Up-regulation of small intestinal interleukin-17 immunity in untreated coeliac disease but not in potential coeliac disease or in type 1 diabetes.

Up-regulation of interleukin (IL)-17 in small intestinal mucosa has been reported in coeliac disease (CD) and in peripheral blood in type 1 diabetes (T1D). We explored mucosal IL-17 immunity in different stages of CD, including transglutaminase antibody (TGA)-positive children with potential CD, children with untreated and gluten-free diet-treated CD and in children with T1D. Immunohistochemistry was used for identification of IL-17 and forkhead box protein 3 (FoxP3)-positive cells and quantitative polymerase chain reaction (qPCR) for IL-17, FoxP3, retinoic acid-related orphan receptor (ROR)c and interferon (IFN)-γ transcripts. IL-1ß, IL-6 and IL-17 were studied in supernatants from biopsy cultures. Expression of the apoptotic markers BAX and bcl-2 was evaluated in IL-17-stimulated CaCo-2 cells. The mucosal expression of IL-17 and FoxP3 transcripts were elevated in individuals with untreated CD when compared with the TGA-negative reference children, children with potential CD or gluten-free diet-treated children with CD (P < 0·005 for all IL-17 comparisons and P < 0·01 for all FoxP3 comparisons). The numbers of IL-17-positive cells were higher in lamina propria in children with CD than in children with T1D (P < 0·05). In biopsy specimens from patients with untreated CD, enhanced spontaneous secretion of IL-1ß, IL-6 and IL-17 was seen. Activation of anti-apoptotic bcl-2 in IL-17-treated CaCo-2 epithelial cells suggests that IL-17 might be involved in mucosal protection. Up-regulation of IL-17 could, however, serve as a biomarker for the development of villous atrophy and active CD.

Children with screening-detected coeliac disease show increased levels of nitric oxide products in urine.

AIM: Increased concentration of nitric oxide (NO) metabolites, nitrite and nitrate, in the urine is a strong indication of ongoing small intestinal inflammation, which is a hallmark of the enteropathy of coeliac disease (CD). It has previously been shown that children with symptomatic, untreated CD have increased levels of NO oxidation products in their urine. The aim of this study was to investigate whether screening-detected, asymptomatic coeliac children display the same urinary nitrite/nitrate pattern. METHODS: In a multicenter screening study, serum samples were collected from 7208 12-year-old children without previously diagnosed CD. Sera were analysed for anti-human tissue transglutaminase (tTG) of isotype IgA. Small bowel biopsy was performed in antibody-positive children, yielding 153 new cases of CD. In the screening-detected individuals, the sum of nitrite and nitrate concentrations in the urine was analysed and used as an indicator of NO production. For comparison, 73 children with untreated, symptomatic CD were studied. RESULTS: The nitrite/nitrate levels in children with screening-detected CD and those with untreated symptomatic CD did not differ significantly. Both groups had significantly increased urinary nitrite/nitrate concentrations compared to the children with normal small bowel biopsy (p < 0.001). CONCLUSION: Children with screening-detected CD have increased production of NO just as children with untreated symptomatic CD. High NO metabolite levels in the urine may indicate a pathogenetic feature of CD and be a marker of major clinical importance.

Are children from Crete abandoning a Mediterranean diet?

INTRODUCTION: Mediterranean countries such as Greece have experienced rapid social change in the last decade. These community changes affect nutritional habits and there is a tendency for the traditional healthy Mediterranean diet to be abandoned. METHODS: The parents of children from one rural Greek village on Crete (Neapolis), and one rural village in Sweden (Kisa) were invited to their primary health care centers for an interview and to fill in a validated nutrition questionnaire, KidMed. RESULTS: There were no differences (p = 0.48) in total KidMed score between the Cretan and Swedish children, adjusted for gender and age. However, there were some significant differences in scores on certain KidMed questions. Parents of the Cretan children reported significantly higher daily use of olive oil at home and more regular nut consumption, but also more commercially baked goods or pastries for breakfast. The parents of Swedish children reported significantly higher use of cereals, grains or bread for breakfast. The mean BMIs were similar for the Cretan (Neapolis mean 16.8, 95% CI 13.5-23.0) and for the Swedish children (Kisa mean 17.4, 95% CI 13.7-25.5) CONCLUSION: The results suggest the possibility of changing nutritional habits, measurable among young children in rural areas. The study raises the question of whether Cretan children may have abandoned some aspects of the traditional Mediterranean diet. It may also be that Swedish children have changed their diet in favor of a more Mediterranean food choice. The major limitation of the study is the small sample size, and further, larger studies are warranted.

Radiation risk and cost-benefit analysis of a paediatric radiology procedure: results from a national study.

