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.     

Effect of a gluten free diet on osteopenia in adults with newly diagnosed coeliac disease.

BACKGROUND/AIMS: Calcium and vitamin D malabsorption in coeliac disease predispose to skeletal demineralisation. This study aimed to determine bone mineral density in patients studied in the first year after diagnosis of coeliac disease, and to detect changes in bone mineral density over the subsequent year. METHODS: Lumbar spine and femoral neck bone mineral density was measured in 21 adults with coeliac disease, diagnosed and started on a gluten free diet during the preceding year, with dual energy x ray absorptiometry and repeated after 12 months. RESULTS: Bone mineral density was significantly lower in patients than in paired controls (matched for age and sex), at lumbar spine (0.819 g/cm2 compared with 1.021 g/cm2, p < 0.001 Wilcoxon signed rank test) and femoral neck (0.663 g/cm2 compared with 0.794 g/cm2, p < 0.001). Repeat measurement after 12 months demonstrated that patients had a significant gain in bone mineral density at lumbar spine (16.6%/year), and femoral neck (15.5%/year, p < 0.002, Wilcoxon signed rank test at both sites), whereas no significant change in bone mineral density was detected in controls. CONCLUSIONS: Treatment of coeliac disease with a gluten free diet is associated with a significant increase in bone mineral density, although patients still had lower bone mineral density than controls.     

Exocrine pancreatic function in children with coeliac disease before and after a gluten free diet.

This study was designed to determine the extent of pancreatic insufficiency in untreated coeliac disease and whether pancreatic secretion is impaired after a prolonged gluten free period. Three groups of patients were studied: group A comprised 44 patients, mean (SD) age 4.0 (3.1) years, with coeliac disease and total or subtotal atrophy of the intestinal mucosa; group B comprised 67 patients, mean age 4.4 (3.0) years, with coeliac disease but with normal morphology of the intestinal villi (after 12.9 months of a gluten free diet); group C comprised 49 control subjects, mean age 3.2 (3.0) years, with normal jejunal histology. In all subjects exocrine pancreatic function was determined by the secretin-caerulein test; bicarbonate concentration and lipase, phospholipase, and chymotrypsin activity were measured after an intravenous injection of secretin 1 clinical unit (CU) + caerulein 75 ng/kg body weight. Faecal chymotrypsin concentration was also assayed. No significant difference was found between values of the duodenal output of pancreatic enzymes and bicarbonate obtained in the three groups; however, 10 of 44 untreated coeliac patients showed tryptic or lipolytic activity, or both, below the normal limit for our laboratory. The mean value of the faecal chymotrypsin concentration was significantly lower in untreated than in treated coeliac patients (p less than 0.0001) or in control subjects (p less than 0.0001). It is concluded that untreated coeliac patients may have pancreatic deficiency independent of a decrease in enterohormone release. No primary or secondary pancreatic insufficiency was found in coeliac patients where the intestinal mucosa had returned to normal.     

Acute gluten challenge in treated adult coeliac disease: a morphometric and enzymatic study.

Using a Quinton hydraulic biopsy tube, jejunal biopsies were obtained from 10 patients with adult coeliac disease in remission and four healthy volunteers before and after administration of gluten fraction III into the proximal duodenum. The biopsies taken at hourly intervals for four hours, were analysed for changes in brush border enzymes, light microscopic appearances, and villous and crypt population counts. The results indicate that mucosal damage occurs within three to four hours of gluten administration with significant falls in brush border enzyme concentrations and villous population counts. The absence of any change in control biopsies indicates that gluten sensitivity is specific to the mucosa of patients with coeliac disease, the timing of the changes being consistent with a type III immune response or direct toxicity. Some recovery of the brush border enzymes but not the villous population was evident 24 hours after gluten administration while the crypt population showed evidence of a compensatory hyperplastic reaction.     

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.     

