Treated Celiac Disease Patients Research Articles

Diagnostic accuracy of red blood cell distribution width-to-lymphocyte ratio for celiac disease

Abstract Background. Celiac disease (CD) is significantly underdiagnosed, despite significant efforts made in the last decades to increase its diagnostic rate. This has lead to a high need for developing new diagnostic strategies. Our aim was to evaluate the diagnostic performance of two routine hematologic indices for CD. Methods. In a prospective observational study, 34 newly diagnosed CD patients, 34 age-sex matched controls with irritable bowel syndrome (IBS) and 16 treated CD patients were assessed regarding the differences in mean lymphocyte count (LY), red blood cell distribution width (RDW) and their ratio (RDW/LY). Results. Elevated RDW (>14) and lymphopenia (<1.5 x 10e9/L) were more frequently seen in newly diagnosed CD patients compared to IBS control group and treated CD patients. Newly diagnosed CD patients had significantly higher mean values of RDW/LY - 10.09, compared to 7.72 in the CD-treated group and 6.79 in IBS controls (p<0.01). Subgroup analysis revealed that RDW/LY was higher in patients with destructive histology (Marsh≥3a), 10.54 vs. 7.99. For a value over 7, RDW/LY had a sensitivity of 88.24% (95% CI 72.55-96.70%) and AUROC of 0.785 (95% CI 0.683- 0.887). Conclusions. RDW/LY ratio is a widely available tool which could be used routinely in clinical practice for CD screening.

Risk of Autoimmune Endocrinopathies in Treated Celiac Disease Patients in Olmsted County: 2017 Category Award (Small Intestine)

Introduction: The incidence of celiac disease (CD) is increasing in both pediatric and adult population of Olmsted County, Minnesota. It is known that patients with autoimmune endocrinopathies (AE) like diabetes mellitus type1 (DM1) and thyroid disorders (TD) [Hashimoto Thyroiditis (HT) and Graves' disease (GD)] are at increased risk of developing CD. On the other hand there is conflicting data on the risk of AE in treated CD patients. Our study aims to assess the incidence of DM1 and TD in treated CD patients and determine if length of gluten exposure affects the subsequent risk of AE in treated CD.Table: Table. Patient characteristicsMethods: Using the Rochester Epidemiology Project, we retrospectively reviewed the Mayo Clinic and Olmsted Medical Center medical records from January 1997 to December 2015. We identified all CD cases of patients aged 80 years or younger at the time of diagnosis. Incidence rates were calculated and patient's demographics were abstracted. This study was approved by the IRB of Mayo Clinic and Olmsted County. Results: We identified a total of 274 (< 18y=117, >18y=157) patients with CD diagnosis. The majority were females 183 (67%) with mean age of 8.8± 4.4y and 44.5± 17 y in children and adults respectively. A total of 48 (17%) subjects had a diagnosis of AE before their CD diagnosis; 20 (42%) with DM1 and 28 (58%) with HT. Among the 226 CD patients without a baseline AE, only 9 (4%) patients were diagnosed with AE after their CD diagnosis with a median follow-up of 6.7y (range: 0-22.5), this led to an AE event rate of 4.87 per 1,000 person-years of follow-up. Of those 9 patients, 7 were children and 2 adults. The mean age at the time of AE diagnosis was 12 ±1.7y and 64.5 ±24.3y in children and adults respectively. The 2(1%) adults patients developed HT, whereas among the 7(3%) children 1 developed DM1 and 6 TD (1 GD and 5 HT). The median duration period between CD diagnosis and the development of AE was shorter in pediatrics than adults [25m (6-65) vs 65m (59-71)]. Conclusion: In both age groups, a 6.7y follow up period led to an AE event rate of 4.87 per 1,000 person-years of follow-up among CD patients. Higher incidence and earlier development of AE in children suggests the need for screening (mainly HT) and close monitoring after CD diagnosis.

Lack of immunogenicity of hydrolysed wheat flour in patients with coeliac disease after a short-term oral challenge.

