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Background Little information is available on the effect of a follow-up strategy in celiac disease patients during gluten-free diet. Aims To assess 5 year time course of t-transglutaminase antibodies (t-TG) in celiac disease patients enrolled in a community based follow-up program. Methods Annual t-TG testing and periodical clinic visit in 2245 patients. Results Proportion of patients with negative t-TG progressively increased from 83% to 93% during the 5-year follow-up: poor adherence to gluten-free diet (HR 4.764), long duration of gluten-free diet (HR 0.929) and female gender (HR 1.472) were independently associated with serological outcome. In individual patients, 69% tested t-TG “persistently negative”, 1% “persistently positive” and 30% “intermittently negative or positive”. By applying mathematical modelling to t-TG conversion rates observed in this latter group at beginning and end of the follow-up program, the predicted proportion of t-TG negative population increased from 90% to 95% over 5 years. Conclusions Time-course of t-TG serology in the community fluctuates in 1/3 of celiac disease patients suggesting inconstant adherence to gluten-free diet and need of follow-up strategy. Periodical serological and clinical follow-up is a viable and efficacious strategy to promote adherence to gluten-free diet as inferred from time-course of t-TG serology.

Celiac disease (CD) is an enteropathy characterized by a Th1-type immune response to the dietary gluten. The transcriptional mechanisms or factors that control Th1 cell development in this condition remain to be elucidated. The aim of this study was to analyze in CD the expression of interferon (IFN) regulatory factor (IRF)-1, a transcription factor that regulates the differentiation and function of Th1 cells. Duodenal biopsies were taken from children with untreated CD and control children, and analyzed for IRF-1 by Southern blotting of reverse-transcriptase PCR products and Western blotting. IRF-1 DNA-binding activity was assessed by electrophoretic shift mobility assay. The effect of gliadin stimulation on IRF-1 induction was investigated in an ex vivo organ culture of treated CD biopsies. Enhanced IRF-1 was seen in untreated CD in comparison with controls. This was evident at both the RNA and protein level. Furthermore, untreated CD samples exhibited stronger nuclear accumulation and DNA-binding activity of IRF-1 than controls. In contrast, IRF-2, a transcriptional repressor that binds the same DNA element and competes with IRF-1, was expressed at the same level in nuclear proteins extracted from both untreated CD and control patients. In explant cultures of treated CD biopsies, gliadin enhanced both IRF-1 RNA and protein. This effect was prevented by a neutralizing IFN-gamma antibody. Furthermore, stimulation of normal duodenal biopsies with IFN-gamma enhanced IRF-1. These data indicate that IRF-1 is a hallmark of the gliadin-mediated inflammation in CD and suggest that IFN-gamma/IRF-1 signaling pathway can play a key role in maintaining and expanding the local Th1 inflammatory response in this disease.

OBJECTIVES: The molecular and functional properties of small intestinal tissue transglutaminase are largely unknown despite growing interest because of its role in celiac disease (CD). In this study, we aimed to evaluate tissue transglutaminase expression and enzymatic activity in bioptic fragments obtained from the duodenum of untreated individuals with CD and from control subjects. METHODS: Analysis of tissue transglutaminase mRNA expression was performed by reverse transcription-polymerase chain reaction (RT-PCR). The presence of the enzyme in bioptic fragments as well as in homogenates from CD patients and controls was revealed by immunohistochemistry and Western blot, respectively, using the antitissue transglutaminase CUB 7402 clone. To evaluate in situ transglutaminase activity, sections of bioptic fragments were incubated in the presence of 5 mmol/L CaCl(2) with 5-(biotinamido)pentylamine or, alternatively, with a biotinylated glutamine-containing hexapeptide (TVQQEL) and the biotinylated 31-43 A-gliadin-derived peptide. RESULTS: Tissue transglutaminase mRNA levels were 1.0-fold higher (p < 0.05) in CD patients than in controls. Immunohistochemistry and in situ demonstration of enzymatic activity in celiac mucosa clearly showed an increased expression of active tissue transglutaminase in the extracellular matrix of the subepithelial region and in the enterocytes. Staining of the biotinylated 31-43 A-gliadin peptide in the same area of tissue transglutaminase suggested the presence of lysine-donor substrates in intestinal mucosa. CONCLUSIONS: Tissue transglutaminase is more expressed and active in defined areas of the small intestinal mucosa from patients with CD. The presence in the celiac mucosa of proteins able to act as amine-donor substrates suggests that tissue transglutaminase-mediated post-translational modification of proteins cross-linked with gliadin peptides may represent a pathogenic mechanism of CD.

