Structural and mechanistic analysis of Ca2+-dependent regulation of transglutaminase 2 activity using a Ca2+-bound intermediate state.

Mammalian transglutaminases, a family of Ca2+-dependent proteins, are implicated in a variety of diseases. For example, celiac disease (CeD) is an autoimmune disorder whose pathogenesis requires transglutaminase 2 (TG2) to deamidate select glutamine residues in diet-derived gluten peptides. Deamidation involves the formation of transient γ-glutamyl thioester intermediates. Recent studies have revealed that in addition to the deamidated gluten peptides themselves, their corresponding thioester intermediates are also pathogenically relevant. A mechanistic understanding of this relevance is hindered by the absence of any structure of Ca2+-bound TG2. We report the X-ray crystallographic structure of human TG2 bound to an inhibitory gluten peptidomimetic and two Ca2+ ions in sites previously designated as S1 and S3. Together with additional structure-guided experiments, this structure provides a mechanistic explanation for how S1 regulates formation of an inhibitory disulfide bond in TG2, while also establishing that S3 is essential for γ-glutamyl thioester formation. Furthermore, our crystallographic findings and associated analyses have revealed that i) two interacting residues, H305 and E363, play a critical role in resolving the thioester intermediate into an isopeptide bond (transamidation) but not in thioester hydrolysis (deamidation); and ii) residues N333 and K176 stabilize preferred TG2 substrates and inhibitors via hydrogen bonding to nonreactive backbone atoms. Overall, the intermediate-state conformer of TG2 reported here represents a superior model to previously characterized conformers for both transition states of the TG2-catalyzed reaction.

The Evolution of Transglutaminases Underlies the Origin and Loss of Cornified Skin Appendages in Vertebrates.

Transglutaminases (TGMs) cross-link proteins by introducing covalent bonds between glutamine and lysine residues. These cross-links are essential for epithelial cornification which enables tetrapods to live on land. Here, we investigated which evolutionary adaptations of vertebrates were associated with specific changes in the family of TGM genes. We determined the catalog of TGMs in the main clades of vertebrates, performed a comprehensive phylogenetic analysis of TGMs, and localized the distribution of selected TGMs in tissues. Our data suggest that TGM1 is the phylogenetically oldest epithelial TGM, with orthologs being expressed in the cornified teeth of the lamprey, a basal vertebrate. Gene duplications led to the origin of TGM10 in stem vertebrates, the origin of TGM2 in jawed vertebrates, and an increasing number of epithelium-associated TGM genes in the lineage leading to terrestrial vertebrates. TGM9 is expressed in the epithelial egg tooth, and its evolutionary origin in stem amniotes coincided with the evolution of embryonic development in eggs that are surrounded by a protective shell. Conversely, viviparous mammals have lost both the epithelial egg tooth and TGM9. TGM3 and TGM6 evolved as regulators of cornification in hair follicles and underwent pseudogenization upon the evolutionary loss of hair in cetaceans. Taken together, this study reveals the gain and loss of vertebrate TGM genes in association with the evolution of cornified skin appendages and suggests an important role of TGM9 in the evolution of amniotes.

Opportunities for Improving Biopsy and Non-Biopsy-Based Diagnosis of Celiac Disease.

The accumulating data regarding a non-biopsy diagnosis of celiac disease has led to its adoption in certain scenarios, although debate on whether and when to use non-biopsy criteria in clinical practice is ongoing. Despite the growing popularity and evidence basis for a biopsy-free approach to diagnosis in the context of highly elevated serologies, there will continue to be a role for a biopsy in some groups. This review summarizes the current evidence supporting a non-biopsy approach and arguments supporting continued reliance on biopsy, and focuses on opportunities to improve both approaches.

New Insights on Genes, Gluten, and Immunopathogenesis of Celiac Disease.

Celiac disease (CeD) is a gluten-induced enteropathy that develops in genetically susceptible individuals upon consumption of cereal gluten proteins. It is a unique and complex immune disorder to study as the driving antigen is known and the tissue targeted by the immune reaction can be interrogated. This review integrates findings gained from genetic, biochemical, and immunologic studies, which together have revealed mechanisms of gluten peptide modification and HLA binding, thereby enabling a maladapted anti-gluten immune response. Observations in human samples combined with experimental mouse models have revealed that the gluten-induced immune response involves CD4+ T cells, cytotoxic CD8+ T cells, and B cells; their cross-talks are critical for the tissue-damaging response. The emergence of high-throughput technologies is increasing our understanding of the phenotype, location, and presumably function of the gluten-specific cells, which are all required to identify novel therapeutic targets and strategies for CeD.