A gluten-free diet is currently the only reliable therapeutic strategy that is approved for coeliac disease (CD). For many patients, however, compliance remains inadequate. To investigate the immunogenicity of wheat flour that was pre-treated with selected lactobacilli and fungal proteases (hydrolysed wheat gluten) in coeliac patients. The immunogenicity of hydrolysed wheat gluten was evaluated both in vitro in intestinal T cell lines (TCLs) and in vivo in treated CD patients after a short-term gluten challenge. Twenty treated CD patients were enrolled and equally randomised into two groups. The patients ate bread that was prepared with hydrolysed wheat flour or natural wheat flour (10 g of gluten/d for 3 days). The interferon (INF)-γ responses to natural gliadin and a 33-mer peptide were assessed by the enzyme-linked immunospot (ELISPOT) assay on peripheral blood mononuclear cells (PBMCs) both before and 6 days after the start of the challenge. Hydrolysed wheat was not able to activate the TCLs from the coeliac intestinal mucosa. Consistent with the in vitro results, no significant increase in INF-γ secretion was observed in patients who consumed hydrolysed wheat flour. Conversely, the consumption of natural wheat gluten mobilised INF-γ secreting cells in the blood (P<.05). We confirm that fermentation of wheat flour with sourdough lactobacilli and fungal proteases is capable of abolishing the T cell immunogenicity of gluten in coeliac patients. Our data also validate the short-term oral challenge as a useful tool for testing the efficacy of novel therapeutic approaches.

The human digestive tract has proteases capable of gluten hydrolysis

ObjectiveTo identify, purify, and characterize the proteins responsible for glutenase activity in the feces of healthy subjects and patients with celiac disease (CD). MethodsSixteen subjects were included in this study; 8 were healthy with no known food intolerances, and 8 were treated CD patients on a gluten-free diet. Fecal samples were homogenized, and precipitated proteins were purified by chromatography. Glutenase activity was evaluated by bioassays, zymography, and high-performance liquid chromatography with immunogenic 33-mer, 19-mer, and 13-mer gliadin peptides. ResultsThe gastrointestinal elastase 3B (CEL3B), elastase 2A (CEL2A), and carboxypeptidase A1 (CBPA1) enzymes degraded human gluten. These proteins fully hydrolyzed 13-mer and 19-mer gliadin peptides that trigger immune-mediated enteropathy in individuals genetically predisposed to CD and partially digested a 33-mer. Feces from patients with CD showed more glutenase activity than feces from individuals without CD (171–466% higher). Peptidase activity against the gliadin peptides also increased in patients with CD. ConclusionThe digestive tracts of patients with CD and healthy subjects have enzymatic machinery needed for gluten degradation. Patients with CD showed more gluten hydrolysis than did healthy individuals, although, in both cases, a fraction of 33-mer peptide remained intact. Gliadin peptides derived from gastrointestinal digestion, especially the 33-mer, can potentially be used by commensal microbiota from both CD-positive and CD-negative individuals, and differences in bacterial hydrolysis can modify its immunogenic capacity.

Serum Alkylresorcinols as Biomarkers of Dietary Gluten Exposure in Celiac disease

