How dietary advanced glycation end products could facilitate the occurrence of food allergy.

BACKGROUND: Food allergy (FA) is one of the most common chronic conditions in children with an increasing prevalence facilitated by the exposure to environmental factors in predisposed individuals. It has been hypothesized that the increased consumption of ultra-processed foods, containing high levels of dietary advanced glycation end products (AGEs), could facilitate the occurrence of FA. OBJECTIVE: We sought to provide preclinical and clinical evidence on the potential role of AGEs in facilitating the occurrence of FA. METHODS: Human enterocytes, human small intestine organ culture, and PBMCs from children at risk for allergy were used to investigate the direct effect of AGEs on gut barrier, inflammation, TH2 cytokine response, and mitochondrial function. Intake of the 3 most common glycation products in Western diet foods, Nε-(carboxymethyl) lysine, Nε-(1-carboxyethyl) lysin, and Nδ-(5-hydro-5- methyl-4-imidazolone-2-yl)-ornithine (MG-H1), and the accumulation of AGEs in the skin were comparatively investigated in children with FA and in age-matched healthy controls. RESULTS: Human enterocytes exposed to AGEs showed alteration in gut barrier, AGE receptor expression, reactive oxygen species production, and autophagy, with increased transepithelial passage of food antigens. Small intestine organ cultures exposed to AGEs showed an increase of CD25+ cells and proliferating crypt enterocytes. PBMCs exposed to AGEs showed alteration in proliferation rate, AGE receptor activation, release of inflammatory and TH2 cytokines, and mitochondrial metabolism. Significant higher dietary AGE intake and skin accumulation were observed children with FA (n = 42) compared with age-matched healthy controls (n = 66). CONCLUSIONS: These data, supporting a potential role for dietary AGEs in facilitating the occurrence of FA, suggest the importance of limiting exposure to AGEs children as a potential preventive strategy against this common condition.

IL-10-producing regulatory cells impact on celiac disease evolution.

Celiac Disease (CD) is a T-cell mediated disorder caused by immune response to gluten, although the mechanisms underlying CD progression are still elusive. We analyzed immune cell composition, plasma cytokines, and gliadin-specific T-cell responses in patients with positive serology and normal intestinal mucosa (potential-CD) or villous atrophy (acute-CD), and after gluten-free diet (GFD). We found: an inflammatory signature and the presence of circulating gliadin-specific IFN-γ+ T cells in CD patients regardless of mucosal damage; an increased frequency of IL-10-secreting dendritic cells (DC-10) in the gut and of circulating gliadin-specific IL-10-secreting T cells in potential-CD; IL-10 inhibition increased IFN-γ secretion by gliadin-specific intestinal T cells from acute- and potential-CD. On GFD, inflammatory cytokines normalized, while IL-10-producing T cells accumulated in the gut. We show that IL-10-producing cells are fundamental in controlling pathological T-cell responses to gluten: DC-10 protect the intestinal mucosa from damage and represent a marker of potential-CD.

Status of vitamins and minerals in children with screening-identified celiac disease: A case-control study.

OBJECTIVES: Micronutrient deficiencies characterize classical "late-diagnosed" celiac disease (CeD). This study aimed to identify the prevalence of micronutrient deficiencies among children with "early-diagnosed" screening-identified CeD to determine the clinical value of routine testing for deficiencies in those patients. METHODS: A case-control study was conducted on screening-identified CeD patients diagnosed during a mass screening study (84 patients, mean age 11.3 ± 2.6 years). The controls (443 children, mean age 10.8 ± 2.5 years) were negative for celiac disease serological screening. Hemoglobin, serum levels of iron, ferritin, folate, vitamin B12, vitamin A, vitamin E, 25-OH vitamin D, zinc, and selenium were measured. RESULTS: The mean serum levels of hemoglobin, iron, ferritin, vitamin D, zinc, copper, and selenium were significantly lower in CeD patients than in healthy controls (hemoglobin 12.56 vs. 13.02 g/dL [p = 0.04]; iron 10.61 vs. 17.6 µmol/L [p < 0.001], ferritin 25.7 vs. 48.3 µg/L [p < 0.001], vitamin D 29.1 vs. 37.5 nmol/L, zinc 11.9 vs. 21.7 µmol/L, copper 18.9 vs. 32.5 µmol/L, selenium 1.04 vs. 1.36 µmol/L; p < 0.001). Patients with celiac and severe intestinal damage (Marsh IIIb and IIIc) had significantly lower serum ferritin and vitamin A levels than patients with mild intestinal damage (Marsh II and IIIa) (ferritin 15 vs. 22 µg/L, p < 0.025; vitamin A 0.85 vs. 1.35 µmol/L, p = 0.007). CONCLUSION: Micronutrient deficiencies are still detectable in "early-diagnosed" screening-identified CeD cases, a clinically relevant result that strongly supports efforts for screening and early diagnosis of CeD.

