After oxidation, α-helix ratio and sulfhydryl content of MFP declined along with the increased carbonyl and dityrosine levels. Bromophenol blue bound in MFP under 1 mM H2 O2 slightly increased. Polymers together with attenuated myosin heavy chain in protein pattern were observed. Compared with non-oxidized MFP, storage modulus (G') of MFP subjected to 1 mM H2 O2 for 4 hr increased while that of MFP exposed to 16 mM H2 O2 declined. When treated with microbial transglutaminase (MTG), mildly oxidized (1 mM H2 O2 , 2 hr) MFP showed higher G' while heavily oxidized (16 mM H2 O2 , 2 hr) MFP had lower G' than control. Oxidation showed pronounced influences on physiochemical and gelling properties of JS MFP and the oxidation extent affected MTG role on it. PRACTICAL APPLICATIONS: Protein oxidation occurs extensively in muscle and exerts great influences on muscle food quality. JS is widely used for producing gel food in China. Its muscle also contains oxidation initiators such as H2 O2, hemoglobin and lipids, increasing the susceptibility to protein oxidation. Results of the study exposed the effects of hydroxyl radical oxidation on physiochemical and gelling properties of JS MFP. It provides strategic support to improve gel properties of MFP by manipulating oxidation.

Micropipette Tip-Based Immunoassay with Electrochemical Detection of Antitissue Transglutaminase to Diagnose Celiac Disease Using Staples and a Paper-Based Platform.

In this work, 1-200 µL polypropylene micropipette tips were used as platforms for performing immunoassays after converting their inner surfaces on a capture zone for the analyte of interest. We have used a micropipette-tip immunoelectroanalytical platform for the detection of antitissue transglutaminase (IgA), the main biomarker for celiac disease. Modification of the tip wall with poly-l-lysine allowed adsorption of tissue transglutaminase (tTG), which will capture later anti-tTG (IgA) antibodies developed in celiac-affected people. A sandwich-type format was followed, incubating simultaneously the analyte and the detection antibody, labeled with horseradish peroxidase. With this new application for an extremely common lab material, we can perform quantitative analysis by dispensing the liquid into a low-cost and miniaturized staple-based paper electrochemical platform. The analytical signal was the reduction of the enzymatically oxidized substrate, recorded chronoamperometrically (i-t curve). The intensity of the current obtained at a fixed time after the application of the cathodic potential followed a linear relationship with anti-tTG (IgA) concentration. The relative standard deviation obtained for immunoassays performed in different tips indicates the adequate precision of this new methodology, which is very promising for decentralized analysis. Negative and positive controls produced results that were in accordance with those obtained with spectrophotometric enzyme linked-immunosorbent assays.

Multi-omics Biomarker Pipeline Reveals Elevated Levels of Protein-glutamine Gamma-glutamyltransferase 4 in Seminal Plasma of Prostate Cancer Patients.

Seminal plasma, because of its proximity to prostate, is a promising fluid for biomarker discovery and noninvasive diagnostics. In this study, we investigated if seminal plasma proteins could increase diagnostic specificity of detecting primary prostate cancer and discriminate between high- and low-grade cancers. To select 147 most promising biomarker candidates, we combined proteins identified through five independent experimental or data mining approaches: tissue transcriptomics, seminal plasma proteomics, cell line secretomics, tissue specificity, and androgen regulation. A rigorous biomarker development pipeline based on selected reaction monitoring assays was designed to evaluate the most promising candidates. As a result, we qualified 76, and verified 19 proteins in seminal plasma of 67 negative biopsy and 152 prostate cancer patients. Verification revealed a prostate-specific, secreted and androgen-regulated protein-glutamine gamma-glutamyltransferase 4 (TGM4), which predicted prostate cancer on biopsy and outperformed age and serum Prostate-Specific Antigen (PSA). A machine-learning approach for data analysis provided improved multi-marker combinations for diagnosis and prognosis. In the independent verification set measured by an in-house immunoassay, TGM4 protein was upregulated 3.7-fold (p = 0.006) and revealed AUC = 0.66 for detecting prostate cancer on biopsy for patients with serum PSA ≥4 ng/ml and age ≥50. Very low levels of TGM4 (120 pg/ml) were detected in blood serum. Collectively, our study demonstrated rigorous evaluation of one of the remaining and not well-explored prostate-specific proteins within the medium-abundance proteome of seminal plasma. Performance of TGM4 warrants its further investigation within the distinct genomic subtypes and evaluation for the inclusion into emerging multi-biomarker panels.

Identification of Betamethasone-Regulated Target Genes and Cell Pathways in Fetal Rat Lung Mesenchymal Fibroblasts.

