In vivo gluten ingestion in coeliac disease

Studies to determine the nature of the cereal component toxic to patients with coeliac disease have concentrated on wheat due to its nutritional importance. A number of in vitro studies have indicated the presence of one or more coeliac-disease-activating epitopes with the N terminus of the A gliadin molecule. In vivo challenge with three synthetic peptides subsequently indicated the toxicity of a peptide corresponding to amino acid 31-49 of A gliadin. Changes induced by this peptide included a decrease in the ratio of villous height to crypt depth, a decrease in enterocyte surface cell height and an increase in intra-epithelial lymphocyte count. There was evidence of mRNA for the pro-inflammatory cytokincs interferon gamma and interleukin 2 within 2 h of the in vivo challenge. We examined electron-microscopically biopsies from patients with coeliac disease challenged with gluten to determine the subcellular sites where gliadin co-localised with T-cell receptor subunits and HLA antigens. There were differences in co-localisation patterns between treated and untreated coeliac patients. These differences and the different patterns of gliadin staining within enterocytes of coeliac patients and controls, observed by others, suggest that gliadin may be metabolised via a different immunogenic pathway in coeliac disease. This might result in an abnormal presentation to the immune system, triggering a pathogenic, rather than a tolerogenic response.     

Frontal cortical perfusion abnormalities related to gluten intake and associated autoimmune disease in adult coeliac disease: Tc-ECD brain SPECT study

OBJECTIVE: Since brain perfusion abnormalities have been described by single-photon emission computed tomography in some autoimmune diseases, the aim of the present study was to evaluate the incidence of perfusion abnormalities by brain single-photon emission computed tomography in a group of coeliac disease patients, and to investigate whether gluten intake and associated autoimmune diseases may be considered risk factors in causing cerebral impairment. METHODS: Thirty-four adult coeliac patients (16 on a gluten-free diet and 18 on a gluten-containing diet, 18 (53%) with autoimmune diseases) underwent 99mTc-ethyl cysteinate dimer brain single-photon emission computed tomography and qualitative evaluation of brain perfusion was performed together with a semiquantitative estimation using the asymmetry index. Ten subjects on our database, matched for sex, age and ethnic group, who were proved normal by histology ofjejunal mucosa (four males and six females; median age 39 years, range 27-55 years), were included as control group. RESULTS: Twenty-four out of 34 patients (71%) showed brain single-photon emission computed tomography abnormalities confirmed by abnormal regional asymmetry index (>5%; range 5.8-18.5%). Topographic comparison of the brain areas showed that the more significant abnormalities were localised in frontal regions, and were significantly different from controls only in coeliac disease patients on unrestricted diet. The prevalence of single-photon emission computed tomography abnormalities was similar in coeliac disease patients with (74%) and without (69%) associated autoimmune disease. CONCLUSIONS: Abnormalities of brain perfusion seem common in coeliac disease. This phenomenon is similar to that previously described in other autoimmune diseases, but does not appear to be related to associated autoimmunity and, at least in the frontal region, may be improved by a gluten-free diet.     

Clinical response to gluten withdrawal is not an indicator of coeliac disease

Objective. Although the diagnosis of coeliac disease requires specific histological and serological findings, patients considered to be affected by coeliac disease only on the basis of clinical improvement after gluten withdrawal are commonly referred to our outpatient clinic. The objective of this study was to investigate whether the clinical response of gastrointestinal symptoms to gluten withdrawal and subsequent dietary re-introduction could be an indicator of the presence of coeliac disease. Material and methods. From December 1998 to January 2007, 180 patients on a gluten-free diet because of a diagnosis of coeliac disease not based on proper diagnostic criteria came to our out-patient clinic. In 112 of these patients, gluten was re-introduced into their diet. Subsequent duodenal biopsies and endomysial antibodies confirmed the diagnosis of coeliac disease in 51 of them. The relationship between improvement/worsening of symptoms and withdrawal/re-introduction of dietary gluten was analysed. Results. Gastrointestinal symptoms improved in 64.7% of coeliac patients and 75.0% of non-coeliac patients after gluten withdrawal (χ² test, p=NS). Gluten re-introduction was followed by clinical exacerbation in 71.4% of coeliac patients and 54.2% of non-coeliac patients (χ² test, p=NS). The positive predictive value for clinical improvement after gluten withdrawal was 36%; the positive predictive value for clinical exacerbation after gluten re-introduction was 28%. Conclusions. Clinical response to either withdrawal or re-introduction of dietary gluten has no role in the diagnosis of coeliac disease.     