Early diet and the risk of coeliac disease. An update 2024 position paper by the ESPGHAN special interest group on coeliac disease.

This position paper by the European Society for Paediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) Special Interest Group on Coeliac Disease (SIG-CD) presents an update to the 2016 recommendations concerning early diet and the risk of coeliac disease (CD). This update adheres to the policy that mandates reviewing guidelines every 5 years, particularly when new data emerge. The 2024 statements and recommendations are essentially similar to the 2016 recommendations. Breastfeeding, whether any amount, exclusive, or of any duration, does not reduce the risk of developing CD. Introducing gluten into an infant's diet at any time between completed 4 months (≥17 weeks) and 12 months of age does not affect the cumulative incidence of CD, although earlier introduction may lead to earlier seroconversion and CD. In observational studies involving cohorts with a known risk for CD, consuming a high amount of gluten compared to a low amount during weaning and in the subsequent childhood years-specifically the first 2-3 years, and even up to 5 years in some studies-was associated with an increased risk for CD. However, the specific optimal amounts of gluten consumption remain undetermined due to insufficient evidence on safe thresholds, and the impact of restricting gluten in the diet of healthy children of unknown risk for CD is unknown. Thus, any recommendation on the gluten amount is currently unjustifiable for the general population and infants with known HLA risk types. There is no specific guidance on the type of gluten-containing foods to be introduced at weaning.

Prediction Models for Celiac Disease Development in Children From High-Risk Families: Data From the PreventCD Cohort.

BACKGROUND & AIMS: Screening for celiac disease (CD) is recommended in children with affected first-degree relatives (FDR). However, the frequency of screening and at what age remain unknown. The aims of this study were to detect variables influencing the risk of CD development and develop and validate clinical prediction models to provide individualized screening advice. METHODS: We analyzed prospective data from the 10 years of follow-up of the PreventCD-birth cohort involving 944 genetically predisposed children with CD-FDR. Variables significantly influencing the CD risk were combined to determine a risk score. Landmark analyses were performed at different ages. Prediction models were created using multivariable Cox proportional hazards regression analyses, backward elimination, and Harrell's c-index for discrimination. Validation was done using data from the independent NeoCel cohort. RESULTS: In March 2019, the median follow-up was 8.3 years (22 days-12.0 years); 135/944 children developed CD (mean age, 4.3 years [range, 1.1-11.4]). CD developed significantly more often in girls (P = .005) and in Human Leukocyte Antigen (HLA)-DQ2 homozygous individuals (8-year cumulative incidence rate of 35.4% vs maximum of the other HLA-risk groups 18.2% [P < .001]). The effect of homozygosity DR3-DQ2/DR7-DQ2 on CD development was only present in girls (interaction P = .04). The prediction models showed good fit in the validation cohort (Cox regression 0.81 [0.54]). To calculate a personalized risk of CD development and provide screening advice, we designed the Prediction application https://hputter.shinyapps.io/preventcd/. CONCLUSION: Children with CD-FDR develop CD early in life, and their risk depends on gender, age and HLA-DQ, which are all factors that are important for sound screening advice. These children should be screened early in life, including HLA-DQ2/8-typing, and if genetically predisposed to CD, they should get further personalized screening advice using our Prediction application. TRIAL REGISTRATION NUMBER: ISRCTN74582487 (https://www.isrctn.com/search?q=ISRCTN74582487).

Tolerogenic IL-10-engineered dendritic cell-based therapy to restore antigen-specific tolerance in T cell mediated diseases.

