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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.

Adolescent↗

Early biochemical responses of the small intestine of coeliac patients to wheat gluten.

The pathogenesis of coeliac disease has been investigated by studying the response of small intestinal hydrolases in patients with coeliac disease subject to gluten challenge. Small intestinal biopsies taken before and two and a half hours after a gluten challenge in five patients with coeliac disease who had been maintained on a gluten free diet were examined by a combination of electron and light microscopy, organ culture, pulse chase biosynthetic labelling, SDS-PAGE and autoradiography. Before the challenge, the small intestinal biopsies showed nearly normal morphology. Two and a half hours after the challenge there was deterioration in villus architecture, distortion of microvillus structure, disorganisation of the intermicrovillus pit region, an increase in lysosome like bodies in the apical cytoplasm of the luminal enterocytes and pronounced hypertrophy of the rough endoplasmic reticulum of these cells. SDS-PAGE of small intestinal biopsies from four treated coeliac patients before gluten challenge revealed normal microvillus membrane and hydrolase composition. There was a generalised reduction but no specific alteration in the pattern of polypeptide synthesis in the mucosa of the small intestine in these subjects two and a half hours after the gluten challenge. These results suggest that the generalised reduction in small intestinal brush border enzymes in coeliac patients is not the primary pathogenetic mechanism and represents a secondary effect.

Adult↗

Animal model of gluten induced enteropathy in mice.

The aim of our experiments was to produce a local T cell mediated immune response to gliadin in the mouse small intestine as a possible animal model of gluten sensitive enteropathy, coeliac disease. BALB/c and BDF1 mice were immunised systemically with gliadin in complete Freund's adjuvant. The jejunal mucosa was challenged by feeding a gluten containing diet, and villus and crypt lengths, crypt cell production rate, and intraepithelial lymphocyte counts were determined to assess mucosal cell mediated immunity. In some animals permeability and local immunity were modulated by concurrent intestinal anaphylaxis or a graft versus host reaction. There were no changes in the jejunal mucosa of BALB/c mice fed a gluten containing diet after having been parenterally immunized. When, however, mice were parenterally immunised with gliadin, fed a gluten containing diet, rendered hypersensitive to helminth antigen by infection with the nematode parasite Nippostrongylus brasiliensis, and challenged intravenously to produce intestinal anaphylaxis crypt cell production rate was significantly higher than in ovalbumin immunized controls at 12 days after parasite challenge. Finally, graft versus host reaction was induced in BDF1 mice that had been parenterally immunised with gliadin and were on a gluten containing diet. Two weeks later these mice had significantly longer crypts and a higher crypt cell production rate and intraepithelial lymphocyte count than control, unimmunized mice with graft versus host reaction. We conclude that active immunization with gliadin does not in itself produce intestinal cell mediated immunity to gliadin contained in the diet, or enteropathy. Additional factors, such as those occurring during intestinal anaphylaxis (increase intestinal permeability), or during graft versus host reaction (enhanced antigen presentation), seem to be necessary for the full expression of a jejunal mucosal reaction.

Anaphylaxis↗

Gluten specific, HLA-DQ restricted T cells from coeliac mucosa produce cytokines with Th1 or Th0 profile dominated by interferon gamma.

Coeliac disease is precipitated in susceptible subjects by ingestion of wheat gluten or gluten related prolamins from some other cereals. The disease is strongly associated with certain HLA-DQ heterodimers, for example, DQ2 (DQ alpha 1*0501, beta 1*0201) in most patients and apparently DQ8 (DQ alpha 1*0301, beta 1*0302) in a small subset. Gluten specific T cell clones (TCC) from coeliac intestinal lesions were recently established and found to be mainly restricted by HLA-DQ2 or HLA-DQ8. Antigen induced production of cytokines was studied in 15 TCC from three patients, 10 being DQ2 and five DQ8 restricted. Cell culture supernatants were prepared by stimulation with gluten peptides in the presence of DQ2+ or DQ8+ Epstein-Barr virus transformed B cells as antigen presenting cells (APC). Supernatants were analysed for cytokines by bioassays, ELISA, and CELISA. Cellular cytokine mRNA was analysed semi-quantitatively by slot blotting and polymerase chain reaction (PCR). All TCC were found to secrete interferon (IFN) gamma, often at high concentrations (> 2000 U/ml); some secreted in addition interleukin (IL) 4, IL 5, IL 6, IL 10, tumour necrosis factor (TNF), and transforming growth factor (TGF) beta. The last TCC thus displayed a Th0-like cytokine pattern. However, other TCC produced IFN gamma and TNF but no IL 4, or IL 5, compatible with a Th1-like pattern. In conclusion, most DQ8 restricted TCC seemed to fit with a Th0 profile whereas the DQ2 restricted TCC secreted cytokines more compatible with a Th1 pattern. The TCC supernatants induced upregulation of HLA-DR and secretory component (poly-Ig receptor) in the colonic adenocarcinoma cell line HT-29.E10, most probably reflecting mainly the high IFN gamma concentrations. This cytokine, particularly in combination with TNF alpha, might be involved in several pathological features of the coeliac lesion. The characterised cytokine profiles thus support the notion that mucosal T cells activated in situ by gluten in a DQ restricted fashion play a central part in the pathogenesis of coeliac disease.