Incidence of small-intestinal mucosal abnormalities and of clinical coeliac disease in the relatives of children with coeliac disease

Evidence is presented of a higher than normal incidence both of clinical coeliac disease and of small-intestinal mucosal abnormalities in relatives of children with coeliac disease. In such relatives the incidence of mucosal abnormality may differ from the incidence of clinical coeliac disease. The data show an absence of any simple Mendelian pattern of inheritance: in place of the hypothesis that inheritance is through a dominant gene of reduced penetrance, it is argued that the pathogenesis of coeliac disease is multifactorial, the genetic basis of susceptibility being polygenic and interacting with environmental factors. On this hypothesis the relative contributions of inheritance and environment to liability to the clinical condition are estimated, the genetic component being 45% +/- 9. Environmental factors appear more important in the development of mucosal abnormality.     

Cellular hypersensitivity to gluten derived peptides in coeliac disease.

Wheat gluten derived antigens have been tested for their ability to inhibit the migration of leucocytes from healthy subjects and patients with coeliac disease. Three preparations of a water soluble fraction (Frazer's fraction III, FIII) of partial peptic tryptic digests of wheat gluten had different effects in a direct (one stage) assay. Subfractions B and B2 caused migration inhibition of leucocytes from patients with treated coeliac disease but not of leucocytes from healthy volunteers or patients with Crohn's disease or ulcerative colitis. This migration inhibition seems to be specific for gluten fractions because maize zein fraction B, beta-lactoglobulin and ovalbumin did not cause it. The sensitivity of coeliac leucocytes to fraction B is not related to factors present in coeliac serum as the migration of leucocytes from healthy individuals preincubated with coeliac sera was not inhibited. Puromycin diminished inhibition by fraction B, which was active at 1.2 micrograms/ml in an indirect (two stage) migration inhibition assay; this is consistent with a process involving elaboration of lymphokine(s). More highly purified fractions of B2, P1-P4 were prepared by reverse phase high performance liquid chromatography (HPLC) and showed differing potency in direct and indirect assays, with P4 being the most active fraction. Inhibition of migration by gluten derived peptides appears to result from the release of lymphokine by leucocytes specifically from coeliac patients.     

Dietary analysis in symptomatic patients with coeliac disease on a gluten-free diet: the role of trace amounts of gluten and non-gluten food intolerances.

BACKGROUND: Whereas many people with coeliac disease (CD) are asymptomatic when consuming a gluten-free diet (GFD), a proportion continues to experience symptoms. The reasons for this are unclear. METHODS: Thirty-nine adult members of The Coeliac Society of New South Wales, all of whom had persistent gastrointestinal symptoms despite adhering to a GFD, were evaluated. Dietary analysis indicated that 22 (56%) were consuming a GFD as defined by the WHO/FAO Codex Alimentarius (Codex-GFD), in which foods containing up to 0.3% of protein from gluten-containing grains can be labelled as 'gluten free'. The remaining 17 were following a no detectable gluten diet (NDG)-GFD, as defined by Food Standards Australia. All subjects were required to follow a NDG-GFD during the study. Those in whom symptoms persisted after changing from a Codex-GFD and those who entered the study already on a NDG-GFD began an elimination diet followed by open and double-blind challenges to identify specific non-gluten food or food chemical intolerances. RESULTS: Of 22 patients who switched to a NDG-GFD symptoms resolved in 5 (23%) and were reduced in 10 others (45%). Thirty-one subjects commenced the elimination diet. Symptomatic improvement was experienced in 24 (77%). Subsequent food or food chemical challenges resulted in a mean of five positive challenges per individual. Diarrhoea was the most commonly provoked symptom, followed by headache, nausea, and flatulence. Symptoms were especially provoked by amine, salicylate and soy. CONCLUSION: The consumption of trace amounts of gluten, traditionally allowed in a Codex-GFD, may be responsible for the continuing symptoms seen in some patients with CD. Further investigation for non-gluten food intolerances should follow if symptoms persist after adherence to a NDG-GFD.     