Tolerogenic dendritic cells play a critical role in promoting antigen-specific tolerance via dampening of T cell responses, induction of pathogenic T cell exhaustion and antigen-specific regulatory T cells. Here we efficiently generate tolerogenic dendritic cells by genetic engineering of monocytes with lentiviral vectors co-encoding for immunodominant antigen-derived peptides and IL-10. These transduced dendritic cells (designated DCIL-10/Ag) secrete IL-10 and efficiently downregulate antigen-specific CD4+ and CD8+ T cell responses from healthy subjects and celiac disease patients in vitro. In addition, DCIL-10/Ag induce antigen-specific CD49b+LAG-3+ T cells, which display the T regulatory type 1 (Tr1) cell gene signature. Administration of DCIL-10/Ag resulted in the induction of antigen-specific Tr1 cells in chimeric transplanted mice and the prevention of type 1 diabetes in pre-clinical disease models. Subsequent transfer of these antigen-specific T cells completely prevented type 1 diabetes development. Collectively these data indicate that DCIL-10/Ag represent a platform to induce stable antigen-specific tolerance to control T-cell mediated diseases.

Antibody Profile, Gene Expression and Serum Cytokines in At-Risk Infants before the Onset of Celiac Disease.

Immunological events that precede the development of villous atrophy in celiac disease (CeD) are still not completely understood. We aimed to explore CeD-associated antibody production (anti-native gliadin (AGA), anti-deamidated gliadin (DGP) and anti-tissue transglutaminase (anti-tTG)) in infants at genetic risk for CeD from the Italian cohorts of the PREVENT-CD and Neocel projects, as well as the relationship between antibody production and systemic inflammation. HLA DQ2 and/or DQ8 infants from families with a CeD case were followed from birth. Out of 220 at-risk children, 182 had not developed CeD by 6 years of age (CTRLs), and 38 developed celiac disease (CeD). The profiles of serum cytokines (INFγ, IL1ß, IL2, IL4, IL6, IL10, IL12p70, IL17A and TNFα) and the expression of selected genes (FoxP3, IL10, TGFß, INFγ, IL4 and IL2) were evaluated in 46 children (20 CeD and 26 CTRLs). Among the 182 healthy CTRLs, 28 (15.3%) produced high levels of AGA-IgA (AGA+CTRLs), and none developed anti-tTG-IgA or DGP-IgA, compared to 2/38 (5.3%) CeD infants (Chi Sq. 5.97, p = 0.0014). AGAs appeared earlier in CTRLs than in those who developed CeD (19 vs. 28 months). Additionally, the production of AGAs in CeD overlapped with the production of DGP and anti-tTG. In addition, gene expression as well as serum cytokine levels discriminated children who developed CeD from CTRLs. In conclusion, these findings suggest that the early and isolated production of AGA-IgA antibodies is a CeD-tolerogenic marker and that changes in gene expression and cytokine patterns precede the appearance of anti-tTG antibodies.

ESPGHAN Position Paper on Management and Follow-up of Children and Adolescents With Celiac Disease.

OBJECTIVES: To gather the current evidence and to offer recommendations for follow-up and management. METHODS: The Special Interest Group on Celiac Diseases of the European Society of Paediatric Gastroenterology Hepatology and Nutrition formulated ten questions considered to be essential for follow-up care. A literature search (January 2010-March 2020) was performed in PubMed or Medline. Relevant publications were identified and potentially eligible studies were assessed. Statements and recommendations were developed and discussed by all coauthors. Recommendations were voted upon: joint agreement was set as at least 85%. RESULTS: Publications (n = 2775) were identified and 164 were included. Using evidence or expert opinion, 37 recommendations were formulated on: The need to perform follow-up, its frequency and what should be assessed, how to assess adherence to the gluten-free diet, when to expect catch-up growth, how to treat anemia, how to approach persistent high serum levels of antibodies against tissue-transglutaminase, the indication to perform biopsies, assessment of quality of life, management of children with unclear diagnosis for which a gluten-challenge is indicated, children with associated type 1 diabetes or IgA deficiency, cases of potential celiac disease, which professionals should perform follow-up, how to improve the communication to patients and their parents/caregivers and transition from pediatric to adult health care. CONCLUSIONS: We offer recommendations to improve follow-up of children and adolescents with celiac disease and highlight gaps that should be investigated to further improve management.