BackgroundTherapy for celiac disease (CD) mainly relies on following a gluten‐free diet (GFD); however, a serum marker for gluten intake has yet to be established.AimsTo evaluate the utility of alkylresorcinol concentrations for detecting gluten intake in studies of human and mouse.MethodsAlkylresorcinol concentrations were compared among treated CD patients (n=34), untreated CD patients (n=36), and controls (n=33). Furthermore, 7 additional CD patients whose serum samples were available at diagnosis and after GFD were evaluated. In mice studies, alkylresorcinol concentrations were compared in the serum of 5 mice fed a regular chow and 10 mice fed lifelong with a gluten‐free chow. In addition, the effect of adding gluten on changes of alkylresorcinol concentrations was also evaluated.ResultsTotal alkylresorcinol concentrations were significantly lower in treated CD patients (median [IQR], 3 (2–8) nmol/L), compared to untreated CD patients (median [IQR], 32 [11–74] nmol/L; P&lt;.0001) or healthy controls (median [IQR], 54 [23–112] nmol/L; P&lt;.0001). Moreover, alkylresorcinol concentrations in CD patients significantly decreased after introduction of a GFD (median, 34 nmol/L at diagnosis vs. 5 nmol.L after GFD, p=0.02). In the mice, median (IQR) total alkylresorcinol concentrations in serum samples of mice fed lifelong with a gluten‐free chow was 1.8 (1.6–2.3) nmol/L, which was further significantly increased to 16 (11–22) nmol/L after 8 days of feeding with the gluten free chow that had gluten added to it. (p=.008).ConclusionSerum alkylresorcinol concentrations could be a useful marker for dietary gluten in CD.Support or Funding InformationNo funding to report.

Intestinal, Systemic, and Oral Gluten-related Alterations in Patients With Nonceliac Gluten Sensitivity.

Nonceliac gluten sensitivity (NCGS) is an emergent condition, the framework of which is yet unclear, whereas the diagnosis is suggested only by gluten-dependent symptoms after excluding wheat allergy and celiac disease (CD). Our goal was to highlight intestinal, systemic, and oral alterations to clarify the NCGS pathogenesis and identify new diagnostic tools. A total of 60 NCGS patients, 20 untreated CD, 20 treated CD, and 20 healthy volunteers were recruited. The differential diagnosis among gluten-related disorders was performed by serological, allergy, and histologic tools. NCGS patients were also subjected to antigliadin antibody (AGA) detection and HLA typing. All participants underwent an oral mucosa patch test for gluten (GOMPT), whereas an oral provocation test (OPT) for gluten was performed in 26 NCGS patients. About 6/60 (10%) NCGS patients showed IgG AGA-positive results, whereas 45/60 (75%) patients carried HLA-DQ2 and/or HLA-DQ8 genes. GOMPT showed positive results in 45/60 (75%) NCGS patients, 3/20 (15%) untreated CD patients, 5/20 (25%) treated CD patients, and in no healthy volunteers. No significant difference was found between the severity of symptoms reported by NCGS patients subjected to OPT with gluten-containing croissants and those who underwent OPT with gluten-free croissants. GOMPT seems to be a specific tool for NCGS diagnosis, although further investigations are needed to overcome limits due to the small population studied and to contextualize GOMPT false-positive results.

Ancestry-based stratified analysis of Immunochip data identifies novel associations with celiac disease.

To identify candidate genes in celiac disease (CD), we reanalyzed the whole Immunochip CD cohort using a different approach that clusters individuals based on immunoancestry prior to disease association analysis, rather than by geographical origin. We detected 636 new associated SNPs (P<7.02 × 10-07) and identified 5 novel genomic regions, extended 8 others previously identified and also detected 18 isolated signals defined by one or very few significant SNPs. To test whether we could identify putative candidate genes, we performed expression analyses of several genes from the top novel region (chr2:134533564-136169524), from a previously identified locus that is now extended, and a gene marked by an isolated SNP, in duodenum biopsies of active and treated CD patients, and non-celiac controls. In the largest novel region, CCNT2 and R3HDM1 were constitutively underexpressed in disease, even after gluten removal. Moreover, several genes within this region were coexpressed in patients, but not in controls. Other novel genes like KIF21B, REL and SORD also showed altered expression in active disease. Apart from the identification of novel CD loci, these results suggest that ancestry-based stratified analysis is an efficient strategy for association studies in complex diseases.