Intestinal permeability in coeliac disease: the response to gluten withdrawal and single-dose gluten challenge.

Intestinal permeability has been studied in 21 patients with coeliac disease in relapse and after gluten withdrawal using an oral test of intestinal permeability based on the simultaneous oral administration of two probe molecules. The increased absorption of the larger molecule (cellobiose) and the decreased absorption of the smaller (mannitol) found in untreated coeliac disease both returned to normal within five months of starting treatment, the abnormality in cellobiose absorption correcting more rapidly than that of mannitol. After exposure to a single oral dose of gluten, the intestinal permeability of six patients with treated coeliac disease became transiently abnormal with an increased absorption of cellobiose, returning to normal within one week. The possible structural and functional implications of these findings are discussed. The cellobiose/mannitol ratio appears to be of value in assessing the response to gluten withdrawal in coeliac disease, and also in monitoring patients who are already established on a gluten free diet by detecting dietary lapses and 'non-responding coeliac disease'. It may also offer an alternative to jejunal biopsy in patients subjected to gluten challenge.     

Genetics of the immune response to gluten in coeliac disease

Accumulating evidence indicates that coeliac disease (CD) is a multifactorial disorder where several heritable factors in conjunction with environmental factors are involved in the disease development. Gluten proteins are a critical environmental factor as the presence of disease in affected individuals is strictly dependent on dietary gluten exposure. Most likely coeliac patients have genetically shaped immune responses to gluten proteins that cause intestinal pathology. Many of the CD genes thus supposedly encode variants of proteins with immune functions. HLA has been identified as a major genetic factor, but yet no further genes have been identified. There probably exist several predisposing non-HLA genes, but available data indicate that the heritable contribution by each of them can be small. Combined genetic and functional studies will hopefully identify additional predisposing genes in the future.     

Cell-mediated immunity to gluten within the small intestinal mucosa in coeliac disease.

Jejunal biopsies from controls and coeliac patients were maintained in organ culture in the presence of gluten fraction III. The culture media were assayed for evidence of lymphokine activity in a migration inhibition test using normal peripheral blood leucocytes. Significant inhibition of migration was produced by media from untreated coeliac patients compared with controls (P less than 0.005) or treated coeliac patients (P less than 0.001), indicating the production of a leucocyte migration inhibition factor (LIF) by untreated coeliac mucosa in response to gluten fraction III. The degree of inhibition correlated with the preculture interepithelial lymphocyte count in the coeliac biopsies (P less than 0.02). In six coeliac patients studied when on a normal diet and on a gluten-free diet, LIF was produced while on a normal diet, but not while on a gluten-free diet. These results suggest that a local cell-mediated immune reaction to gluten is present in the mucosa of patients with untreated coeliac disease but that this is reversed by treatment with a gluten-free diet.     

Variability of gluten intolerance in treated childhood coeliac disease.

Fifty children consecutively attending a clinic for coeliac disease co-operated in a trial; 10 found to have flat mucosa were excluded. Forty children of mean age 9.8 years, whose duodenal or jejunal mucosa had returned to normal or near normal appearance after a mean of 5.8 years on gluten-free diets, were put back on normal diets. In 37, mucosal occurred in a mean of 16.9 months (four to 74 months). Four of the 37 had serial biopsies, in which mucosal enzymes (particularly lactase) fell and interepithelial lymphocyte counts rose before the mucosal morphology was regarded as definitely 'coeliac'. Three children had normal mucosal appearance after 58 to 73 months on normal diets, one of whom showed temporary mucosal abnormalities, another having occasionally low enzymes, in both suggesting underlying gluten sensitivity. Lactase suppression and raised IEL counts appear to be sensitive indicators of gluten intolerance. In our experience, a diagnosis of coeliac disease based on severe mucosal damage and a satisfactory response to a gluten-free but milk-containing diet implies a very strong likelihood of permanent or prolonged gluten intolerance, but with a striking variability in its expression.     