Pivotal Role of Inflammation in Celiac Disease.

Celiac disease (CD) is an immune-mediated enteropathy triggered in genetically susceptible individuals by gluten-containing cereals. A central role in the pathogenesis of CD is played by the HLA-restricted gliadin-specific intestinal T cell response generated in a pro-inflammatory environment. The mechanisms that generate this pro-inflammatory environment in CD is now starting to be addressed. In vitro study on CD cells and organoids, shows that constant low-grade inflammation is present also in the absence of gluten. In vivo studies on a population at risk, show before the onset of the disease and before the introduction of gluten in the diet, cellular and metabolic alterations in the absence of a T cell-mediated response. Gluten exacerbates these constitutive alterations in vitro and in vivo. Inflammation, may have a main role in CD, adding this disease tout court to the big family of chronic inflammatory diseases. Nutrients can have pro-inflammatory or anti-inflammatory effects, also mediated by intestinal microbiota. The intestine function as a crossroad for the control of inflammation both locally and at distance. The aim of this review is to discuss the recent literature on the main role of inflammation in the natural history of CD, supported by cellular fragility with increased sensitivity to gluten and other pro-inflammatory agents.

Inflammation Is Present, Persistent and More Sensitive to Proinflammatory Triggers in Celiac Disease Enterocytes.

Celiac disease (CD) is a chronic inflammatory disease caused by a genetic predisposition to an abnormal T cell-mediated immune response to the gluten in the diet. Different environmental proinflammatory factors can influence and amplify the T cell-mediated response to gluten. The aim of this manuscript was to study the role of enterocytes in CD intestinal inflammation and their response to different proinflammatory factors, such as gliadin and viruses. Intestinal biopsies from CD patients on a gluten-containing (GCD-CD) or a gluten-free diet (GFD-CD) as well as biopsies from potential CD patients (Pot-CD) before the onset of intestinal lesions and controls (CTR) were used to investigate IL-1ß and IL-6 mRNA levels in situ. Organoids from CD patients were used to test the levels of NF-κB, ERK, IL-6, and IL-1ß by Western blot (WB), ELISA, and quantitative PCR. The Toll-like receptor ligand loxoribine (Lox) and gliadin peptide P31-43 were used as proinflammatory stimuli. In CD biopsies inflammation markers IL-1ß and IL-6 were increased in the enterocytes, and also in Pot-CD before the onset of the intestinal lesion and in GFD-CD. The inflammatory markers pNF-κB, pERK, IL-1ß, and IL-6 were increased and persistent in CD organoids; these organoids were more sensitive to P31-43 and Lox stimuli compared with CTR organoids. Taken together, these observations point to constitutive inflammation in CD enterocytes, which are more sensitive to inflammatory stimuli such as food components and viruses.

A New Butyrate Releaser Exerts a Protective Action against SARS-CoV-2 Infection in Human Intestine.

Butyrate is a major gut microbiome metabolite that regulates several defense mechanisms against infectious diseases. Alterations in the gut microbiome, leading to reduced butyrate production, have been reported in patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. A new butyrate releaser, useful for all the known applications of butyrate, presenting physiochemical characteristics suitable for easy oral administration, (N-(1-carbamoyl-2-phenyl-ethyl) butyramide (FBA), has been recently developed. We investigated the protective action of FBA against SARS-CoV-2 infection in the human small intestine and enterocytes. Relevant aspects of SARS-CoV-2 infection were assessed: infectivity, host functional receptor angiotensin-converting enzyme-2 (ACE2), transmembrane protease serine 2 (TMPRSS2), neuropilin-1 (NRP1), pro-inflammatory cytokines expression, genes involved in the antiviral response and the activation of Nf-kB nuclear factor (erythroid-derived 2-like) 2 (Nfr2) pathways. We found that FBA positively modulates the crucial aspects of the infection in small intestinal biopsies and human enterocytes, reducing the expression of ACE2, TMPRSS2 and NRP1, pro-inflammatory cytokines interleukin (IL)-15, monocyte chemoattractant protein-1 (MCP-1) and TNF-α, and regulating several genes involved in antiviral pathways. FBA was also able to reduce the number of SARS-CoV-2-infected cells, and ACE2, TMPRSS2 and NRP1 expression. Lastly, through the inhibition of Nf-kB and the up-regulation of Nfr2, it was also able to reduce the expression of pro-inflammatory cytokines IL-15, MCP-1 and TNF-α in human enterocytes. The new butyrate releaser, FBA, exerts a preventive action against SARS-CoV-2 infection. It could be considered as an innovative strategy to limit COVID-19.