Carcinomas assemble a filamentous CXCL12-keratin-19 coating that suppresses T cell-mediated immune attack.

Cancer immunotherapy frequently fails because most carcinomas have few T cells, suggesting that cancers can suppress T cell infiltration. Here, we show that cancer cells of human pancreatic ductal adenocarcinoma (PDA), colorectal cancer, and breast cancer are coated with transglutaminase-2 (TGM2)-dependent covalent CXCL12-keratin-19 (KRT19) heterodimers that are organized as filamentous networks. Since a dimeric form of CXCL12 suppresses the motility of human T cells, we determined whether this polymeric CXCL12-KRT19 coating mediated T cell exclusion. Mouse tumors containing control PDA cells exhibited the CXCL12-KRT19 coating, excluded T cells, and did not respond to treatment with anti-PD-1 antibody. Tumors containing PDA cells not expressing either KRT19 or TGM2 lacked the CXCL12-KRT19 coating, were infiltrated with activated CD8+ T cells, and growth was suppressed with anti-PD-1 antibody treatment. Thus, carcinomas assemble a CXCL12-KRT19 coating to evade cancer immune attack.

Membrane Association of the Short Transglutaminase Type 2 Splice Variant (TG2-S) Modulates Cisplatin Resistance in a Human Hepatocellular Carcinoma (HepG2) Cell Line.

Hepatocellular carcinoma (HCC) is a heterogeneous malignancy with complex carcinogenesis. Although there has been significant progress in the treatment of HCC over the past decades, drug resistance to chemotherapy remains a major obstacle in its successful management. In this study, we were able to reduce chemoresistance in cisplatin-resistant HepG2 cells by either silencing the expression of transglutaminase type 2 (TG2) using siRNA or by the pre-treatment of cells with the TG2 enzyme inhibitor cystamine. Further analysis revealed that, whereas the full-length TG2 isoform (TG2-L) was almost completely cytoplasmic in its distribution, the majority of the short TG2 isoform (TG2-S) was membrane-associated in both parental and chemoresistant HepG2 cells. Following the induction of cisplatin toxicity in non-chemoresistant parental cells, TG2-S, together with cisplatin, quickly relocated to the cytosolic fraction. Conversely, no cytosolic relocalisation of TG2-S or nuclear accumulation cisplatin was observed, following the identical treatment of chemoresistant cells, where TG2-S remained predominantly membrane-associated. This suggests that the deficient subcellular relocalisation of TG2-S from membranous structures into the cytoplasm may limit the apoptic response to cisplatin toxicity in chemoresistant cells. Structural analysis of TG2 revealed the presence of binding motifs for interaction of TG2-S with the membrane scaffold protein LC3/LC3 homologue that could contribute to a novel mechanism of chemotherapeutic resistance in HepG2 cells.

Infection-driven activation of transglutaminase 2 boosts glucose uptake and hexosamine biosynthesis in epithelial cells.

Transglutaminase 2 (TG2) is a ubiquitously expressed enzyme with transamidating activity. We report here that both expression and activity of TG2 are enhanced in mammalian epithelial cells infected with the obligate intracellular bacteria Chlamydia trachomatis. Genetic or pharmacological inhibition of TG2 impairs bacterial development. We show that TG2 increases glucose import by up-regulating the transcription of the glucose transporter genes GLUT-1 and GLUT-3. Furthermore, TG2 activation drives one specific glucose-dependent pathway in the host, i.e., hexosamine biosynthesis. Mechanistically, we identify the glucosamine:fructose-6-phosphate amidotransferase (GFPT) among the substrates of TG2. GFPT modification by TG2 increases its enzymatic activity, resulting in higher levels of UDP-N-acetylglucosamine biosynthesis and protein O-GlcNAcylation. The correlation between TG2 transamidating activity and O-GlcNAcylation is disrupted in infected cells because host hexosamine biosynthesis is being exploited by the bacteria, in particular to assist their division. In conclusion, our work establishes TG2 as a key player in controlling glucose-derived metabolic pathways in mammalian cells, themselves hijacked by C. trachomatis to sustain their own metabolic needs.