Base Sequence↗

Cellular infiltrate of the jejunum after re-introduction of dietary gluten in children with treated coeliac disease.

Jejunal lamina propria plasma cells and eosinophils and intraepithelial lymphocytes were raised in coeliac children on gluten-containing diets, but only intraepithelial lymphocytes were increased in patients on gluten-free diets. In contrast, lamina propria lymphocytes were reduced in children with coeliad disease on gluten-containing diets but were normal in paitents on gluten-free diets. In children with coeliac disease who were studied serially, lamina propria plasma cells and eosinophils and intraepithelial lymphocytes increased, and lamina propria lymphocytes decreased, within three months of the reintroduction of gluten to the diet. These observations are essentially similar to those made in the adult form of the disease and suggest that more than one type of immunological reaction is involved in the pathogenesis of the jejunal lesion.

Adolescent↗

Immunological phenomena in the jejunum and serum after reintroduction of dietary gluten in children with treated coeliac disease.

Jejunal mucosal immunoglobulin-containing cells of all three major classes (IgA, IgM, IgG) were increased in coeliac children on gluten-containing diets but only IgM cell numbers were raised in those on gluten-free diets. Patients with subtotal villous atrophy had greater numbers of immunoglobulin-containing cells than patients with normal mucosa. In previously treated patients studied before and after three months on a gluten-containing diet ther was an increase in all three classes of cell, IgM containing cells showing the greatest proportional rise. Basement membrane staining with anti-IgA serum occurred in coeliacs and was most intense in untreated patients. Apart from one patient with very low levels of serum IgA, serum immunoglobulins did not differ from normal. However, after reintroduction of gluten to the diet a significant fall in serum IgM concentrations occurred compared with levels in the same patients while on gluten-free diets. It seems probable that both IgA and IgM systems are important in the immunopathogenesis of the small intestinal lesion of childhood coeliac disease.

Adolescent↗

Immunological assay for the diagnosis of coeliac disease: interaction between purified gluten fractions.

Increased leukocyte migration inhibition factor (LIF) production by coeliac patients' lymphocytes is a reliable immunologic test for gluten-sensitive coeliac disease providing the small peptide subfractions of gluten are used as mitogens. To investigate discrepancies in results obtained in various published studies, subfractions (B2 and B3) of crude gluten peptic tryptic digest [Frazer's fraction III (F3)] were used singly and in combination as mitogens in coeliac patients and compared in various doses with F3. There was significant increased production of LIF with B2 or B3 when tested separately as compared to a mixture of B2 +/- B3 or to F3. LIF production with B2 or B3 was 23.7 +/- 6.9%; with B2 + B3, it was 10.65 +/- 14.7% (t = 4.05; P less than 0.0005); and with F3, it was 9.2 +/- 10.2% (t = 5.8; P less than 0.0005). There was no difference in the response between B2 + B3 and F3 (t = 0.4; P = 0.32). Studies of LIF production following stimulation by various doses and combinations of gluten mitogens in the same patient gave the best results with B2 or B3 in 10 microgram doses, with a tendency to less LIF production with higher dosages or combinations of B2 + B3 or F3, even to abolition of LIF production in some coeliac cases. The LIF assay is a reliable immunologic test for gluten sensitivity, providing the B2 or B3 subfractions are used as mitogens.

Adolescent↗

Effects of dietary gluten on the hepatotoxic action of galactosamine and/or endotoxin in rats.

