A non-toxic analogue of a coeliac-activating gliadin peptide: a basis for immunomodulation?

BACKGROUND: A-gliadin residues 31-49 (peptide A) binds to HLA-DQ2 and is toxic to coeliac small bowel. Analogues of this peptide, which bind to DQ2 molecules but are non-toxic, may be a potential route to inducing tolerance to gliadin in patients with coeliac disease. METHODS: Toxicity was investigated with small bowel organ culture in six patients with untreated coeliac disease, four with treated coeliac disease and six controls. Analogue peptides comprised alanine substituted variants of peptide A at L31 (peptide D), P36 (E), P38 (F), P39 (G) and P42 (H). RESULTS: Peptides D and E were toxic in biopsies from some patients. Peptides F, G and H were not toxic. CONCLUSIONS: Peptide F, which binds to DQ2 more strongly than peptide A, is not toxic in patients with coeliac disease in-vitro; this could be an initial step towards investigation of the induction of tolerance to gliadin in patients affected by coeliac disease.

Gluten-induced nitric oxide and pro-inflammatory cytokine release by cultured coeliac small intestinal biopsies.

OBJECTIVES: To determine whether there was increased nitric oxide (NO) production from coeliac small intestinal biopsies cultured in vitro with gluten and whether the inhibition of NO production could prevent gluten-induced enterotoxicity. The relationship between NO production with the pro-inflammatory cytokines interferon-gamma (IFN-gamma), tumour necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) was evaluated. DESIGN: Small intestinal biopsies from ten patients with treated coeliac disease and six controls were studied. METHODS: Small intestinal biopsies were taken from each patient and set up in culture with Frazer's fraction III (FFIII), a peptic/tryptic digest of gluten, FFIII plus L-NMMA and L-NMMA alone, culture medium alone and ovalbumin which served as a control protein. The biopsies were cultured for 20 h at 37 degrees C. The supernatants were then collected and analysed for nitrite using the Greiss reaction; cytokine levels were determined using ELISA kits. Enterocyte height was determined by microscopy using a calibrated eyepiece graticule and cryostat sections of the cultured biopsies. RESULTS: Coeliac biopsies cultured with FFIII produced significantly greater nitrite compared to culture medium alone (P< 0.05) and this could be blocked with L-NMMA (P< 0.01). A reduction in enterocyte height was seen in coeliac biopsies cultured with FFIII compared to culture medium alone (P < 0.01) and this was ameliorated but not completely blocked when FFIII was cultured with L-NMMA. These changes were not seen in the controls. There was a significant reduction in IL-1beta levels in the supernatant of coeliac biopsies cultured with FFIII compared to culture medium alone (P< 0.05), but when cultured with FFIII and L-NMMA there was a significant increase in IL-1beta levels (P< 0.05). An increase in IFN-gamma levels was also seen when coeliac biopsies were cultured with FFIII and L-NMMA (P< 0.05). This pattern was not seen with TNF-alpha. CONCLUSIONS: Increased levels of NO can be found when coeliac biopsies are cultured with gluten in an in vitro small intestinal culture system, and NO may play a role in the observed enterotoxicity as the inhibition of NO production ameliorates the enterocyte damage. The results suggest that NO is involved in the regulation of pro-inflammatory cytokines, particularly IL-1beta. This is likely to be one of many pathways leading to the observed mucosal pathology in coeliac disease and demonstrates the close interactions between them.

Binding of gluten-derived peptides to the HLA-DQ2 (alpha1*0501, beta1*0201) molecule, assessed in a cellular assay.

The nature of the immunopathogenic relationship underlying the very strong association of coeliac disease (CD) to the HLA-DQ (A1*0501, B1*0201) genotype is not known, but probably relates to binding of gluten-derived epitopes to the HLA-DQ (alpha1*0501, beta1*0201) heterodimer (DQ2). These epitopes have not yet been defined. In this study we have tested the binding of various gluten-derived peptides to DQ2 in a cellular assay using Epstein-Barr virus (EBV)-transformed B lymphocytes and murine fibroblast transfectants. One of these peptides (peptide A), which has previously been shown to exacerbate the CD lesion in vitro and in vivo, was found to bind to DQ2, albeit only moderately, lending further credence to its possible role in the pathogenesis of CD. The nature of peptide A's binding to DQ2 was explored with truncated and conservative point substituted analogues and compared with the published DQ2 binding motif, the results of which explain the observed level of binding.