Variation in Testing for and Incidence of Celiac Autoimmunity in Canada: A Population-Based Study.

BACKGROUND & AIMS: The incidence of biopsy-confirmed celiac disease has increased. However, few studies have explored the incidence of celiac autoimmunity based on positive serology results. METHODS: A population-based cohort study assessed testing of tissue transglutaminase antibodies (tTG-IgA) in Alberta from 2012 to 2020. After excluding prevalent cases, incident celiac autoimmunity was defined as the first positive tTG-IgA result between 2015 and 2020. Testing and incidence rates for celiac autoimmunity were calculated per 1000 and 100,000 person-years, respectively. Incidence rate ratios (IRRs) were calculated to identify differences by demographic and regional factors. Average annual percent changes (AAPCs) assessed trends over time. RESULTS: The testing rate of tTG-IgA was 20.2 per 1000 person-years and remained stable from 2012 to 2020 (AAPC, 1.2%; 95% confidence interval [CI], -0.5 to 2.9). Testing was higher in female patients (IRR, 1.66; 95% CI, 1.65-1.66), those living in metropolitan areas (IRR, 1.39; 95% CI, 1.38-1.40), and in areas of lower socioeconomic deprivation (lowest compared to highest IRR, 1.24; 95% CI, 1.23-1.25). Incidence of celiac autoimmunity was 33.8 per 100,000 person-years and increased from 2015 to 2020 (AAPC, 6.2%; 95% CI, 3.1-9.5). Among those with tTG-IgA results ≥10 times the upper limit of normal, the incidence was 12.9 per 100,000 person-years. The incidence of celiac autoimmunity was higher in metropolitan settings (IRR, 1.28; 95% CI, 1.21-1.35) and in the least socioeconomically deprived areas compared to the highest (IRR, 1.22; 95% CI, 1.14-1.32). CONCLUSIONS: Incidence of celiac autoimmunity is high and increasing, despite stable testing rates. Variation in testing patterns may lead to underreporting the incidence of celiac autoimmunity in nonmetropolitan areas and more socioeconomically deprived neighborhoods.

Systemic C-peptide supplementation ameliorates retinal neurodegeneration by inhibiting VEGF-induced pathological events in diabetes.

Diabetic retinopathy (DR) is caused by retinal vascular dysfunction and neurodegeneration. Intraocular delivery of C-peptide has been shown to be beneficial against hyperglycemia-induced microvascular leakage in the retina of diabetes; however, the effect of C-peptide on diabetes-induced retinal neurodegeneration remains unknown. Moreover, extraocular C-peptide replacement therapy against DR to avoid various adverse effects caused by intravitreal injections has not been studied. Here, we demonstrate that systemic C-peptide supplementation using osmotic pumps or biopolymer-conjugated C-peptide hydrogels ameliorates neurodegeneration by inhibiting vascular endothelial growth factor-induced pathological events, but not hyperglycemia-induced vascular endothelial growth factor expression, in the retinas of diabetic mice. C-peptide inhibited hyperglycemia-induced activation of macroglial and microglial cells, downregulation of glutamate aspartate transporter 1 expression, neuronal apoptosis, and histopathological changes by a mechanism involving reactive oxygen species generation in the retinas of diabetic mice, but transglutaminase 2, which is involved in retinal vascular leakage, is not associated with these pathological events. Overall, our findings suggest that systemic C-peptide supplementation alleviates hyperglycemia-induced retinal neurodegeneration by inhibiting a pathological mechanism, involving reactive oxygen species, but not transglutaminase 2, in diabetes.

Genomic characterization of vulvar squamous cell carcinoma reveals differential gene expression based on clinical outcome.