This study was done to clarify the effects of dietary wheat gluten on the hepatotoxic action of D-galactosamine (GalN) and endotoxin (Etx). Male Wistar rats fed a high casein or high gluten (supplemented with L-Lys and L-Thr) diet were injected with GalN or Etx, and the plasma glutamate oxaloacetate transaminase, glutamate pyruvate transaminase, and lactase dehydrogenase activities were examined 20 h later. In rats fed the high gluten diet, these enzyme activities were lower than in the high casein group after injection of 800 mg/kg of GalN. But such a difference between the casein and gluten groups was not clear when they were treated with 400 mg/kg of GalN nor observed even after injection of Etx or Etx+GalN (400 mg/kg). Similarly these was no difference in the plasma concentrations of Etx, tumor necrosis factor-alpha, or interferon-gamma in the rats receiving an injection of 800 mg/kg of GalN between both dietary groups. These results suggest that dietary gluten affords protection against hepatic injury by a high dose of GalN but not by a low dose of GalN and/or Etx.

Alanine Transaminase↗

Bone mineral density and importance of a gluten-free diet in patients with celiac disease in childhood.

OBJECTIVES: Celiac disease (CD), a common cause of malabsorption, is known to be associated with disorders of the skeleton, but there are conflicting data about the effect of diet on bone metabolism. The aims of this study were to investigate the prevalence of osteopenia; to identify the relationship between bone mineral density (BMD), serum calcium, and parathyroid hormone levels; and to determine the effect of gluten-free diet on BMD in children with celiac disease. DESIGN: The study included 32 patients with CD (group 1) and 82 healthy controls (group 2). The patients with CD were evaluated under 2 subgroups, ie, 16 patients with recent diagnosis (group 1a) and 16 patients who follow their diet strictly (group 1b). BMD values and concentrations of calcium, phosphorus, alkaline phosphatase, and intact parathyroid hormone were determined on entry to the study and at 12 months in celiac patients. These values were compared with those of healthy control participants. RESULTS: BMD and bone mineral content values in patients with recent diagnosis were found to be significantly lower than the control group. The BMD values in patients with recent diagnosis were significantly increased after a gluten-free diet for 1 year. Osteopenia was found more commonly in patients with recent diagnosis than patients in whom a gluten-free diet had been instituted. At 1-year follow-up, osteopenia was not resolved with the gluten-free diet, and this was especially true in patients without gastrointestinal manifestation. In patients with recent diagnosis (group 1a), the mean calcium level was found to be lower than the patients who follow their diet strictly (group 1b). There was a positive correlation between calcium level and BMD and bone mineral content. CONCLUSIONS: BMD is almost invariably low in newly diagnosed celiac patients in childhood. We therefore recommend that BMD should be evaluated in patients with CD. Strict gluten avoidance promoted a significant increase in BMD. However, values still remained markedly low after 1 year of follow-up in some patients. These patients should be followed for longer periods of time with yearly BMD evaluation, as 1 year of diet therapy was found to be insufficient for osteopenia to be resolved.

Adolescent↗

The release of human pancreatic polypeptide, gastrin, gastric inhibitory polypeptide, and somatostatin in celiac disease related to the histological appearance of jejunal mucosa before and 1 year after gluten withdrawal.

Jejunal biopsies and the postprandial response of pancreatic polypeptide (PP), gastrin, gastric inhibitory polypeptide (GIP), and somatostatin have been examined in nine patients with celiac disease before and 1 year after gluten withdrawal. All presented initially with total villous atrophy of the jejunal mucosa. After gluten withdrawal five showed marked mucosal regeneration on light microscopy examination (responders) and four only moderate or no regeneration (nonresponders). Before treatment the celiac patients had enhanced gastrin response and normal PP response compared with normal controls. After gluten withdrawal the integrated gastrin release was reduced to normal in the responders (275 versus 114; p less than 0.05) but remained elevated in the nonresponders (231 versus 204). Postprandial PP release was similar before and after treatment regardless of the degree of mucosal regeneration. In the responders the integrated release of GIP was increased (180 versus 241; p less than 0.05), and the somatostatin release was enhanced (-2.6 versus 8.4; p less than 0.05) after gluten withdrawal. We conclude that the postprandial release of GIP and somatostatin increases and that the release of gastrin decreases when the intestinal mucosa is regenerated in celiacs on a gluten-free diet. The release of PP after food is not influenced by mucosal regeneration.

