In vitro toxicity of gluten peptides in coeliac disease assessed by organ culture.

BACKGROUND: We have used organ culture to investigate the in vitro toxicity of three oligopeptides corresponding to amino acids 31-49 (peptide A), 202-220 (peptide B), and 3-21 (peptide C) of A-gliadin, Frazer's fraction III (FFIII), and ovalbumin. METHODS: Eight to 14 jejunal biopsy specimens were obtained from each of 8 treated and 7 untreated coeliac patients and 5 normal controls and cultured for 18 h in organ culture with test peptide (1 mg/ml) or medium alone. Mean enterocyte cell heights (ECH) were compared with paired values for specimens grown in medium alone. RESULTS: A significant reduction in the mean of the ECH values for each of the patient groups was observed with peptide A and FFIII in both treated (p = 0.01 and 0.02, respectively) and untreated (p = 0.03 and 0.01) coeliac patients when compared with tissue incubated with medium alone. No significant changes in the mean ECH value were noted in any of the patient groups in tissue incubated with peptide B, peptide C, or ovalbumin as compared with those with medium alone. CONCLUSIONS: These results suggest that peptide A is toxic in vitro to the jejunal mucosa of both treated and untreated coeliac patients, correlating with recent findings that this peptide exacerbates coeliac disease in vivo.

Detection of interferon gamma mRNA in the mucosa of patients with coeliac disease by in situ hybridisation.

In situ hybridisation has been used to study interferon gamma (IFN gamma) mRNA expression in the small intestine of patients with coeliac disease. Sections of jejunal biopsies were obtained from five patients with treated and five with untreated coeliac disease and five disease controls. These sections were hybridised with radiolabelled specific DNA oligonucleotide probes. The lamina propria of untreated coeliac disease patients contained a significantly increased number of IFN gamma producing cells compared with controls but there was no significant difference between the coeliac patients treated with a gluten free diet and controls. The results suggest that IFN gamma may play a part in the immunopathogenesis of coeliac disease.

Effects of interferon-gamma and tumour necrosis factor-alpha on epithelial HLA class-II expression on jejunal mucosal biopsy specimens cultured in vitro.

HLA class-II molecules present antigen to the immune system and are expressed by normal villous enterocytes. Increased expression occurs in inflammatory bowel and coeliac disease, and it is suggested that cytokines may mediate such increased expression. The effects of the cytokines IFN-gamma and TNF-alpha on epithelial HLA class-II expressions have been studied in adult human jejunal mucosal biopsy specimens cultured in vitro. Specimens from nine patients with normal jejunal histology were cultured for 24 h with IFN-gamma, TNF-alpha, or both. Six of nine patients showed increased HLA-DR and HLA-DP expression after culture with the cytokines. We have demonstrated that these cytokines induce increased epithelial HLA class-II expression in adult jejunal mucosal specimens cultured in vitro, a model that most closely resembles the in vivo state. This provides further evidence that such increased expression occurs secondarily to the products of immunologic activation.

Timing of infiltration of T lymphocytes induced by gluten into the small intestine in coeliac disease.

Jejunal biopsy was performed on control subjects and patients with treated and untreated coeliac disease. Monoclonal antibodies to T lymphocyte surface markers were used to quantify T cell phenotypic subsets in the jejunal mucosa. The patients with untreated coeliac disease had significantly more of both suppressor/cytotoxic and helper/inducer T cells in the surface epithelium than either the control subjects or the treated patients. Serial jejunal biopsy specimens were taken from five treated coeliac patients for six hours after a gluten challenge. In four of these five infiltration of the surface epithelium by both T cell phenotypes, together with deterioration in the villus architecture, had occurred both within two hours of having started the challenge. This suggests that T lymphocytes may have a role in the pathogenesis of coeliac disease.

The effect of gluten on HLA-DR in the small intestinal epithelium of patients with coeliac disease.

Frozen sections of control and coeliac jejunal mucosa were examined for HLA-DR staining. In general the staining pattern in the controls and treated coeliac patients was a patchy subapical pattern on villous tips. Untreated coeliac patients gave a different pattern with staining extending to the crypt enterocytes. Gluten challenge in three treated coeliac patients induced an altered staining pattern within 2 h.

Evaluation of a gliadin-containing gluten-free product in coeliac patients.

Gluten-free products based on wheat starch contain gliadin which exacerbates coeliac disease. A cross-over study was carried out in which ten treated coeliac patients ingested six slices daily of home-baked gliadin-containing gluten-free bread for 6 weeks. While jejunal biopsy morphometry and 51Cr-EDTA excretion were similar after the test and control periods, four of the subjects reported diarrhoea associated with the gliadin-contaminated gluten-free product. Wheat starch-based gluten-free products can cause persistent symptoms in treated coeliac patients.