OBJECTIVE: The greatest challenge in the management of vulvar squamous cell carcinoma (VSCC) is treatment of recurrent disease where options for surgery and radiation have been exhausted, or treatment of disease where distant metastasis is present. Identification of mutations differentially expressed between tumor from patients who died of aggressive disease and tumor from patients with an indolent course could reveal novel prognostic indicators and guide development of therapeutic drugs. METHODS: From 202 consecutive patients with VSCC, patients who recurred and died of disease (group A) were identified and matched by age, tumor size, depth of invasion and nodal status with those whose disease did not recur (group B). Tumors from 21 patients were subjected to whole exome sequencing of DNA and RNA, immunohistochemistry (IHC) antibodies of PD-L1 and P16, and in-situ hybridization (ISH) for high-risk HPV. RESULTS: Analysis of DNA and RNA revealed six genes that were strongly differentially expressed between group A and B: TGM3, ACVR2A, ROS1, NFEL2, CCND1 and BCL6. Clinically relevant DNA mutations were significantly greater in group A versus B: 7 vs 2.3 mutations per patient. The most common genomic alterations were mutations in TP53 and the promoter region of TERT. Other common genomic events include alterations of FAT1, CDKN2A, PIK3CA, CCND1, and LRP1B. All samples were MSI stable and tumor mutational burden (TMB) was similar in groups A and B. Most VSCC specimens (81%) were positive for PD-L1. CONCLUSIONS: ACVR2A and TGM3 are significantly under-expressed in tumors with poor outcome, suggesting they may play a role in tumor suppression. Clinical outcome of VSCC appears independent of MSI, TMB, or PD-L1 status.

Monitoring patients with celiac disease on gluten free diet: different outcomes comparing three tissue transglutaminase IgA assays.

OBJECTIVES: Tissue transglutaminase (tTG) IgA antibodies are a hallmark for celiac disease (CD). In CD patients on gluten free diet (GFD) these antibodies are transient. Few studies are available comparing the tTG-IgA assay characteristics for monitoring response to GFD. Since discrepant results were reported in patients on GFD after switching tTG-IgA assays, we conducted a retrospective observational study to monitor GFD response using three different tTG-IgA assays. METHODS: Diagnostic samples from 44 adults and 17 children with CD were included. Of most patients two follow-up samples after introduction of GFD were available. In all samples tTG-IgA were assessed using one fluorochrome-enzyme immuno-assay (FEIA) and two chemiluminescence immuno-assays (CLIA) and intestinal fatty acid binding protein (i-FABP) as surrogate marker for intestinal epithelial damage was measured. RESULTS: Using CLIA assays, normalization of antibody levels was delayed compared to FEIA (p<0.001). Of all samples taken after at least 6 months on GFD with elevated i-FABP indicating intestinal epithelial damage, 40 % had positive tTG-IgA according to the FEIA, 85 and 90 % according to the two CLIA. CONCLUSIONS: Normalization of tTG-IgA in patients on GFD depends on the assay used. Both CLIA appear to be more sensitive in detecting suboptimal treatment response in CD-indicated by elevated i-FABP - when applying the manufacturer's recommended cut-off for the diagnosis of CD.

Longitudinal screening of HLA-risk and HLA-nonrisk children for celiac disease to age 15 years: CiPiS study.

OBJECTIVES: Autoantibodies against tissue transglutaminase (tTG) are serological markers of celiac disease. The aim was to study the applicability of human leukocyte antigen (HLA)-genotyping and tTG autoantibodies in the screening of celiac disease in a longitudinal birth cohort followed to age 15 years. METHODS: Included were 13,860 HLA-DQ-genotyped children at birth and previously invited to a screening at age 3 and 9 years, respectively. HLA-DQB1*02 and/or DQB1*03:02 (HLA-risk) children were compared with non-HLA-DQB1*02 and non-DQB1*03:02 (HLA-nonrisk) children. The present study reinvited 12,948/13,860 (93.4%) children at age 15 years of whom 1056/2374 (44.5%) participated in screening at both age 3 and 9 years. Both immunoglobulin A (IgA) and G (IgG) autoantibodies against tTG were analyzed separately in radiobinding assays. Persistently tTG autoantibody-positive children were examined with intestinal biopsy to confirm the diagnosis of celiac disease. RESULTS: At age 3 years, celiac disease was diagnosed in 56/1635 (3.4%) HLA-risk children compared with 0/1824 HLA-nonrisk children (p < 0.001). By age 9 years, celiac disease was diagnosed in 72/1910 (3.8%) HLA-risk children compared with 0/2167 HLA-nonrisk children (p < 0.001). Screening at age 15 years detected 14/1071 (1.3%) HLA-risk children positive for IgA-tTG and/or IgG-tTG of whom 12/1071 (1.1%) remained persistently positive. Among those, 10/1071 (0.9%, 95% confidence interval: 0.4%-1.7%) HLA-risk children were diagnosed with celiac disease compared with 0/1303 HLA-nonrisk children (p < 0.001) and 5/491 (1.0%) were negative in screenings at both 3 and 9 years of age. CONCLUSIONS: Screening for celiac disease needs to be performed at multiple timepoints to detect all cases but can be restricted to children at HLA-risk.