Adult↗

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.

Biopsy↗

Endomysial antibodies in the diagnosis of celiac disease and the effect of gluten on antibody titers.

Celiac disease, a common chronic gastrointestinal disorder, is gluten induced and is controlled with a gluten-free diet. While the management of CD with a gluten-free diet is quite effective, the diagnosis is rather difficult. The ESPGAN criteria for the diagnosis of CD seems to be tedious and time-consuming. Serological tests for IgA class endomysial antibodies, as detected by indirect immunofluorescence, on human and primate smooth muscles are specific and sensitive markers of celiac disease. Of all the specimens examined, endomysial antibodies were present in patients with gluten-sensitive enteropathy. These antibodies occurred in all active cases of celiac disease, in 90 percent suspected celiac patients where all the ESPGAN criteria has not been fulfilled. This contrasts to the presence of endomysial antibodies in 46 percent of confirmed and 17 percent of suspected celiac patients maintained on a gluten-free diet for various time intervals. Endomysial antibodies also occurred in all cases with chronic diarrhea and gut histology consistent with CD and 8% of asymptomatic family members of CD patients. None of the patients with other gastrointestinal and liver diseases had endomysial antibodies. These studies thus emphasize the specificity and sensitivity of endomysial antibodies for celiac disease.

Animals↗

Nutritional value of wet corn gluten feed for sheep and lactating dairy cows.

Experiments were conducted to determine digestibility of wet corn gluten feed in sheep and effect of wet corn gluten feed on DM intake, milk production, and milk composition by dairy cows. In Trial 1, six wethers were fed wet corn gluten feed at maintenance and ad libitum for determination of nutrient digestibility by the conventional 7-d total collection technique. The sheep ate 1.32 times maintenance at ad libitum intake and no significant differences were found in digestibility due to intake. Digestibilities of DM, organic matter, energy, CP, ether extract, ADF, NDF, and hemicellulose averaged 70.3, 73.0, 73.7, 78.3, 72.8, 51.4, 58.1, and 60.4%, respectively. Mean total digestible nutrients for both treatments was 70.3. In Trial 2, 12 Holstein cows in mid to late lactation were allotted to a 4 x 4 Latin square design. Cows were fed a total mixed ration twice daily. Wet corn gluten feed was fed at 0 (control), 10, 20, and 30% of the total ration DM. There were no significant treatment effects on DM intake, milk yield, or milk composition. Dry matter intake (kg/d), milk production (kg/d), and percent milk fat were 21.4, 22.9, and 3.71, respectively, for control; 21.4, 23.0, and 3.80 for the 10%; 21.0, 23.1, and 3.71 for the 20%; and 21.0, 23.2, and 3.89 for the 30% wet corn gluten feed.

Animal Feed↗

Evaluation of wheat gluten in milk replacers and calf starters.

Two trials were conducted to evaluate wheat gluten as an ingredient in calf feeds. In one trial, Holstein bull calves (n = 120) were assigned for 6 wk to one of five milk replacers, which contained different percentages of CP and different percentages of protein furnished from soluble wheat gluten. Within a given protein percentage, BW gains of calves were not affected by the percentage of protein that was supplied as wheat gluten. Calves fed milk replacer containing 18% CP with 33% wheat gluten gained as much as calves fed replacers containing 20% CP. In another trial, newborn Holstein calves (n = 62) were used. Protein supplements of the calf starters used until 7 wk of age were either soybean meal or spray-dried wheat gluten and soybean meal. No significant differences were observed between the two treatments. Also, no significant carry-over effect occurred when all calves received a common diet from 7 to 10 wk of age.

Animal Feed↗

Effect of supplementation of limiting amino acids on tyrosine toxicity in rats fed wheat gluten diets.

The effect of supplementation of limiting amino acids on rats fed a 10 or 20% wheat gluten diet containing 5% tyrosine has been investigated. Growth-limiting amino acids in a 20% wheat gluten diet were lysine and threonine. When rats were fed a 20% wheat gluten diet containing excess tyrosine, the addition of 1.0% lysine-HCl or 1.0% lysine-HCl and 0.4% threonine completely prevented the development of tyrosine toxicity, and was accompanied by a lowering of free tyrosine concentrations in plasma, liver and muscle. In rats receiving a 10% wheat gluten diet, lysine, threonine and methionine were limiting for growth. The addition of 1.0% lysine-HCl to the 10% wheat gluten plus 5% tyrosine diet failed to alleviate the tyrosine toxicity, but the supplement of 1.0% lysine-HCl and 0.4% threonine could partially alleviate the toxicity. The combined addition of 1.0% lysine-HCl, 0.4% threonine and 0.5% methionine was most effective. The free tyrosine concentrations in plasma, liver and muscle were lowered greatly by the supplementation of three limiting amino acids, but amounts of free tyrosine and phenolic metabolites excreted in the urine did not decrease with addition of these limiting amino acids.