Increase in nitric oxide urinary products during gluten challenge in children with coeliac disease

BACKGROUND: Coeliac disease is a gluten-sensitive enteropathy where pro-inflammatory cytokines and excess nitric oxide (NO) production can contribute to mucosal damage. NO urinary products are elevated in coeliac children on a gluten diet, but it is not known how rapidly this increase develops after gluten exposure. METHODS: Oral gluten challenge was performed in 25 children whose families kept a daily record of gluten intake and symptoms. Blood was analysed monthly for antigliadin (AGA) and endomysium antibodies (EMA). Urine was analysed every second week for NO products, i.e. the sum of nitrite and nitrate was measured with a colorimetric method. We performed a third biopsy when clinical symptoms indicated a relapse. Median age at the post-challenge biopsy was 3.8 (2.7-8.8) years. RESULTS: Signs of morphological or serological relapse were seen in all children. Mean daily gluten intake was 0.10 (range 0.02-0.26) g/kg bodyweight. Median NO level was doubled and significantly higher after 4 weeks of challenge but not after 2 weeks. EMA, but not AGA levels, correlated positively with NO. Intraepithelial lymphocyte count was significantly higher in the post-challenge biopsy, but did not correlate with the NO levels. CONCLUSIONS: NO products in urine increased during gluten challenge. EMA levels reflected severity of mucosal damage, and NO products reflected the inflammatory response, which was doubled after 4 weeks of challenge. The NO analysis is simple and non-traumatic for the child. It can be performed repeatedly during investigation of children with suspected coeliac disease.     

Compliance of adolescents with coeliac disease with a gluten free diet.

A cohort of 123 patients with coeliac disease, diagnosed in the first three years of life and followed up for at least 10 years, was reevaluated during the teenage period in terms of compliance with the diet and clinical state. Mucosal structure and lymphocytes were assessed in small intestinal biopsy specimens obtained from 36 subjects, by computerised image analysis. Of these adolescents with coeliac disease, 65% were adhering to a strict gluten free diet, 11.4% were on a gluten free diet but with occasional gluten intake, and 23.6% were on a gluten containing diet. Clinical symptoms occurred more frequently in patients on a gluten containing diet, but not in patients on a semi-strict diet. Occasional intake of small amounts (0.06-2 g/day) of gluten did not produce increased concentrations of antigliadin antibodies but resulted in an appreciably increased crypt epithelial volume and expanded crypt intraepithelial lymphocyte population.     

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.     

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.     

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.     

Osteoporosis in treated adult coeliac disease.

Forty five women and 10 men with coeliac disease diagnosed in adult life, who were already on a gluten free diet, had serial bone mineral density measurements at the lumbar spine and femoral neck over 12 months. Osteoporosis, defined as a bone mineral density (BMD) < or = 2 SD below the normal peak bone mass was found in 50% of male and 47% of female coeliac patients. Patients with a BMD < or = 2 SD below age and sex matched normal subjects, had a significantly lower body mass index (21.3 kg.m-2 compared with 25.2 kg.m-2, p < 0.02 Wilcoxon rank sum test) and lower average daily calcium intake (860 mg/day compared with 1054 mg/day, p < 0.05 Wilcoxon rank sum test) than patients with normal bone mineral density. In postmenopausal women with coeliac disease there was a strong correlation between the age at menopause and BMD at both the lumbar spine (r = 0.681, p < 0.01, Spearman's rank correlation) and femoral neck (r = 0.632, p < 0.01). No overall loss of bone was shown over the 12 months of follow up, and relative to the reference population there was a significant improvement in BMD at the lumbar spine in women (p < 0.025, paired t test) and at the femoral neck in men (p < 0.05, paired t test). There was a significant negative correlation between the annual percentage change in BMD at the lumbar spine and the duration of gluten free diet (r = -0.429, p<0.01, Spearman's rank correlation), with the largest gain in BMD in patients with most recently diagnosed coeliac disease. Osteoporosis was shown in 47% of patients with treated adult coeliac disease. Recognised risk factors for osteoporosis in the general population including low body mass index, dietary calcium intake, and early menopause are particularly important in coeliac disease. Treatment of coeliac disease with a gluten free diet probably protects against further bone loss, and in the early stages is associated with a gain in bone mineral density.     