Innate and adaptive immunity in self-reported nonceliac gluten sensitivity versus celiac disease

BackgroundImmune mechanisms have been implicated in nonceliac gluten sensitivity (NCGS), a condition characterized by intestinal and/or extraintestinal symptoms caused by the ingestion of gluten in non-celiac/non-wheat allergic individuals. AimsWe investigated innate and adaptive immunity in self-reported NCGS versus celiac disease (CD). MethodsIn the supernatants of ex vivo-cultured duodenal biopsies from 14 self-reported NCGS patients, 9 untreated and 10 treated CD patients, and 12 controls we detected innate cytokines – interleukin (IL)-15, tumor necrosis factor-α, IL-1β, IL-6, IL-12p70, IL-23, IL-27, IL-32α, thymic stromal lymphopoietin (TSLP), IFN-α-, adaptive cytokines – interferon (IFN)-γ, IL-17A, IL-4, IL-5, IL-10, IL-13-, chemokines – IL-8, CCL1, CCL2, CCL3, CCL4, CCL5, CXCL1, CXCL10-, granulocyte colony stimulating factor (G-CSF) and granulocyte-macrophage colony stimulating factor (GM-CSF). ResultsMucosal innate and adaptive cytokines, chemokines and growth factors did not differ between self-reported NCGS, treated CD and controls. On the contrary, IL-6, IL-15, IL-27, IFN-α, IFN-γ, IL-17A, IL-23, G-CSF, GM-CSF, IL-8, CCL1 and CCL4 were significantly higher in untreated CD than in self-reported NCGS, treated CD and controls, while TSLP was significantly lower in untreated CD than in self-reported NCGS, treated CD and controls. ConclusionIn our hands, patients with self-reported NCGS showed no abnormalities of the mucosal immune response.

Effect of Maize Prolamins on Peripheral Blood Mononuclear Cells from Celiac Disease Patients

Celiac disease (CD) is an enteropathy induced by wheat prolamins (gliadins) and in some rare cases by maize prolamins (zeins) possibly due to a similar immune response. The aim was to study the cellular immune response to zeins in comparison to gliadins of peripheral blood mononuclear cells (PBMC) from CD patients. Isolated PBMC from two treated CD patients and three non-CD controls were challenged in vitro with gliadins or zeins and released gamma-interferon (IFN-γ) in culture medium was measured. PBMC were stimulated with gliadin or zein immunogenic peptides or their digested fractions (3-5 kDa).The gliadin peptide G33-mer induced an expected IFN-γ releasing of PBMC from both patients 1 and 2, with a higher level between days 0 and 6 for patients 1 and no differences para patient 2. The zein peptide Z34-mer induced a higher increase of IFN-γ in both CD patients at 0 day, even higher that it of G33-mer for patient 2, and both of them were highly decreased at day 6. Finally, the zein digested fraction induce an IFN-γ release similar to that of gliadin digested fraction in both cases, although negligible for patient 1 and significant for patient 2. In conclusion, the cellular response to zeins was partially similar to it of gliadins after an in vitro challenge.

Application of Deamidated Gliadin Antibodies in the Follow-Up of Treated Celiac Disease.

IntroductionThe role of serological tests such as IgA anti-transglutaminase autoantibodies has become increasingly important in celiac disease (CD) diagnosis. However, the efficiency of these tests for patient follow-up is controversial. We investigated the correlation of 12 different serological tests, including recent deamidated gliadin and actin IgA tests, with villous atrophy (VA) in a retrospective cohort of treated celiac patients.Materials and MethodsSerum samples were collected from 100 treated CD patients who had intestinal biopsy in the course of their follow-up. Antibodies against transglutaminase, deamidated gliadin peptides, and native gliadin were measured, along with IgA anti-actin. The biopsy slides were all blind-reviewed and scored according to Marsh classification.ResultsFor all deamidated gliadin and transglutaminase tests, we found that a positive result was significantly associated with persistence of intestinal VA, with a diagnostic efficacy up to 80%. Furthermore, antibodies titers directly correlated with the degree of VA, indicating a strong link between disease activity and presence of antibodies in the serum. Interestingly, the tests with the highest association with persistent VA were those for deamidated gliadin IgG. Using a test positivity pattern analysis, we were also able to identify several groups of patients with distinct antibody profiles that showed significant differences in intestinal damage and diet compliance.ConclusionsAltogether, these results show that deamidated gliadin antibodies are strongly correlated with VA and should be considered valuable tools in CD follow-up and that multiplex serologic analysis for treated CD represents a promising tool for personalized patient management.