Preterm birth is characterized by severe lung immaturity that is frequently treated antenatally or postnatally with the synthetic steroid betamethasone. The underlying cellular targets and pathways stimulated by betamethasone in the fetal lung are poorly defined. In this study, betamethasone was compared with corticosterone in steroid-treated primary cultures of fetal rat lung fibroblasts stimulated for 6 hours and analyzed by whole-cell transcriptome sequencing and glucocorticoid (GC) receptor (GR) chromatin immunoprecipitation sequencing (ChIP-Seq) analysis. Strikingly, betamethasone stimulated a much stronger transcriptional response compared with corticosterone for both induced and repressed genes. A total of 483 genes were significantly stimulated by betamethasone or corticosterone, with 476 stimulated by both steroids, indicating a strong overlap in regulation. Changes in mRNA levels were confirmed by quantitative PCR for eight induced and repressed target genes. Pathway analysis identified cell proliferation and cytoskeletal/cell matrix remodeling pathways as key processes regulated by both steroids. One target, transglutaminase 2 (Tgm2), was localized to fetal lung mesenchymal cells. Tgm2 mRNA and protein levels were strongly increased in fibroblasts by both steroids. Whole-genome GR ChIP-Seq analysis with betamethasone identified GC response element-binding sites close to the previously characterized GR target genes Per1, Dusp1, Fkbp5, and Sgk1 and near the genes identified by transcriptome sequencing encoding Crispld2, Tgm2, Hif3α, and Kdr, defining direct genomic induction of expression in fetal lung fibroblasts via the GR. These results demonstrate that betamethasone stimulates specific genes and cellular pathways controlling cell proliferation and extracellular matrix remodeling in lung mesenchymal fibroblasts, providing a basis for betamethasone's treatment efficacy in preterm birth.

Anticuerpos antitransglutaminasa no relacionados con la ingesta de gluten / Anti-tissue transglutaminase antibodies not related to gluten intake

INTRODUCCIÓN: Los anticuerpos antitransglutaminasa (ATG) poseen alta especificidad para el diagnóstico de enfermedad celíaca (EC). Sin embargo, se han descrito anticuerpos ATG positivos en pacientes no celíacos. OBJETIVO: Valorar la presencia de anticuerpos ATG positivos no relacionados con la ingesta de gluten. PACIENTES Y MÉTODOS: Revisión retrospectiva de historias clínicas y seguimiento de pacientes con sospecha de EC y con un comportamiento serológico atípico, es decir, anticuerpos ATG positivos a pesar de una dieta sin gluten y disminución de anticuerpos ATG tomando gluten. RESULTADOS: Se incluyeron 9 casos. De ellos, 5 casos tenían afectación histológica Marsh 3 en la biopsia inicial y diagnóstico de EC (grupo A). Se retiró el gluten de la dieta y se retiraron las proteínas de leche de vaca (PLV) por la afectación nutricional. Al reintroducir las PLV aumentaron los ATG y al retirarlas se volvieron a normalizar. Los otros 4 pacientes presentaban una biopsia inicial normal (grupo B): en estos no se retiró el gluten, pero sí las PLV por sospecha de alergia no IgE mediada. Los síntomas desaparecieron y se normalizaron los ATG al retirar las PLV manteniendo dieta con gluten. Todos presentan el haplotipo de susceptibilidad para EC. CONCLUSIONES: En algunos celíacos, la reintroducción de PLV en la dieta tras un período de exclusión induce un aumento de los anticuerpos ATG IgA. Si se han descartado transgresiones con gluten, las PLV pueden causar esta respuesta inmune. Hemos observado también esta respuestaen pacientes con alergia no IgE, mediada por las PLV, portadores del haplotipo de susceptibilidad HLA DQ2/DQ8

INTRODUCTION: Anti-tissue transglutaminase antibodies (tTG) have high specificity for coeliac disease (CD). However, positive anti-tTG antibodies have been described in non-coeliac patients. Aim To assess positive anti-tTG antibodies not related to gluten intake. PATIENTS AND METHODS: Retrospective review and follow up conducted on patients with suspected CD (increase anti-tTG levels and gastrointestinal symptoms) but with atypical serology results, positive anti-tTG with gluten free diet and a decrease in anti-tTG levels despite gluten intake. RESULTS: A total of 9 cases were reviewed in which 5 cases had Marsh 3 involvement in the initial biopsy, and were diagnosed with CD (Group A). They began a gluten free diet and also a cow's milk protein (CMP) free diet because of their nutritional status. When CMP was re-introduced, anti-tTG increased, and returned to normal after the CMP was withdrawn again. The other 4 patients had a normal initial biopsy (Group B). Gluten was not removed from their diet, but they started a CMP free diet because a non IgE mediated CMP allergy was suspected. Symptoms disappeared, and anti-tTG was normal after CMP free diet with gluten intake. All the patients had susceptibility haplotype HLA DQ2/DQ8. CONCLUSIONS: CMP ingestion after an exclusion diet can induce an increase in anti-tTG in some coeliac subjects. CMP can produce this immune response if there were no gluten transgressions. This response has also been observed in non-IgE mediated CMP allergy patients with the susceptibility haplotype HLA DQ2/DQ8

Pitfalls in adult celiac disease diagnosis

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Separation of two microbial transglutaminase isomers from Streptomyces mobaraensis using pH-mediated cation exchange chromatography and their characterization.