Amino Acids↗

Epidemiology of coeliac disease in children in one Croatian county: possible factors that could affect the incidence of coeliac disease and adherence to a gluten-free diet (Part II).

Coeliac disease is a life-long disorder characterised by small-intestinal mucosal damage caused by gluten. The treatment, gluten-free diet, leads to mucosal restoration and reduces the risk of malignant and non-malignant complications. According to our recent results, cumulative incidence in Croatian children is 1:519 life-births. Coeliac disease presents early, mostly with typical symptoms. We investigated possible influences of breast-feeding duration and time of gluten introduction on such presentation of the disease. Study included coeliacs born in Medimurje between 1985 and 1994 (N = 31). Patients' adherence to treatment was also studied. Although short breast-feeding (mean 2.9 months) and early gluten introduction (mean 4.9 months) were shown, no correlation was found between those factors and time of the disease presentation. Concerning adherence to gluten-free diet, only 50% of patients maintained it strictly, 23% irregularly, while 27% were on normal diet. In order to improve it, regular follow-ups are recommended.

Breast Feeding↗

[Usefulness of selected serum cytokines for monitoring compliance with a gluten-free diet in celiac patients].

UNLABELLED: In celiac disease in response to gluten, the immune reaction develops both in the small intestine and in cells of peripheral blood. The profile of cytokines produced by the cells differs between patients in compliance and not, with regard to gluten-free diet. THE AIM: The aim of the study was to evaluate the usefulness of serum profile determination for chosen cytokines in monitoring of gluten-free diet in celiac patients. MATERIAL AND METHODS: The investigation comprised a group of 110 patients with celiac disease, who were divided into two groups: with active form of celiac disease (EmA(+), n=56) and with a non-active form of the disease (EmA (-), n = 54). The chosen cytokine IL-2, IL-4, IL-6, IL-IO and IFN-y levels were determined in patients and soluble receptor For IL-2 (sIL-2R). For determination of the selected cytokines, brand sets of ELISA microplates (Bender Med. Systems) were used according to recommendations of the producer. The obtained results were analysed with statistical methods. RESULTS: Comparison of mean values of chosen cytokines levels in particular groups showed, that in case of sIL-2R and IL-6, they were significantly statistically higher in the group of patients complying to gluten-free diet. Statistical analysis revealed negative correlation between sIL-2R levels and the length of diet compliance in patients with non-active celiac disease and positive correlation between concentrations of sIL-2R and the titre of endomysial antibodies in the group of patients with active form of the disease. CONCLUSIONS: The determination of sIL-2R levels in serum together with endomysial antibodies assessment can be used as a method for monitoring the compliance to gluten-free diet in patients with celiac disease.

Adolescent↗

Enzyme immunoassay for determination of gluten in foods: collaborative study.

A collaborative study was performed in 15 laboratories to validate a monoclonal antibody-based enzyme immunoassay (EIA) for determination of gluten in foods. The study included 13 samples: maize starch, "gluten-free" baking mixes, wheat flours, cookies, cooked meats, and a soup. Gluten was present in these samples at either zero or 0.02 to 10% by weight, i.e., over almost 3 orders of magnitude. The mean assay values for the foods varied from 88 to 105% of the actual amounts. The assay was quantitative for cereal products and the soup with repeatability (RDS-r, relative standard deviation) and reproducibility (RSD-R) of 16-22% and 24-33%, respectively. The assay was semiquantitative for the processed meat products (RSD-r 14 and 26% and RSD-R 46 and 56%), probably because gluten was unevenly distributed in the small (1 g) samples that were analyzed. The ELISA method produced no false positive results, and false negatives obtained with tannin-containing foods could be avoided by use of a modified sample extractant. None of the collaborators reported problems in following the protocol. The method has been adopted official first action by AOAC for determination of wheat gluten in foods.

Antibodies, Monoclonal↗