Identification of a γc Receptor Antagonist That Prevents Reprogramming of Human Tissue-resident Cytotoxic T Cells by IL15 and IL21.

BACKGROUND & AIMS: Gamma chain (γc) cytokines (interleukin [IL]2, IL4, IL7, IL9, IL15, and IL21) signal via a common γc receptor. IL2 regulates the immune response, whereas IL21 and IL15 contribute to development of autoimmune disorders, including celiac disease. We investigated whether BNZ-2, a peptide designed to inhibit IL15 and IL21, blocks these cytokines selectively and its effects on intraepithelial cytotoxic T cells. METHODS: We obtained duodenal biopsies from 9 patients with potential celiac disease (positive results from tests for anti-TG2 but no villous atrophy), 30 patients with untreated celiac disease (with villous atrophy), and 5 patients with treated celiac disease (on a gluten-free diet), as well as 43 individuals without celiac disease (controls). We stimulated primary intestinal intraepithelial CD8+ T-cell lines, or CD8+ T cells directly isolated from intestinal biopsies, with γc cytokines in presence or absence of BNZ-2. Cells were analyzed by immunoblots, flow cytometry, or RNA-sequencing analysis for phosphorylation of signaling molecules, gene expression profiles, proliferation, and levels of granzyme B. RESULTS: Duodenal tissues from patients with untreated celiac disease had increased levels of messenger RNAs encoding IL15 receptor subunit alpha (IL15RA) and IL21 compared with tissues from patients with potential celiac disease and controls. Activation of intraepithelial cytotoxic T cells with IL15 or IL21 induced separate signaling pathways; incubation of the cells with IL15 and IL21 cooperatively increased their transcriptional activity, proliferation, and cytolytic properties. BNZ-2 specifically inhibited the effects of IL15 and IL21, but not of other γc cytokines. CONCLUSIONS: We found increased expression of IL15RA and IL21 in duodenal tissues from patients with untreated celiac disease compared with controls. IL15 and IL21 cooperatively activated intestinal intraepithelial cytotoxic T cells. In particular, they increased their transcriptional activity, proliferation, and cytolytic activity. The peptide BNZ-2 blocked these effects, but not those of other γc cytokines, including IL2. BNZ-2 might be used to prevent cytotoxic T-cell-mediated tissue damage in complex immune disorders exhibiting upregulation of IL15 and IL21.

Diagnosing Coeliac Disease During Mass-Screening of General Paediatric Population: Is Biopsy Avoidable?

OBJECTIVES: Studies evaluating the correlation between tissue transglutaminase immunoglobulin antibody (TGA-IgA) levels and the degree of enteropathy in screening-detected coeliac disease (CD) patients from the general childhood population are scarce. The objectives of our study were to evaluate the correlation between the TGA-IgA titre and the degree of enteropathy and to evaluate whether the no-biopsy approach to diagnose CD in symptomatic patients proposed by the European Society for Pediatric Gastroenterology, Hepatology, and Nutrition could be extended to asymptomatic CD patients diagnosed during mass screening studies. METHODS: The present study is a sub-study of a cross-sectional mass screening study, "Exploring the Iceberg of Coeliacs in Saudi Arabia", conducted among school-aged children (6-15 years) in 2014-2015. The 93 biopsy-confirmed CD patients constituted the study cohort of the present study (mean age 11.4 ±â€Š2.6 years; 24 males). TGA-IgA titres and endomysial antibodies (EMA) at the time of biopsy and grade of enteropathy were assessed, and human leukocyte antigen DQ 2.2/2.5/8 genotyping was performed. RESULTS: Thirty-four patients had TGA-IgA titres >10× upper limit of normal (ULN; 36%); all had villous atrophy with positive EMA and DQ 2.2/2.5/8. The sensitivity and specificity of a TGA-IgA titre >10× ULN in correctly diagnosing CD was 100%. There was a significant positive correlation between the anti-TGA-IgA titre and the severity of enteropathy (P < 0.001). There was no significant difference in the TGA-IgA titre between the asymptomatic and symptomatic CD patients. CONCLUSIONS: Our results provide evidence that a TGA-IgA titre >10× ULN correlates with villous atrophy in CD patients detected by mass screening.