Differences in gluten metabolism among healthy volunteers, coeliac disease patients and first-degree relatives.

Coeliac disease (CD) is an immune-mediated enteropathy resulting from exposure to gluten in genetically predisposed individuals. Gluten proteins are partially digested by human proteases generating immunogenic peptides that cause inflammation in patients carrying HLA-DQ2 and DQ8 genes. Although intestinal dysbiosis has been associated with patients with CD, bacterial metabolism of gluten has not been studied in depth thus far. The aim of this study was to analyse the metabolic activity of intestinal bacteria associated with gluten intake in healthy individuals, CD patients and first-degree relatives of CD patients. Faecal samples belonging to twenty-two untreated CD patients, twenty treated CD patients, sixteen healthy volunteers on normal diet, eleven healthy volunteers on gluten-free diet (GFD), seventy-one relatives of CD patients on normal diet and sixty-nine relatives on GFD were tested for several proteolytic activities, cultivable bacteria involved in gluten metabolism, SCFA and the amount of gluten in faeces. We detected faecal peptidasic activity against the gluten-derived peptide 33-mer. CD patients showed differences in faecal glutenasic activity (FGA), faecal tryptic activity (FTA), SCFA and faecal gluten content with respect to healthy volunteers. Alterations in specific bacterial groups metabolising gluten such as Clostridium or Lactobacillus were reported in CD patients. Relatives showed similar parameters to CD patients (SCFA) and healthy volunteers (FTA and FGA). Our data support the fact that commensal microbial activity is an important factor in the metabolism of gluten proteins and that this activity is altered in CD patients.

Intestinal Microbiota and Celiac Disease: Cause, Consequence or Co-Evolution?

It is widely recognized that the intestinal microbiota plays a role in the initiation and perpetuation of intestinal inflammation in numerous chronic conditions. Most studies report intestinal dysbiosis in celiac disease (CD) patients, untreated and treated with a gluten-free diet (GFD), compared to healthy controls. CD patients with gastrointestinal symptoms are also known to have a different microbiota compared to patients with dermatitis herpetiformis and controls, suggesting that the microbiota is involved in disease manifestation. Furthermore, a dysbiotic microbiota seems to be associated with persistent gastrointestinal symptoms in treated CD patients, suggesting its pathogenic implication in these particular cases. GFD per se influences gut microbiota composition, and thus constitutes an inevitable confounding factor in studies conducted in CD patients. To improve our understanding of whether intestinal dysbiosis is the cause or consequence of disease, prospective studies in healthy infants at family risk of CD are underway. These studies have revealed that the CD host genotype selects for the early colonizers of the infant’s gut, which together with environmental factors (e.g., breast-feeding, antibiotics, etc.) could influence the development of oral tolerance to gluten. Indeed, some CD genes and/or their altered expression play a role in bacterial colonization and sensing. In turn, intestinal dysbiosis could promote an abnormal response to gluten or other environmental CD-promoting factors (e.g., infections) in predisposed individuals. Here, we review the current knowledge of host-microbe interactions and how host genetics/epigenetics and environmental factors shape gut microbiota and may influence disease risk. We also summarize the current knowledge about the potential mechanisms of action of the intestinal microbiota and specific components that affect CD pathogenesis.

Prevalence of abnormal liver function tests in celiac disease and the effect of a gluten-free diet in the US population.