Validation of no-biopsy pathway for the diagnosis of celiac disease in Asian adults: a multicenter retrospective study.

BACKGROUND AND AIM: While European Society of Pediatric Gastroenterology Hepatology and Nutrition advocates a no-biopsy pathway for the diagnosis of celiac disease (CeD) in children if IgA anti-tissue transglutaminase antibody (anti-tTG ab) titer is ≥10-fold upper limit of normal (ULN) and have a positive IgA anti-endomysial antibody (EMA); the data for anti-tTG Ab titer-based diagnosis of CeD in adults is still emerging. We planned to validate if IgA anti-tTG Ab titer ≥10-fold predicts villous abnormalities of modified Marsh grade ≥2 in Asian adult patients with CeD. METHODS: We recruited 937 adult patients with positive anti-tTG Ab from two databases, including AIIMS Celiac Clinic and Indian National Biorepository. The diagnosis of definite CeD was made on the basis of a positive anti-tTG Ab and the presence of villous abnormalities of modified Marsh grade ≥2. RESULTS: Of 937 adult patients with positive anti-tTG Ab, 889 (91.2%) showed villous abnormalities of modified Marsh grade ≥2. Only 47.6% of 889 adults with CeD had anti- tTG Ab titers of ≥10-fold. The positive predictive value (PPV) and specificity of anti tTG Ab titer ≥10-fold for predicting modified Marsh grade ≥2 were 99.8% and 98%, respectively. At anti-tTG Ab titer ≥11-fold, specificity and PPV were 100% for predicting villous abnormalities of modified Marsh grade ≥2. CONCLUSIONS: Approximately 50% of adults with CeD may benefit from the no biopsy pathway, reducing the health burden and risks of gastroscopy/anesthesia.

Outcome in pediatric celiac disease is independent of the diagnostic approach in patients with high antibody levels.

OBJECTIVES: European Society for Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) guidelines enable the diagnosis of celiac disease (CD) without biopsies in patients with immunoglobulin A (IgA)-antibodies against tissue transglutaminase (TGA-IgA) ≥ 10× the upper limit of normal (ULN) and positivity of endomysial antibodies in a second blood sample. Limited data exist comparing the biopsy versus the nonbiopsy diagnostic approach regarding long-term outcomes in CD patients. Our study aimed to investigate the influence of the diagnostic approach on adherence to gluten-free diet (GFD), serological remission (defined as normalization of TGA-IgA during follow-up (FU)) and clinical remission in CD patients with TGA-IgA ≥ 10× ULN. METHODS: Retrospective multicenter study. Patients with CD and TGA-IgA ≥ 10× ULN at diagnosis were included in the study. Patients with confirmed diagnosis by biopsy were compared to patients diagnosed by nonbiopsy approach using univariate analysis, Kaplan-Meier survival curve, and logistic regression models. RESULTS: A total of 282 CD patients (192 [68.1%] in the biopsy group; 90 [31.9%] in the nonbiopsy group) were analyzed. The median time to normalization of TGA-IgA was 16.5 months [interquartile range, IQR: 13, 28] in the biopsy and 15 months [IQR: 12, 26] in the nonbiopsy group; p = 0.14). Rates of normalized TGA-IgA at first to third-year FU were comparable between both groups. Adherence to GFD did not seem to be influenced by the diagnostic approach. CONCLUSIONS: The nonbiopsy approach is not inferior to the biopsy approach in terms of adherence to GFD and serological remission in patients with CD.

Discovery of novel 1H-benzo[d]imidazole-4,7-dione based transglutaminase 2 inhibitors as p53 stabilizing anticancer agents in renal cell carcinoma.