Seronegative Villous Atrophy in Children: Clinical and Immunohistochemical Features.

OBJECTIVES: Villous atrophy (VA) is not pathognomonic of celiac disease (CD). We aimed at reporting distribution, clinical, and immunohistochemical features of seronegative VA (SNVA) in a pediatric population. METHODS: We retrospectively collected data from patients who underwent intestinal biopsies between 2010 and 2017 and showed VA without serum CD-associated autoantibodies. Marsh-Oberhuber grading was used. Density of intraepithelial lymphocytes (IELs) expressing CD3 or TCRγδ+ receptor and of lamina propria CD25+ cells was assessed by immunohistochemistry. Intestinal deposits of anti-tissue tranglutaminase2 (anti-TG2) were also investigated by double immunofluorescence. RESULTS: Over a 7-year period, 64 out of 1282 patients with VA had negative serum CD serology. Diagnoses were: inflammatory bowel diseases (IBD) (21/64), Gastro-Esophageal Reflux Disease (GERD) (12/64), food allergy (8/64), infections (7/64, of which 3 HIV infections), immune deficiency (3/64), short bowel syndrome (3/64), congenital diarrhea (2/64), other/inconclusive diagnosis (8/64). Forty-four, 15, and 5 showed Marsh 3a, 3b, and 3c lesion, respectively. The latter category included 2 patients with Crohn disease, 2 with immunodeficiencies, 1 with lymphohistiocytosis. In 41/46 (89%) patients, mononuclear CD25+ cells were above the cut-off, indicating mucosal inflammation but only 18/46 (39%) had IELs and TCRγδ + IELs above limits of normality. In 10 of 46 (22%) patients, a positive immunofluorescence indicated the presence of anti-TG2 mucosal antibodies. CONCLUSIONS: SNVA is not rare representing up to 5% of the cases of VA. Most patients have a Marsh 3a lesion. Immunohistochemical analysis may be helpful in excluding CD, whereas the finding of mucosal anti-TG2, particularly with a weak staining, shows no absolute specificity for CD.

Deamidated Gliadin Antibodies: Do They Add to Tissue Transglutaminase-IgA Assay in Screening for Celiac Disease?

OBJECTIVES: Use of deamidated gliadin peptide (DGP) test kits as adjunctive to tissue-transglutaminase-IgA (TTG-IgA) for the diagnosis of celiac disease (CD) has been a controversial issue. The objectives of our study were to evaluate the diagnostic performance of DGP antibodies compared with TTG-IgA and to evaluate the correlation between DGP-antibody titers and degree of enteropathy. METHODS: We included children who underwent endoscopy and biopsies because of positivity of any of the serology tests in the "celiac profile" (TTG-IgA, DGP-IgA, and DGP-IgG) from 2012 to 2019. We divided children into clinically suspected cases of CD (group 1) and asymptomatic cases screened as they were from a high-risk group (group 2). RESULTS: Group 1 constituted 52 children and group 2 included 81 children (76 type-1 diabetes [T1D]). The sensitivity and positive-predictive value (PPV) of DGP-IgG in group 1 (90%, 98%) and group 2 (91%, 85.5%) were comparable with TTG-IgA (98%, 92% in group 1; 100%, 80% in group 2). By adding DGP-IgG to TTG-IgA, the performance of TTG-IgA has improved marginally in group 1 (sensitivity 100%, PPV 92.3%). All cases with DGP-IgG titer 2 times ULN in group 1, and >4 times ULN in group 2 had villous atrophy. All T1D patients with TTG IgA >10 times ULN had villous atrophy. CONCLUSIONS: DGP-IgG assay did not add to the performance of TTG-IgA. DGP-IgG titer correlated with enteropathy. The diagnosis of CD can be made in asymptomatic T1D child with TTG-IgA titer >10 times ULN and positive endomyseal antibodies.

Pediatric Celiac Disease Patients Show Alterations of Dendritic Cell Shape and Actin Rearrangement.