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.

Oesophageal and gastric carcinoma in the Republic of Yemen.

We conducted a preliminary survey on 3064 patients who underwent upper gastrointestinal endoscopy at the Al-Thawra Hospital in Sana'a, Republic of Yemen, between January and December 1991. The age/sex distribution, demographic features and social habits with respect to cigarette and water-pipe smoking and Qat chewing were compared for patients with oesophageal and gastric cancers (n = 183). A preponderance of women with carcinoma of the mid-oesophageal was noted, previously only recorded in areas of high prevalence. Unlike Western populations, smoking and alcohol consumption were not significant risk factors. A high frequency of Qat chewing and water-pipe smoking was found for both men and women and for a group with tumours of the gastro-oesophageal junction or cardia (chi 2 = 2.646, P > 0.05). Numbers were insufficient to identify independent effects of each factor individually. Dietary habits alone were insufficient to account for the excess of affected females. A case-control study is now underway to investigate further the role of dietary factors, social habits, demographic features and Helicobacter pylori infection on the development of upper gastrointestinal cancer in the Yemen.

Pitfalls in diagnosing coeliac disease.

AIMS: To highlight the pitfalls in the diagnosis of coeliac disease and to make recommendations for its diagnosis and the management of refractory cases with equivocal histology. METHODS: Six patients, referred since 1989 with a diagnosis of coeliac disease based on duodenal biopsy specimens taken at endoscopy, and who failed to respond to a gluten-free diet were studied. All patients were subjected to peroral jejunal biopsy. Morphometric analysis of villus height:crypt depth ratios, surface enterocyte cell heights, and intraepithelial counts was used to aid in the assessment of equivocal histology. RESULTS: Subsequent small intestinal biopsy specimens both taken when the patients were following a gluten-free diet and after gluten challenge were normal in all cases. Morphometric analysis and intraepithelial counts were normal. CONCLUSIONS: Misinterpretation of the original slides was often due to poor sample quality and tangential sectioning. Failure to respond to a gluten-free diet should always raise doubt regarding the initial diagnosis, especially when the findings are normal. For correct diagnosis at least three distal duodenal biopsy specimens should be taken simultaneously, and these should be of an adequate size and correctly orientated. Review by a histopathologist experienced in gastrointestinal diagnosis is essential in difficult cases. Quantitative morphometric analysis is helpful in equivocal cases, and jejunal suction biopsy, following a gluten challenge, may be necessary in patients refractory to treatment.

Hepatitis E--an important cause of imported non-A, non-B hepatitis among migrant workers in Qatar.

In 1985, Glynn et al. [Journal of Medical Virology 17:371-375] reported on epidemic viral hepatitis in Qatar and concluded that 72% (91/126) had acute enterically transmitted non-A, non-B viral hepatitis (ET-NANBH). Most of the patients (98%) presented within 8 weeks of arrival in Qatar and were migrant workers from the Indian subcontinent. The data was reanalysed for evidence of infection with hepatitis E virus (HEV). Seventy-eight of 91 (86%) of stored sera were still suitable for analysis since collection in 1981. A newly described enzyme immunoassay (EIA) for both IgG and IgM anti-HEV was used (Abbott Laboratories, Delkenheim, Germany); 59/78 (76%) were positive for either or both assays. All but two were from the Indian subcontinent. The data suggest that HEV was the major cause of ET-NANBH in Qatar in 1981, particularly among newly arrived migrant workers from the Indian subcontinent.

Sarcoidosis presenting as multiple subcutaneous nodules.

We report a young West Indian man who presented with non-specific constitutional symptoms and widespread subcutaneous nodules which were non-diagnostic on histology. The diagnosis of sarcoidosis was made on the basis of progressive bilateral hilar lymphadenopathy, interstitial pulmonary infiltration, a raised serum angiotensin-converting-enzyme level and a granulomatous hepatitis. All symptoms and signs improved dramatically on corticosteroid therapy.