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

Vaccination and Other Antigen-Specific Immunomodulatory Strategies in Celiac Disease

Background: Celiac disease (CD) results from an alteration in the oral tolerance to dietary gluten. The response to gluten is normally tightly regulated and involves the secretion of TGF-β and IL-10 from different subtypes of regulatory T cells (Tregs). Interestingly, in addition to proinflammatory cytokines, the inflamed CD mucosa also contains high levels of T cell-derived IL-10 compared with treated CD patients or normal donors. Furthermore, most studies describe an increase in the number of Foxp3+ Tregs in the small intestinal mucosa in CD patients compared to controls. This paradoxical condition suggests that regulatory mechanisms might operate to counterbalance the abnormal gliadin-triggered immune activation in untreated mucosa. Indeed, addition of exogenous IL-10 to mucosal cultures from treated CD patients can suppress gliadin-induced T cell activation. Considering the central role of adaptive immunity in CD, the development of strategies to stimulate these mechanisms is a primary goal of efforts to restore gluten tolerance. Key Messages: Different immunomodulatory strategies have been explored. NexVax2, a desensitizing vaccine that uses three dominant gluten peptides administered subcutaneously to induce a tolerogenic response in CD patients, is under development. Alternatively, the potential of substituted, cyclic or dimeric peptide analogues as blockers to prevent HLA from binding to the immunodominant gliadin epitopes has been demonstrated in vitro. In line with these results, we recently found that modified (transamidated) gliadins influenced the immune response in intestinal biopsy samples from CD patients with overt disease by drastically reducing the production of IFN-γ. Notably, in a mouse model, transamidated gliadins reverted the phenotype of the gliadin-inducible immune response from an inflammatory phenotype to an anti-inflammatory phenotype. Conclusions: Various approaches are currently under investigation to recover gluten tolerance based on the use of both modified and native antigen molecules. More specific studies are now required to test the efficacy of such strategies for preventing CD.

Sa1303 The Gluten-Free Diet Produces an Increased Body Mass Index and an Abnormal Waist Circumference Which Is Not Related to the Composition of Food Intake

Background/Aims: A gluten-free diet (GFD) is the only available long-life therapy for celiac disease (CD). Clinical presentation is diverse with few patients with underweight, while the great majority has either normal or overweight. In the general population, overweight causes increase in the waist circumference (WC), an anthropometric measurement associated to the cardiometabolic risk. However, this association has not been established in CD patients. Moreover, there are several evidences that suggest that these anthropometric changes are due to an unbalanced GFD as consequence of an inadequate food selection by patients. Our aims were double: (1) to determine body mass index (BMI) at diagnosis of CD, and (2) to assess changes in the BMI induced by treatment, and the WC and composition the food intake at least two-years after initiation of a GFD. Materials and methods: From July to November 2013, all adult patients with CD attending the ambulatory celiac disease clinic were enrolled in the study if they were on a GFD for at least 2 years. BMI (normal: 18.524.9 kg/m2) and WC (normal: women < 80 cm; men < 94 cm) were determined according to conventional measurements. The composition of food intake was estimated according to conventional formulas based on a food questionnaire by an expert nutritionist. Results: 56 consecutive patients (52 women; median age 48 years [range: 20-65]; time on a GFD: 5 years [range: 2-15]) were enrolled in the study. At diagnosis, 59% of patients had normal BMI, 25% had overweight or were obese, and 16% had underweight. Compared with findings at diagnosis, 93% of GFD treated CD patients had a significantly increased BMI (21 kg/m2 [range: 14-36] vs. 24 kg/m2 [range: 17-37], respectively; p<0.0001). Migration to a higher category was shown in 41% of patients. Twenty four out of 56 patients (43%) showed an abnormally high WC with an highly significant correlation with the final BMI ( r=0.86; 95% CI: 0.77-0.92; p<0.0001). Our dietary analysis has showed that 71% and 86% of patients did not cover the daily recommendation of vegetables and fruits, and that 48% and 71% reported a higher cereal and simple sugar consumption than recommended, respectively. Nevertheless, anthropometric measures did not correlated with the composition of food intake. Conclusions: Our study evidences that a long-term GFD significantly increases BMI generating a migration to higher categories. The WC, an anthropometric surrogate of cardiovascular risk, correlated with overweight and is abnormal in 43% of patients. According to our observations, these anthropometric findings did not seem to be associated to the inadequate composition of food intake.