Undetected celiac disease (CD) can cause serious complications and excessive mortality (1)(2)(3). Noninvasive tests could be useful in physicians’ offices to identify patients for intestinal biopsies (4). Serum IgA anti-endomysium (AEA) and anti-human transglutaminase (anti-htTG) antibody assays are the most widely used laboratory tests for CD (5). Autoantibody determinations have been simplified by the use of rapid testing (6)(7), but when saliva specimens were evaluated for AEA testing, they were found to be not suitable (8). To evaluate the potential utility of saliva as a sample for anti-htTG testing, we compared salivary IgA anti-htTG (measured by ELISA with a dot-blot method and an AEA test) with serum IgA anti-htTG antibodies (measured by ELISA and an AEA test) in patients with CD and in controls. Salivary IgA anti-htTG activity was analyzed for the IgA secretory chain by use of a monoclonal antibody. The recognition patterns of the saliva samples were compared with those for the serum samples and with the pattern for a gut-derived IgA monoclonal antibody to htTG from one CD patient, using two transglutaminase deletion mutants, one recognized and one not by the patients’ sera (9). We studied 47 untreated CD patients (29 females and 18 males; median age, 19 years; range, 2–52 years) diagnosed in 2002–2003 according to ESPGHAN criteria (10) and 47 patients with celiac disease (26 females and 21 males; median age, 19 years; range, 4–50 years) on a gluten-free diet (GFD) for at least 12 months. We recruited 51 healthy controls with no history of gastrointestinal or autoimmune diseases (25 females and 26 males; median age, 14 years; range, 6–50 years) and identified samples from 49 individuals suffering from various gastrointestinal diseases (18 with Crohn disease, 10 with ulcerative colitis, 10 with milk allergy, 6 with acute …

OBJECTIVES: The association between celiac disease and insulin dependent diabetes mellitus (IDDM) is well established. Rectal gluten challenge has been used in patients with celiac disease and in first degree relatives as a tool to assess the mucosal immune response to gluten. The aim of this study was to assess the mucosal immune response to gluten in IDDM children by rectal gluten challenge. METHODS: Rectal biopsy specimens were obtained from 19 children with IDDM before and 6 h after rectal challenge with 2 g of a peptic tryptic digest of gliadin. A total of 16 treated celiac patients and 10 control subjects were also investigated. Epithelium and lamina propria CD3(+) and gamma delta(+) lymphocytes were counted with reference to a standard reference area of muscularis mucosae (10(4) microm(2)). RESULTS: After a local instillation of gliadin, a significant (>mean + 1 SD) percentage increment of lamina propria and epithelium CD3(+) and of lamina propria and epithelium gamma delta(+) lymphocytes was observed in five IDDM children, as compared to 11 and 13 celiac patients and one and two controls, respectively. A discriminant analysis allowed correct classification of 100% of patients with celiac disease and controls. The same analysis classified four of 19 IDDM children in the group of celiac patients. The positivity was associated with normal serology (antigliadin antibody, antiendomysial antibody, and antitissue transglutaminase antibodies) and a morphologically normal jejunal mucosa. All four patients had HLA-DQ alleles associated with celiac disease. CONCLUSIONS: Approximately 20% of IDDM children react to rectal instillation of gliadin. Long term follow-up is necessary to establish whether these subjects are at increased risk for developing celiac disease.

BACKGROUND: Studies on intestinal T cell clones from the mucosa of patients with coeliac disease have led to the identification of immunogenic gliadin epitopes. One is HLA-DQ8 restricted, its recognition by T cells being increased by introduction of negatively charged residues operated by tissue transglutaminase. AIM: To test HLA-DQ8 restricted epitope in both native (QYPSGQGSFQPSQQNPQA) and deamidated (QYPSGEGSFQPSQENPQA) forms in an organ culture system of treated coeliac mucosa from HLA-DQ8 positive and HLA-DQ8 negative patients. PATIENTS AND METHODS: Jejunal biopsies obtained from 10 patients with coeliac disease (six HLA-DQ8 positive and four HLA-DQ8 negative) were cultured in vitro with a peptic-tryptic digest (PT) of gliadin, or with the native (peptide A) or deamidated (peptide B) peptide. Intraepithelial CD3(+) and lamina propria total CD25(+) and CD3(+)CD25(+) cells were counted, lamina propria intercellular adhesion molecule 1 (ICAM-1) expression was evaluated, as well as that of Fas molecules on epithelial cells. RESULTS: In HLA-DQ8 positive, but not in HLA-DQ8 negative, coeliacs the density of intraepithelial CD3(+) cells, lamina propria total CD25(+), and CD3(+)CD25(+) cells, as well as expression of ICAM-1 and Fas molecules were significantly increased in biopsies cultured with PT, peptide A, or peptide B compared with biopsies cultured in medium alone. CONCLUSION: These data show that the DQ8 restricted gliadin peptide is immunogenic only in the intestinal mucosa of HLA-DQ8 positive coeliac patients in both native and deamidated forms.