Is gluten challenge necessary for the diagnosis of coeliac disease in young children?

Sixty-seven children under 2 years of age presenting with a classic clinical picture of coeliac disease with a severe small-intestinal mucosal lesion were investigated. All improved clinically and histologically on a gluten-free diet. During gluten challenge the mucosal damage recurred in 64 (95.5%) children, thus fulfilling the criteria for coeliac disease formulated by the European Society for Paediatric Gastroenterology and Nutrition. Three (4.5%) children had no mucosal relapse 2 years or more after return to a gluten-containing diet. These children were classified as having transient gluten intolerance. The low frequency of non-relapsers in the present study calls into question the practice of performing gluten challenge.     

Similar prevalence of coeliac disease in children and middle-aged adults in a district of Sweden.

Coeliac disease in children and adults is considered to be a variety of the same disorder. This gains epidemiological support in the present study, which reports on the observed prevalence of coeliac disease in an area of Sweden (population, 140 500). On 1 July 1981, the prevalence rate was found to be 104/100 000 (1:960) among children, and the same figure, 106/00 000 (1:950), was found for coeliac disease unaccompanied by dermatitis herpetiformis in the middle-aged population. The figures were obtained in patients seeking medical are and thus represent minimum rates, and it is likely that the actual prevalence of coeliac disease in Sweden will prove still higher.     

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.     

Increased rectal nitric oxide in coeliac disease after local challenge with gluten

BACKGROUND: Coeliac disease is an inflammatory disorder characterized by reversible atrophy of small intestinal villi following the ingestion of gluten. Earlier studies indicate that the inflammatory response to gluten may occur also very distally in the gastrointestinal tract. The aim of this study was to evaluate whether rectal challenge with gluten would trigger an increased local production of the gas nitric oxide (NO), a novel marker of intestinal inflammation. METHODS: Rectal challenge with partially digested gluten was performed in 20 patients with treated coeliac disease and in 13 healthy controls. Luminal levels of NO were measured in the rectum at 0, 8 and 24 h using a chemiluminescence technique. RESULTS: In patients with coeliac disease mean rectal NO increased from 235+/-90 parts per billion (ppb) at 0 h to 4965+/-1653 ppb at 24 h (P < 0.005). In the control group there was no significant increase. One control subject responded with high NO levels at 24 h and the same individual tested positive for anti-endomysium IgA antibodies. Subsequent duodenal biopsing showed substantial villusatrophy. CONCLUSIONS: Rectal challenge with gluten results in increased luminal levels of NO in a group of patients with treated coeliac disease. Further studies are needed to evaluate the role of NO in coeliac disease and the potential usefulness of rectal NO measurements in aiding diagnosis of this intestinal disorder.     

Intestinal immunoglobulins in children with coeliac disease

The numbers of immunoglobulin-containing cells in jejunal biopsy specimens of 19 children with active coeliac disease aged 0·5 to 16·5 years were studied by direct immunofluorescence. Intestinal juice immunoglobulins were measured in 14 of these patients.

The number of IgA-containing cells was twice and the number of IgM-containing cells 2·5 times that of age-matched controls. There were also more IgG-, IgE-, and IgD-containing cells in the jejunal mucosa of the coeliac patients, but the absolute numbers of these cells were low. The immunoglobulin content of the intestinal juice was not altered in coeliacs.

A follow-up biopsy specimen was available from seven patients kept on a strict gluten-free diet for one to four months. A significant fall in the numbers of immunoglobulin-containing cells was seen, and they did not differ at that time from the controls. Two patients were followed until full normalization of the jejunal structure and they had normal numbers of immunoglobulin-containing cells.

In children with coeliac disease in contrast to adult coeliacs, the study shows that the IgA-producing system is quantitatively stimulated during gluten challenge. The rapid drop in the numbers of immunoglobulin-containing cells after gluten withdrawal suggests that there is no quantitative abnormality in the local immunoglobulin-producing system of the gut in coeliac disease.

     

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.     

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.     

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.