Sa1304 Bone Mineral Density in Celiac Disease

Background/Aims: A gluten-free diet (GFD) is the only available long-life therapy for celiac disease (CD). Clinical presentation is diverse with few patients with underweight, while the great majority has either normal or overweight. In the general population, overweight causes increase in the waist circumference (WC), an anthropometric measurement associated to the cardiometabolic risk. However, this association has not been established in CD patients. Moreover, there are several evidences that suggest that these anthropometric changes are due to an unbalanced GFD as consequence of an inadequate food selection by patients. Our aims were double: (1) to determine body mass index (BMI) at diagnosis of CD, and (2) to assess changes in the BMI induced by treatment, and the WC and composition the food intake at least two-years after initiation of a GFD. Materials and methods: From July to November 2013, all adult patients with CD attending the ambulatory celiac disease clinic were enrolled in the study if they were on a GFD for at least 2 years. BMI (normal: 18.524.9 kg/m2) and WC (normal: women < 80 cm; men < 94 cm) were determined according to conventional measurements. The composition of food intake was estimated according to conventional formulas based on a food questionnaire by an expert nutritionist. Results: 56 consecutive patients (52 women; median age 48 years [range: 20-65]; time on a GFD: 5 years [range: 2-15]) were enrolled in the study. At diagnosis, 59% of patients had normal BMI, 25% had overweight or were obese, and 16% had underweight. Compared with findings at diagnosis, 93% of GFD treated CD patients had a significantly increased BMI (21 kg/m2 [range: 14-36] vs. 24 kg/m2 [range: 17-37], respectively; p<0.0001). Migration to a higher category was shown in 41% of patients. Twenty four out of 56 patients (43%) showed an abnormally high WC with an highly significant correlation with the final BMI ( r=0.86; 95% CI: 0.77-0.92; p<0.0001). Our dietary analysis has showed that 71% and 86% of patients did not cover the daily recommendation of vegetables and fruits, and that 48% and 71% reported a higher cereal and simple sugar consumption than recommended, respectively. Nevertheless, anthropometric measures did not correlated with the composition of food intake. Conclusions: Our study evidences that a long-term GFD significantly increases BMI generating a migration to higher categories. The WC, an anthropometric surrogate of cardiovascular risk, correlated with overweight and is abnormal in 43% of patients. According to our observations, these anthropometric findings did not seem to be associated to the inadequate composition of food intake.

PTH-117 Thymic Stromal Lymphopoietin Is Primarily Reduced In Refractory Coeliac Disease Duodenal Mucosa

IntroductionThymic stromal lymphopoietin (TSLP), a cytokine released by enterocytes and gut dendritic cells, promotes the development of Foxp3+ regulatory T cells and at the same time inhibits the development of...

Increase in Neuroendocrine Cells in the Duodenal Mucosa of Patients with Refractory Celiac Disease