Microbial transglutaminase (MTGase) derived from Streptomyces mobaraensis has been widely used in the food, biotechnology and medicine fields. The lot-to-lot consistency and product stability of MTGase must be ensured. The structure and charge variants of MTGase can influence its bioactivity. In this study, MTGase isomers (MTG I1 and MTG I2) were found during the separation of MTGase by pH-mediated cation-exchange chromatography. MTG I1 and MTG I2 had the same molecular weight and N-terminal amino acid sequences, but they showed charge heterogeneity. The affinity of MTG I2 for substrates was higher than that of MTG I1, and the thermal stability and the acid-base tolerance of MTG I1 were significantly higher than that of MTG I2. Therefore, the ratio of MTG I1/MTG I2 was positively correlated with the stability of MTGase. The buffer pH and the ionic strength of the eluent had significant effects on the separation of MTG I1 and MTG I2, and the elution gradient steepness and column load showed little effect on the separation of the MTG I1 and MTG I2 peaks. We built a stable and repeatable separation method for MTG I1 and MTG I2. MTG I1 could transform into MTG I2, but MTG I2 was unable to transform into MTG I1, making the transformation of MTG I1 to MTG I2 was irreversible. When MTG I2 was removed from the MTGase, a portion of the MTG I1 could transform into MTG I2. Therefore, one way to increase the stability of MTGase was to reduce the transformation of MTG I1 to MTG I2.

Biofluid Biomarkers in Huntington's Disease.

Huntington's disease (HD) is a chronic progressive neurodegenerative condition where new markers of disease progression are needed. So far no disease-modifying interventions have been found, and few interventions have been proven to alleviate symptoms. This may be partially explained by the lack of reliable indicators of disease severity, progression, and phenotype.Biofluid biomarkers may bring advantages in addition to clinical measures, such as reliability, reproducibility, price, accuracy, and direct quantification of pathobiological processes at the molecular level; and in addition to empowering clinical trials, they have the potential to generate useful hypotheses for new drug development.In this chapter we review biofluid biomarker reports in HD, emphasizing those we feel are likely to be closest to clinical applicability.

On-chip dual enzyme activity assay to investigate regulation of the transamidase and kinase activities of transglutaminase 2.

Transglutaminase 2 (TGase2), a multifunctional enzyme exhibiting both transamidase and kinase activity, is involved in a variety of cellular processes and diseases. However, details of the regulation of TGase2 have not been reported due to the lack of a suitable assay to examine both activities on the same platform under near-physiologic conditions. Thus, we developed an on-chip dual enzyme activity assay for TGase2 to simultaneously monitor the transamidase and kinase activities. Reaction mixtures specific for each enzyme activity were applied onto osteopontin arrays, and enzyme activity was monitored by sequential probing with Cy5-strepavidin and Pro-Q Diamond stain. This approach was used to determine the optimal concentrations of ATP, Mg2+, and Ca2+ for dual-activity assays. The optimized assay was then used to investigate regulation of TGase2 transamidase and kinase activities by various cofactors that could potentially affect its conformation. Monothiol- and disulfide-containing compounds differentially regulated TGase2 transamidase and kinase activities. Acetylation of TGase2 activated the kinase activity but had no effect on the transamidase activity. Phosphorylation and dephosphorylation of TGase2 reciprocally regulated the transamidase and kinase activities. The new approach described here is thus useful for screening potential regulators of TGase2 transamidase and kinase and investigating the pathogenesis of TGase2-associated diseases.

Immunohistologic analysis of the duodenal bulb: a new method for celiac disease diagnosis in children.

BACKGROUND AND AIMS: Anti-tissue transglutaminase antibodies (anti-tTG) have simplified celiac disease (CD) diagnosis. However, in atypical forms of CD, intestinal biopsy sampling is still required. This prospective study investigates whether histologic analysis of the duodenal bulb combined with intestinal IgA anti-tTG deposit immunoassay makes CD diagnosis possible in at-risk children with low concentrations of serum anti-tTG. METHODS: Histologic and intestinal IgA anti-tTG deposit immunoassays were used. RESULTS: Two hundred forty-five symptomatic children positive for serum anti-tTG (>7 U/mL) were enrolled and divided into 3 groups: extensive duodenal atrophy (n = 209), with IgA anti-tTG deposits throughout the duodenum and high serum anti-tTG concentrations (157 ± 178 U/mL); bulb duodenal atrophy (n = 22), with widespread IgA anti-tTG deposits in 9 and in the bulb alone in 13 and low serum anti-tTG concentrations (13.9 ± 8.7 U/mL); and normal duodenum (n = 14), with widespread IgA anti-tTG deposits in 8 and in the bulb alone in 6 and low serum anti-tTG concentrations (10.6 ± 6.2 U/mL). All patients in the first 2 groups were diagnosed with CD and 8 from the third group. All improved after 1 year of gluten-free diet. Bulb duodenal analysis led to a 12% (30/245) increase in CD diagnosis. No CD-related lesions were observed in the 30 control subjects. CONCLUSIONS: In children at risk for CD, bulb duodenum biopsy sampling is essential to identify villous atrophy and detect IgA anti-tTG deposits even in absence of intestinal lesions. These mucosal autoantibodies could well represent a new standard for diagnosing CD.