Overexpression of transglutaminase 2 (TGase 2; TG2) has been implicated in the progression of renal cell carcinoma (RCC) through the inactivation of p53 by forming a protein complex. Because most p53 in RCC has no mutations, apoptosis can be increased by inhibiting the binding between TG2 and p53 to increase the stability of p53. In the present study, a novel TG2 inhibitor was discovered by investigating the structure of 1H-benzo[d]imidazole-4,7-dione as a simpler chemotype based on the amino-1,4-benzoquinone moiety of streptonigrin, a previously reported inhibitor. Through structure-activity relationship (SAR) studies, compound 8j (MD102) was discovered as a potent TG2 inhibitor with an IC50 value of 0.35 µM, p53 stabilization effect and anticancer effects in the ACHN and Caki-1 RCC cell lines with sulforhodamine B (SRB) GI50 values of 2.15 µM and 1.98 µM, respectively. The binding property of compound 8j (MD102) with TG2 was confirmed to be reversible in a competitive enzyme assay, and the binding interaction was expected to be formed at the ß-sandwich domain, a p53 binding site, in the SPR binding assay with mutant proteins. The mode of binding of compound 8j (MD102) to the ß-sandwich domain of TG2 was analyzed by molecular docking using the crystal structure of the active conformation of human TG2. Compound 8j (MD102) induced a decrease in the downstream signaling of p-AKT and p-mTOR through the stabilization of p53 by TG2 inhibition, resulting in tumor cell apoptosis. In a xenograft animal model using ACHN cancer cells, oral administration and intraperitoneal injection of compound 8j (MD102) showed an inhibitory effect on tumor growth, confirming increased levels of p53 and decreased levels of Ki-67 in tumor tissues through immunohistochemical (IHC) tissue staining. These results indicated that the inhibition of TG2 by compound 8j (MD102) could enhance p53 stabilization, thereby ultimately showing anticancer effects in RCC. Compound 8j (MD102), a novel TG2 inhibitor, can be further applied for the development of an anticancer candidate drug targeting RCC.

Analysis on promotive effect of rocking culture on keratinocyte differentiation in three-dimensional reconstitution human epidermis.

A three-dimensional culture system of keratinocytes achieves cornification as a terminal differentiation that can mimic the formation of stratified epidermis. At the onset of keratinocyte differentiation, air-exposure treatment is essential for promotion. We have previously reported that the stimulation of differentiation is accompanied by down-regulation of the transcriptional activity of the hypoxia-inducible factor (HIF) and also found that rocking treatment of cultured keratinocytes in the submerged condition restored their differentiation. A comparative study between with and without rocking was then carried out to investigate the characteristics of the recovered differentiation by morphological and biochemical analysis. In addition, transcriptome analysis revealed the expected similar pattern between air-exposure and rocking culture, including HIF-regulating transcripts. Furthermore, the promotive effect of rocking treatment was impaired under hypoxic culture conditions (1% O2). We showed that the restored promotion of differentiation by rocking culture is mainly due to the abrogation of transcriptional events by hypoxia.

Substrate profiling of human transglutaminase 1 using cDNA display and next-generation sequencing.

Human transglutaminase 1 (TG1) modulates skin development, while its involvement in diseases remains poorly understood, necessitating comprehensive exploration of its substrate interactions. To study the substrate profile of TG1, an in vitro selection system based on cDNA display technology was used to screen two peptide libraries with mutations at varying distance from the reactive glutamine. Next-generation sequencing and bioinformatics analysis of the selected DNA pools revealed a detailed TG1 substrate profile, indicating preferred and non-preferred amino acid sequences. The peptide sequence, AEQHKLPSKWPF, was identified showing high reactivity and specificity to TG1. The position weight matrix calculated from the per amino acid enrichment factors was employed to search human proteins using an in-house algorithm, revealing six known TG1 substrate proteins with high scores, alongside a list of candidate substrates currently under investigation. Our findings are expected to assist in future medical diagnoses and development of treatments for skin disorders.

Diagnostic accuracy of a novel point-of-care test for simultaneous detection of anti-transglutaminase IgA and anti-deamidated gliadin IgG antibodies.