One of the diagnostic hallmarks of the histological lesions associated with celiac disease is the extensive infiltration of the small intestinal epithelium by CD8+ T cells of unknown Ag specificity. In this study, we report recognition of the gliadin-derived peptide (A-gliadin 123–132) by CD8+ T lymphocytes from celiac patients. A-gliadin 123–132-specific IFN-γ production and cytotoxic activity were detected in PBMCs derived from patients on gluten-free diet, but not from either celiac patients on gluten-containing diet or healthy controls. In contrast, A-gliadin 123–132-specific cells were isolated from small intestine biopsies of patients on either gluten-free or gluten-containing diets. Short-term T cell lines derived from the small intestinal mucosa and specific for the 123–132 epitope recognized human APC pulsed with either whole recombinant α-gliadin or a partial pepsin-trypsin gliadin digest. Finally, we speculate on a possible mechanism leading to processing and presentation of class I-restricted gliadin-derived epitopes in celiac disease patients.

BACKGROUNDCoeliac disease is characterised by increased epithelial renewal associated with a mucosal T cell response to gliadin. Keratinocyte growth factor (KGF) is produced by cytokine activated gut stromal cells and may be a link between mucosal T cell activation in untreated coeliac disease and epithelial hyperplasia.AIMSTo characterise expression of KGF in coeliac disease.METHODSKGF transcripts in coeliac disease were measured by quantitative competitive reverse transcription-polymerase chain reaction (RT-PCR) and localised using in situ hybridisation. KGF production by gluten reactive CD4+ T cell clones was examined. In addition, KGF transcripts were measured following ex vivo challenge of coeliac biopsies with a peptic-tryptic digest of gliadin.RESULTSKGF transcripts were elevated in coeliac biopsies compared with normal controls but were not different from non-coeliac disease controls. By in situ hybridisation, KGF mRNA containing cells were present in the upper half of the lamina propria, most abundantly just under the epithelium. There was no signal from cells within the epithelium. Gluten reactive T cell clones did not make KGF. In vitro challenge of coeliac biopsies generated a strong interferon γ response but a specific KGF response could not be detected because of an extremely high number of KGF transcripts in all cultured biopsies.CONCLUSIONSKGF is overexpressed in coeliac biopsies and in tissues with non-coeliac enteropathy. No evidence was found for KGF production by intraepithelial lymphocytes or lamina propria T cells.

In order to study the role of oxidative stress in celiac disease, protein carbonyl groups, thiobarbituric acid-reactive substance and pentosidine were evaluated in the plasma of nine patients with asymptomatic celiac disease and in a control group (n = 25). Plasma alpha-tocopherol, retinol and lipids were determined in the same samples. The levels of markers of oxidative stress derived from both protein (carbonyl groups) and lipids (thiobarbituric acid-reactive substances) were significantly higher in celiac disease patients, whereas lipoproteins and alpha-tocopherol were significantly lower. These data indicate that in celiac disease, even when asymptomatic, a redox imbalance persists; this is probably caused by an absorption deficiency, even if slight. Dietary supplementation with antioxidant molecules may offer some benefit and deserves further investigation.

The biological effects of interferon (IFN)-gamma rely mainly on the activity of the transcription factor signal transducer and activator of transcription (STAT) 1 and the intracellular levels of suppressor of cytokine signaling (SOCS)-1, a negative regulator that controls the amplitude and duration of STAT-1 activation. IFN-gamma is a key mediator of the immunopathology in celiac disease (CD, gluten-sensitive enteropathy). Thus we have investigated STAT-1 signaling and SOCS-1 expression in this condition. As expected, high local concentrations of IFN-gamma were invariably seen in duodenal biopsies from CD patients in comparison to controls. On the basis of immunohistochemistry, STAT-1 phosphorylation, nuclear localization, and DNA-binding activity, STAT-1 activation was consistently more pronounced in CD compared with controls. Despite samples from CD patients containing abundant SOCS-1 mRNA, SOCS-1 protein was expressed at the same level in CD patients and controls. In explant cultures of CD biopsies, gliadin induced the activation of STAT-1 but not SOCS-1. Furthermore, inhibition of STAT-1 prevented the gliadin-mediated induction of ICAM-1 and B7-2. These data suggest that persistent STAT-1 activation can contribute to maintaining and expanding the local inflammatory response in CD.