Several immune-mediated gastrointestinal disorders, including celiac disease (CD), are associated with neuroendocrine cell hyperplasia. However, neuroendocrine cells have never been explored in refractory CD (RCD). Serial duodenal sections from 17 patients with RCD (6 type 1 and 11 type 2), 16 uncomplicated CD patients before and after gluten-free diet, 14 patients with potential CD, 27 patients with non-CD villous atrophy, i.e., common variable immunodeficiency (n=12), Whipple's disease (n=10) and giardiasis (n=5), and 16 healthy subjects were processed for the immunohistochemical detection of chromogranin A (CgA), serotonin, and somatostatin. Mucosal tryptophan hydroxylase (TpH)-1 and serotonin-selective reuptake transporter (SERT) transcripts were measured by quantitative reverse transcription-PCR. Serum CgA and 24-h urine 5-hydroxyindoleacetic acid (5-HIAA) were assessed. Biopsies from treated CD patients were cultured with serotonin or peptic tryptic digest of gliadin (PT-gliadin), and interferon (IFN)-γ was detected by ELISA in culture supernatants. Epithelial cells positive for CgA and serotonin, but not somatostatin, were significantly increased in RCD. Raised mucosal transcripts of TpH-1, but not SERT, were found in RCD. On biopsies from treated CD patients, serotonin upregulated IFN-γ production at levels comparable to those induced by PT-gliadin. Serum CgA, but not urine 5-HIAA, was increased in RCD. No significant difference was found between RCD type 1 and type 2 in terms of neuroendocrine cells, mucosal TpH-1 transcripts, and serum CgA. Serotonin-producing neuroendocrine cells are increased in RCD mucosa. IFN-γ upregulation induced by serotonin suggests that this monoamine may have a role in sustaining the local inflammatory response in CD.

Gene Expression Profile of Peripheral Blood Monocytes: A Step towards the Molecular Diagnosis of Celiac Disease?

AimCeliac disease (CD) is a multifactorial autoimmune disease induced by ingestion of gluten in genetically predisposed individuals. Despite technological progress, the diagnosis of CD is still based on duodenal biopsy as it was 50 years ago. In this study we analysed the expression of CD-associated genes in small bowel biopsies of patients and controls in order to explore the multivariate pathway of the expression profile of CD patients. Then, using multivariant discriminant analysis, we evaluated whether the expression profiles of these genes in peripheral blood monocytes (PBMs) differed between patients and controls.ParticipantsThirty-seven patients with active and 11 with treated CD, 40 healthy controls and 9 disease controls (Crohn’s disease patients) were enrolled.ResultsSeveral genes were differentially expressed in CD patients versus controls, but the analysis of each single gene did not provided a comprehensive picture. A multivariate discriminant analysis showed that the expression of 5 genes in intestinal mucosa accounted for 93% of the difference between CD patients and controls. We then applied the same approach to PBMs, on a training set of 20 samples. The discriminant equation obtained was validated on a testing cohort of 10 additional cases and controls, and we obtained a correct classification of all CD cases and of 91% of the control samples. We applied this equation to treated CD patients and to disease controls and obtained a discrimination of 100%.ConclusionsThe combined expression of 4 genes allows one to discriminate between CD patients and controls, and between CD patients on a gluten-free diet and disease controls. Our results contribute to the understanding of the complex interactions among CD-associated genes, and they may represent a starting point for the development of a molecular diagnosis of celiac disease.

Direct cloning and tetramer staining to measure the frequency of intestinal gluten‐reactive T cells in celiac disease

Knowledge of the frequency of disease-driving CD4⁺ T cells in lesions of chronic human inflammatory diseases is limited. In celiac disease (CD), intestinal gluten-reactive CD4⁺ T cells, which recognize gluten peptides only in the context of the disease-associated HLA-DQ molecules, are key pathogenic players. By cloning CD4⁺ T cells directly from intestinal biopsies of CD patients, we found that 0.5-1.8% of CD4⁺ T cells were gluten reactive. About half of the gluten-reactive T cells were specific for either the immuno-dominant DQ2.5-glia-α1a or DQ2.5-glia-α2 epitopes, suggesting that direct visualization of gluten-specific T cells could be possible. Assessed by flow cytometry, tetramer-positive T cells were present in 10/10 untreated CD patients with a frequency of 0.1-1.2% of CD4⁺ T cells. Gluten-specific T cells were also detectable in most treated CD patients (7/10). Moreover, the frequency of gluten-specific T cells correlated with the degree of histological damage in the gut mucosa as scored by Marsh-grading, and also with serum IgA anti-transglutaminase 2 antibody levels. Tetramer staining of gluten-reactive T cells in biopsy material is a useful tool for future studies of such cells in CD and could also potentially serve as a diagnostic supplement in selected cases.