A national study was performed to investigate radiation doses and associated risks to patients during X-ray fluoroscopy-guided small intestinal biopsies in the investigation of coeliac disease. Thermoluminescent dosemeters (TLD) and questionnaires were sent to 42 of the 43 paediatric departments in Sweden performing these biopsies. During the study period (2 x 3 weeks) 257 biopsies were recorded, representing about 10% of annually performed paediatric investigations. The results show that the absorbed dose during biopsy ranged from 0.04 mGy to 23.8 mGy (mean 1.87 mGy). The fluoroscopy time ranged from 2 s to 663 s (mean 60 s). The collective dose from the procedure amounts to 4.7 manSv year(-1). Thus, the annual excess cancer mortality, including severe hereditary effects, can be estimated at 0.6-0.7 cases per year. However, significant dose saving can be obtained by proper choice of sedation and biopsy equipment.

The effects of oats on the function of gut microflora in children with coeliac disease.

BACKGROUND: Faecal short chain fatty acids (SCFAs) are produced by the gut microflora. We have previously reported high faecal SCFA levels in children with coeliac disease (CD), indicating alteration in gut microfloral metabolism. Data accumulated over recent decades by us and others suggest that wheat-free oats can safely be included in a gluten-free diet (GFD). However, concerns have been raised with respect to the safety of oats in a subset of coeliacs. AIM: To describe faecal SCFA patterns in children with newly diagnosed CD treated for 1 year with a GFD with or without oats. METHODS: This report is part of a randomised, double-blind study on the effect of a GFD containing oats (GFD-oats) vs. a standard GFD (GFD-std). Faecal samples were received from 34 children in the GFD-oats group and 37 in the GFD-std group at initial diagnosis and/or after 1 year on a GFD. Faecal SCFAs were analysed. RESULTS: The GFD-std group had a significantly lower total faecal SCFA concentration at 12 months compared with 0 months (P < 0.05). In contrast, total SCFA in the GFD-oats group remained high after 1 year on the GFD. The children in the GFD-oats group had significantly higher acetic acid (P < 0.05), n-butyric acid (P < 0.05) and total SCFA concentration (P < 0.01) after 1-year diet treatment compared to the GFD-std group. CONCLUSIONS: Our results indicate that oats do affect the gut microflora function, and that some coeliac children receiving oats may develop gut mucosal inflammation, that may present a risk for future complications.

IgA endomysium antibodies--an early predictor for celiac disease in children without villous atrophy.

AIM: To evaluate possible differences between children with anti-endomysium antibodies (EMA) positivity and normal small bowel mucosa and children with positive EMA and an enteropathy diagnosed as celiac disease (CD). METHODS: Children with suspected CD and positive EMA (>or=1/10) undergoing small bowel biopsy during 1996 to 2002, were investigated (n=133). Data registered were: year and month of birth, timing of the first biopsy, sex, heredity for CD, dermatitis herpetiformis and diabetes mellitus and outcome of the anti-gliadin antibody test (AGA). The case group, with EMA positivity and normal histology (n=39; 59% female, mean age at the first biopsy 7.3 years, range 1.4-16), was compared with the disease control group, with positive EMA and a biopsy suggestive and further on diagnosed as CD (n=94; 56% female; mean age 7.6 years at the first biopsy, range 0.70-17). RESULTS: AGA positivity and heredity for CD were found to predict the outcome of a pathological jejunal mucosa. Nineteen of the 39 children in the case group were rebiopsied of whom 11 had developed an enteropathy during a follow-up period of 2-7 years (median 4.5 years). CONCLUSIONS: EMA positivity in the absence of small bowel enteropathy could be a very early predictor for later overt CD, and necessitates further follow-up, especially if the child is AGA positive and there is a family history of CD.

Combinations of common chronic paediatric diseases deviate the immune response in diverging directions.

The cytokine pattern of T lymphocytes has not been characterized in children with combinations of paediatric immunological disorders. We describe cytokine secretion in children with type 1 diabetes, coeliac disease and allergy and combinations of two of these diseases after stimulation with 'disease-specific' antigens. Peripheral blood mononuclear cells (PBMC) were collected from 68 children with type 1 diabetes, allergy or coeliac disease, two of these diseases in combination or none of these diseases. Using the enzyme-linked immunospot (ELISPOT) technique, interferon (IFN)-gamma and interleukin (IL)-4 were analysed from fresh PBMC spontaneously and after in vitro stimulation with antigens associated with one or more of these diseases (insulin, gluten, birch and cat extract, beta-lactoglobulin, ovalbumin and phytohaemagglutinin) in order to divide T helper (Th)1- from Th2-like lymphocytes. Stimulation with birch and cat extract caused increased IL-4 secretion in allergic children. A low IFN-gamma response to insulin was found in type 1 diabetic children, whereas allergic children responded to insulin by increased IL-4 secretion. Children suffering from both type 1 diabetes (Th1-prone) and allergy (Th2-prone) reacted distinctly to general mitogen stimulation. Children suffering from two Th1-dominated diseases (type 1 diabetes and coeliac disease) showed hardly any response to either food or inhalation allergens. Our results indicate an important interplay between common immunological diseases in children. The combination of two Th1-deviated diseases is associated with a suppressed immune response, whereas a combination of Th1- and Th2-dominated diseases appears to increase the general immune response.