BACKGROUND: Point-of-care tests (POCTs) may have a role in detecting undiagnosed cases of Celiac disease (CD). We assessed the diagnostic accuracy of a novel POCT, compared with the conventional serological methods, for simultaneous anti-transglutaminase (tTG) IgA and anti-deamidated gliadin (DGP) IgG antibody detection. Furthermore, we evaluated the effect of different biological matrices (whole blood and serum) on test performance. METHODS: Serum and whole blood from celiac or suspected celiac patients who underwent duodenal biopsy were assayed for the presence of anti-tTG IgA and anti-DGP IgG both with the reference standard methods (Thermo Fisher Scientific, Uppsala, Sweden) and with the POCT (PRIMA Lab SA, Balerna, Switzerland). RESULTS: 266 sera (101 negative and 165 positive) and 60 whole blood samples (34 positive and 26 negative) were included in the study. POCT for anti-DGP IgG showed a sensitivity of 84.3% and a specificity of 90.1%, with positive (PPV) and negative predictive values (NPV) of 91.07% and 82.73%. POCT for anti-tTG IgA showed a sensitivity of 98.31% and a specificity of 98.02%, with a PPV and NPV of 98.31% and 98.02%. Test accuracies were 86.94% and 98.17%, respectively. The agreement of the results between the two different matrices showed a strong correlation rate: 95% for anti-DGP IgG and 100% for anti-tTG IgA. CONCLUSION: The anti-tTG IgA/anti-DGP IgG-based POCT showed good diagnostic accuracy with comparable sensitivities and specificities to reference standard methods in detecting CD in symptomatic patients and could be considered as a mass screening test before referring to conventional serology.

Dietary adherence is not dependent on the mode of diagnosis in children with coeliac disease.

AIM: To compare the adherence to gluten-free diet between children with serology-based and biopsy-proven coeliac disease. METHODS: Medical records were retrospectively reviewed in 257 Swedish children diagnosed with coeliac disease between 2012 and 2019 at a tertiary hospital. Adherence to a gluten-free diet was systematically assessed by trained dietitians at follow-up. Mixed models were used to analyse the dietary adherence by mode of diagnosis (serology-based vs. biopsy-proven). RESULTS: After mean 6.3 (SD 2.4) years, there was neither a difference in the dietary adherence over time depending on the mode of diagnosis (OR 0.64 [95% confidence interval (CI) 0.26, 1.60], p = 0.342), nor if coeliac disease was detected in screening studies (OR 0.74 [95% CI 0.25, 2.17], p = 0.584) or in risk-groups (OR 1.01 [95% CI 0.26, 3.91], p = 0.991) compared to clinically detected diagnosis. Non-adherence to a gluten-free diet increased with age (OR 1.19 [95% CI 1.06, 1.33], p = 0.003). There was no difference in the proportion of patients improving their dietary adherence from non-adherent to adherent over time (p = 0.322). CONCLUSION: Mode of diagnosis did not influence the dietary adherence in Swedish children with coeliac disease, although adherence to a gluten-free diet was inversely associated with increasing age.

No COVID-19 pandemic impact on incidence and clinical presentation of celiac disease in Italian children.

AIM: We aimed to evaluate the impact of Coronavirus Disease-19 (COVID-19) pandemic on the incidence and clinical presentation of celiac disease (CD) in children. METHODS: The diagnoses of CD were compared between the COVID-19 pandemic (from April 2020 to March 2022) and the pre-pandemic period (from April 2018 to March 2020) in three Italian Paediatric Gastroenterology centres (Varese, Como, Catanzaro). Electronic patient records were reviewed and additional information were collected through parental interview. The diagnosis of CD was made according to ESPGHAN criteria. SARS-CoV-2 infection was diagnosed based on pre-vaccination positive serum antibodies or nasopharyngeal swabs. Z test and chi-square were used for statistical analysis. RESULTS: The overall number of paediatric diagnosis of CD did not differ between the two years pre-pandemic and pandemic periods (177 and 172 cases) in the three Italian participating centres. Clinical presentation of CD was also similar throughout the study periods. SARS-CoV-2 infection has been documented in 10.6% of children but only in 5.8% of these occurred before CD diagnosis. CONCLUSION: Different to what reported for other autoimmune diseases, the incidence and presenting symptoms of CD in our paediatric population did not change during the COVID-19 pandemic compared to the previous 2 years.