Analysis and clinical effects of gluten in coeliac disease.

The prolamin working group coordinates research on laboratory gluten analysis in food and on clinical evaluation of patient sensitivity to prolamins. As an observer organization to the Codex Alimentarius Commission, the group summarizes current data on analysis and effects of gluten in coeliac disease. All types of gliadin, the ethanol-soluble fraction of gluten, contain the coeliac-active factor. However, coeliac toxicity and immunogenicity (humoral and cellular) of various prolamins are not identical in coeliac patients. There are no conclusive data on the threshold of gluten sensitivity of coeliac patients. Information as to the long-term risk to coeliac patients exposed to small doses of gliadin is lacking. Therefore, every effort should be made to keep the diet of coeliac patients as gluten-free as possible. The prolamin group is currently evaluating a new enzyme-linked immunosorbent assay (ELISA) protocol for gluten analysis that could serve as a basis for further Codex regulations. The group recommends adherence to a single Codex limit for gluten-free foods. The current limit of 200 ppm gluten is questionable and requires reconsideration based on new information that will be available soon.     

Cell-mediated immunity to gluten fraction III in adult coeliac disease

A migration inhibition test was used to assess sensitisation of blood leucocytes, and thus cell-mediated immunity, to gluten fraction III in controls and patients with coeliac disease. Migration indices were significantly less (indicating sensitisation) in untreated and in treated patients than in controls, and significantly less in treated patients than in untreated patients. At a concentration of 1 mg/ml gluten fraction III, 13% of untreated patients and 54% of treated patients had migration indices in the sensitised range. At 2 mg/ml gluten fraction III, sensitisation was demonstrated in 8% of untreated patients and 48% of treated patients. After starting a gluten free diet, migration indices fell into the sensitised range in all patients followed. After at least nine months on a gluten free diet, migration indices were significantly higher in those patients with a normal interepithelial lymphocyte count than in those patients with a raised interepithelial lymphocyte count. Cell-mediated immunity to gluten fraction III can be detected in the peripheral blood of certain patients with coeliac disease. Detectable sensitivity is related to the time on a gluten free diet, and the interepithelial lymphocyte count.     

Serum antibodies to gliadin in coeliac disease after gluten withdrawal

Serum gliadin antibodies of the IgA and IgG classes were determined by the diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA) in 10 adult patients with villous atrophy of the small-intestinal mucosa. After introduction of a gluten-free diet a gradual decrease in serum gliadin antibody levels occurred, reaching statistical significance at 3 months of treatment for the IgA class (p less than 0.01) and at 6 months for the IgG class (p less than 0.05). A decrease of serum gliadin antibody levels after gluten withdrawal was related to an improvement of the intestinal mucosa and should thus be indicative of whether the patient is following the dietary recommendations. However, determination of gliadin antibody levels cannot replace small-intestinal biopsy, as there are a few patients in whom the antibody levels are not related to the morphology of the gut mucosa.     

Gastric emptying of realistic meals with and without gluten in patients with coeliac disease. Effect of jejunal mucosal recovery

BACKGROUND: Few data are available on disturbed gastric emptying in patients with coeliac disease. The aims of the study were to investigate (a) the presence of delayed gastric emptying: (b) the acute effect on gastric emptying of gliadin; and (c) the effect of jejunal recovery on gastric emptying of meals with or without gluten in such patients. METHODS: We measured gastric emptying of two meals in 16 patients with coeliac disease; one meal contained gliadin. Results were compared with those obtained in 24 controls. In 12 patients, both measurements were repeated after mucosal recovery. Statistical analysis was performed using the analysis of variance for repeated measurements and Student's t test. Mean +/- 1 s(mean) (standard error of the mean) are shown. RESULTS: No difference was found in fasting and in maximal antral sections after the two meals. On entry, gastric emptying was significantly (P < 0.001) delayed compared to controls both after the meal containing gluten (326.9 +/- 12.4 min versus controls 213.5 +/- 11.5) and after the gluten-free meal (315.3 +/- 16.7 min). After jejunal recovery, emptying of the meal containing gluten remained unchanged (337 +/- 18.9 min), whereas emptying of the gluten-free meal was significantly shortened (280.6 +/- 10.5 min; P < 0.001). CONCLUSIONS: In coeliac disease there is an impairment of gastric emptying which is at least partially reversible. This suggests either an immunological disorder or that unabsorbed meal constituents are responsible for an ileal-brake effect.     