Celiac disease (CD) is a frequent intestinal inflammatory disease occurring in genetically susceptible individuals upon gluten ingestion. Recent studies point to a role in CD for genes involved in cell shape, adhesion and actin rearrangements, including a Rho family regulator, Rho GTPase-activating protein 31 (ARHGAP31). In this study, we investigated the morphology and actin cytoskeletons of peripheral monocyte-derived dendritic cells (DCs) from children with CD and controls when in contact with a physiological substrate, fibronectin. DCs were generated from peripheral blood monocytes of pediatric CD patients and controls. After adhesion on fibronectin, DCs showed a higher number of protrusions and a more elongated shape in CD patients compared with controls, as assessed by immunofluorescence actin staining, transmitted light staining and video time-lapse microscopy. These alterations did not depend on active intestinal inflammation associated with gluten consumption and were specific to CD, since they were not found in subjects affected by other intestinal inflammatory conditions. The elongated morphology was not a result of differences in DC activation or maturation status, and did not depend on the human leukocyte antigen (HLA)-DQ2 haplotype. Notably, we found that ARH-GAP31 mRNA levels were decreased while RhoA-GTP activity was increased in CD DCs, pointing to an impairment of the Rho pathway in CD cells. Accordingly, Rho inhibition was able to prevent the cytoskeleton rearrangements leading to the elongated morphology of celiac DCs upon adhesion on fibronectin, confirming the role of this pathway in the observed phenotype. In conclusion, adhesion on fibronectin discriminated CD from the controls' DCs, revealing a gluten-independent CD-specific cellular phenotype related to DC shape and regulated by RhoA activity.

Progression of Celiac Disease in Children With Antibodies Against Tissue Transglutaminase and Normal Duodenal Architecture.

BACKGROUND & AIMS: Potential celiac disease is characterized by positive results from serologic tests for tissue transglutaminase antibodies (anti-TG2) but normal duodenal architecture (Marsh stages 0-1). There is controversy over the best way to manage these patients. We investigated risk factors associated with the development of villous atrophy in children with potential celiac disease. METHODS: We performed a prospective study of 280 children (ages 2-18 years) in Italy with suspected celiac disease, followed for up to 12 years (range, 18-150 months; median 60 months). The subjects had 2 consecutive positive results from tests for anti-TG2, tested positive for the endomysial antibody (anti-EMA), had total serum levels of immunoglobulin A in the normal range, normal duodenal architecture (Marsh stages 0-1) in 5 biopsies, and HLA DQ2- or DQ8-positive haplotypes. The children underwent serologic tests and clinical analyses every 6 months and a small bowel biopsy was taken every 2 years. A total of 210 patients of the original cohort were assessed at the 9-year follow-up evaluation. We performed multivariate analyses of clinical, genetic, and histologic data to identify factors associated with progression to villous atrophy. RESULTS: During the follow-up period, 42 (15%) of 280 children developed villous atrophy, whereas 89 (32%) children no longer tested positive for anti-TG2 or anti-EMA. The cumulative incidence of progression to villous atrophy was 43% at 12 years. In multivariate analysis, the baseline factors most strongly associated with development of villous atrophy were numbers of γδ intraepithelial lymphocyte cells followed by age and homozygosity for the HLA DQB1*02. In discriminant analysis, these baseline factors identified 80% of the children who developed baseline atrophy. CONCLUSIONS: In a long-term study of 280 children with suspected celiac disease (based on anti-TG2 and anti-EMA) on gluten-containing diets, the cumulative incidence of progression to villous atrophy was 43% over a 12-year period. We identified factors that can be used to identify children at highest risk for villous atrophy. This approach might be used to determine whether children with suspected celiac disease should immediately start a gluten-free diet or be monitored on their regular diet.

The intestinal expansion of TCRγδ+ and disappearance of IL4+ T cells suggest their involvement in the evolution from potential to overt celiac disease.

Celiac disease (CD) is characterized by a spectrum of intestinal inflammatory lesions. Most patients have villous atrophy (overt-CD), while others have a morphologically normal mucosa, despite the presence of CD-specific autoantibodies (potential-CD). As the mechanism responsible for villous atrophy is not completely elucidated, we investigated biomarkers specific for the different celiac lesions. Phenotype and cytokine production of intestinal mucosa cells were analyzed by flow cytometry in gut biopsies of children with overt- or potential-CD and in healthy controls. Density of TCRγδ+ T cells was found markedly enhanced in intestinal mucosa of children with overt-CD compared to potential-CD or controls. By contrast, very few IL4+ T cells infiltrated the mucosa with villous atrophy compared to morphologically normal mucosa. IL4+ T cells were classical CD4+ T-helper cells (CD161- ), producing or not IFN-γ, and negative for IL17A. Our study demonstrated that the transition to villous atrophy in CD patients is characterized by increased density of TCRγδ+ T cells, and concomitant disappearance of IL4+ cells. These findings suggest that immunomodulatory mechanisms are active in potential-CD to counteract the inflammatory cascade responsible of villous atrophy. Further studies are required to validate the use of IL4+ and TCRγδ+ T cells as biomarkers of the different CD forms.

Increased frequency of regulatory T cells in pediatric inflammatory bowel disease at diagnosis: a compensative role?

BACKGROUND: Regulatory T cells (Tregs) play a critical role in maintaining immune homeostasis. We investigated two main types of Tregs, the CD4+FOXP3+ and IL-10+ Tr1, in pediatric subjects with inflammatory bowel disease (IBD) both at diagnosis and after the clinical remission. METHODS: Peripheral blood Tregs were analyzed in 16 children with Crohn's disease (CD), 19 with ulcerative colitis (UC), and 14 healthy controls (HC). Two cocktails of fluoresceinated antibodies were used to discriminate between CD4+FOXP3+ and Tr1. RESULTS: We observed in both CD and UC groups a higher frequency of Tr1 at diagnosis compared to controls, which decreased at follow-up compared to diagnosis, in particular in UC. Similarly, in UC patients the percentage of CD4+FOXP3+ Tregs markedly decreased at follow-up compared to the same patients at diagnosis and compared to HC. The expression of CTLA-4 in CD4+FOXP3+ Tregs increased in both groups at clinical remission. CONCLUSION: This study shows that IBD children present at diagnosis an increased frequency of circulating Tregs, probably as a compensative reaction to tissue inflammation. During the clinical remission, the Treg frequency diminishes, and concomitantly, their activation status increases. Notwithstanding, the high Treg density at diagnosis is not sufficient to counteract the inflammation in the childhood IBD.

European Society Paediatric Gastroenterology, Hepatology and Nutrition Guidelines for Diagnosing Coeliac Disease 2020.

OBJECTIVES: The ESPGHAN 2012 coeliac disease (CD) diagnostic guidelines aimed to guide physicians in accurately diagnosing CD and permit omission of duodenal biopsies in selected cases. Here, an updated and expanded evidence-based guideline is presented. METHODS: Literature databases and other sources of information were searched for studies that could inform on 10 formulated questions on symptoms, serology, HLA genetics, and histopathology. Eligible articles were assessed using QUADAS2. GRADE provided a basis for statements and recommendations. RESULTS: Various symptoms are suggested for case finding, with limited contribution to diagnostic accuracy. If CD is suspected, measurement of total serum IgA and IgA-antibodies against transglutaminase 2 (TGA-IgA) is superior to other combinations. We recommend against deamidated gliadin peptide antibodies (DGP-IgG/IgA) for initial testing. Only if total IgA is low/undetectable, an IgG-based test is indicated. Patients with positive results should be referred to a paediatric gastroenterologist/specialist. If TGA-IgA is ≥10 times the upper limit of normal (10× ULN) and the family agrees, the no-biopsy diagnosis may be applied, provided endomysial antibodies (EMA-IgA) will test positive in a second blood sample. HLA DQ2-/DQ8 determination and symptoms are not obligatory criteria. In children with positive TGA-IgA <10× ULN at least 4 biopsies from the distal duodenum and at least 1 from the bulb should be taken. Discordant results between TGA-IgA and histopathology may require re-evaluation of biopsies. Patients with no/mild histological changes (Marsh 0/I) but confirmed autoimmunity (TGA-IgA/EMA-IgA+) should be followed closely. CONCLUSIONS: CD diagnosis can be accurately established with or without duodenal biopsies if given recommendations are followed.