Immunohistochemical analysis of mucosal gamma-interferon production in coeliac disease.

The role of gamma-interferon in the pathogenesis of enteropathies with an immunological basis such as coeliac disease, is unclear. Gamma-interferon immunoreactive lymphocytes were quantified in jejunal biopsies from patients with coeliac disease and from normal controls. In coeliac disease, there was an apparent decrease in the percentage of both intraepithelial (3.5% v 13.5%) and lamina propria (10.3% v 47.2%) lymphocytes expressing gamma-interferon compared with controls. In patients successfully treated with a gluten free diet, the percentage of gamma-interferon immunoreactive intra-epithelial lymphocytes was 10.3%. Intraepithelial lymphocytes were immunonegative for class II major histocompatibility complex, while epithelial cells showed increased expression of this product in coeliac disease. The results show that a relatively large proportion of lymphocytes in normal small bowel express gamma-interferon. They also indicate that in coeliac disease the major increase in the numbers of mucosal lymphocytes is the result of infiltration by lymphocytes not expressing gamma-interferon.     

Reintroduction of gluten in adults and children with treated coeliac disease.

Twenty-eight patients, thought to have coeliac disease and on gluten free diets, were put on a normal diet to confirm their diagnoses. Nineteen had been diagnosed in adult life (ACD) and nine in childhood (CCD). Patients were assessed on jejunal, morphological, and symptomatic parameters. Eighteen patients with ACD relapsed within seven weeks. Nine patients with CCD relapsed at variable times but five took longer than seven weeks, the longest period beint 10 months. Seven patients had no symptoms despite morphological deterioration during challenge and one patient, with ACD, did not relapse and was HLA B8 negative. This patient with ACD had subtotal villous atrophy on two jejunal biopsies and later showed morphological improvement on a gluten free diet. There was no correlation between the relapse time and time spent on a gluten free diet.     

Duration of gluten exposure in adult coeliac disease does not correlate with the risk for autoimmune disorders

BACKGROUND AND AIMS: Duration of gluten exposure seems to predispose adolescents with coeliac disease to autoimmune diseases. In a retrospective cohort study, we assessed the relationship between autoimmune disorders and actual gluten exposure in patients in whom coeliac disease was diagnosed in adult life (> or = 16 years). METHODS: We screened for the presence of autoimmunity in 605 controls (16-84 years) and 422 patients (16-84 years), all of whom had been on gluten withdrawal for at least one year (median follow up 9.5 years). A logistic regression analysis, setting the prevalence of autoimmunity as the dependent variable, was employed to control for independent covariates as predictors of the risk of autoimmunity. RESULTS: The prevalence of autoimmunity was threefold higher (p < 0.00001) in patients than in controls. Mean duration of gluten exposure was 31.2 and 32.6 years for patients with or without autoimmunity. Logistic regression showed that increased age at diagnosis of coeliac disease was related to the prevalence of autoimmune disease while "actual gluten exposure" which takes into account diet compliance, follow up, and age at diagnosis of autoimmune disorders were not predictive for the risk of developing autoimmune diseases (odds ratio 0.82 per year). CONCLUSION: The prevalence of autoimmune diseases in patients with a late coeliac disease diagnosis does not correlate with duration of gluten intake. Early exposure to gluten may modify the immunological response. Gluten withdrawal does not protect patients with a late diagnosis from autoimmune diseases.     

Jejunal mucosal gamma glutamyl transferase activity in coeliac disease.

In view of data suggesting that deficiency of gamma glutamyl transferase (GGT) may be the primary abnormality in the small-intestinal mucosa of patients with coeliac disease, this enzyme was measured in mucosal biopsies from coeliac patients at different stages of treatment. Activity was only slightly reduced in only one of nine well-treated patients, suggesting that deficiency of this enzyme has no primary role in the aetiology of coeliac disease.