Gut microflora associated characteristics in children with celiac disease.

OBJECTIVES: The aim of the study was to investigate the metabolic function of intestinal microflora in children with celiac disease (CD) in order to find out if there is a deviant gut flora in CD patients compared to healthy controls. METHODS: The study group comprised children with CD, consecutively diagnosed according to current criteria given by the European Society for Paediatric Gastroenterology, Hepatology, and Nutrition. Thirty-six children were studied at presentation, i.e., on a normal gluten-containing diet, with clinical symptoms and signs indicative of CD, positive celiac serology markers, and a small bowel biopsy showing severe enteropathy. Forty-seven patients were studied when they had been on a gluten-free diet (GFD) for at least 3 months. For comparison, a group of 42 healthy controls (HC) were studied. The functional status of the intestinal microflora was evaluated by gas-liquid chromatography of short chain fatty acids (SCFAs) in fecal samples. RESULTS: There was a significant difference between untreated CD children and HC as well as between treated CD children and HC regarding acetic, i-butyric, i-valeric acid, and total SCFAs. The propionic and n-valeric acids differed significantly between CD children on GFD and HC. Moreover, there was a strong correlation between i-butyric and i-valeric acids in all study groups. CONCLUSIONS: This is the first study of the SCFA pattern in fecal samples from children with CD. The results indicate that there is a difference in the metabolic activity of intestinal microbial flora in children with CD compared to that in HC. The finding of a different pattern of some SCFAs in celiacs both at presentation and during treatment with GFD indicates that it is a genuine phenomenon of CD not affected by either the diet, the inflammation, or the autoimmune status of the patient.

Breast milk antibodies to foods in relation to maternal diet, maternal atopy and the development of atopic disease in the baby.

Breast milk samples were collected from 152 women during the first week after delivery. The levels of IgG and IgA antibodies to beta-lactoglobulin, ovalbumin and gliadin were assessed with an enzyme-linked immunosorbent assay (ELISA). The breast milk antibody levels did not differ significantly between mothers on a strictly cow's milk and egg-free diet, and mothers taking these foods. Moreover, the colostral food antibody levels did not differ significantly between atopic and non-atopic mothers. Neither was there any correlation between the colostral antibody levels and the development of atopic disease in the baby. I conclude that maternal antigen avoidance during late pregnancy does not affect the food antibody levels in colostrum. High levels of food antibodies in a colostrum sample seem not to offer protection against food allergy in the child.

Skin prick tests may give generalized allergic reactions in infants.

BACKGROUND: Skin prick testing, a widely used method of studying sensitization, is usually considered quick, pedagogic, and relatively inexpensive. Previous studies have shown very few negative reactions and no fatalities. In contrast, both anaphylaxis and death have been reported as a result of intracutaneous tests. OBJECTIVE: To examine detailed case studies of generalized allergic reactions in connection with skin prick testing in order to identify possible risk factors and thereby increase the safety of the test procedure. METHOD: A retrospective study of medical records of six cases with generalized allergic reaction occurring during the study period 1996-1998 at the Pediatric Clinic, University Hospital of Linköping, Sweden. Data about the total number of children tested during the period were collected from the clinic's database. RESULTS: All six cases with generalized reactions were infants <6 months who showed positive skin prick tests to fresh food specimen. Other common features were active eczema and a family history of allergic disease. All infants received prompt treatment and recovered well. The overall rate of generalized reactions was 521 per 100,000 tested children. In the age group <6 months, the corresponding figure was 6,522 per 100,000. CONCLUSION: The risk of generalized reactions after skin prick test with fresh food specimens in young children ought to be acknowledged and should lead to increased precautions when performing the test.

Elevated levels of serum antibodies to the lectin wheat germ agglutinin in celiac children lend support to the gluten-lectin theory of celiac disease.

Lectins recognize carbohydrate moities of glycoproteins and glycolipids, and can elicit several biological effects, including cell agglutination, cell activation and mitogenesis. According to the gluten-lectin theory, celiac lesions represent a response to a toxic lectin, putatively wheat germ agglutinin (WGA). In this study we compared the serum antibody levels IgA, IgG and IgM to WGA and to gliadin in children under investigation for celiac disease (CD), as compared to reference children. We found that the levels of IgA and IgG to WGA as well as gliadin were significantly higher in celiac children on a gluten-containing diet, compared to children on gluten-free diet and reference children. These findings lend support to the concept that WGA is a biologically significant component of gluten. Since WGA can mimic the effects of epidermal growth factor (EGF) at the cellular level, we hypothesize that the crypt hyperplasia seen in celiac children could be due to a mitogenic response induced by WGA.

Allergy prevention by maternal elimination diet during late pregnancy--a 5-year follow-up of a randomized study.

The 209 mothers to be, enrolled in a randomized, prospective, allergy-prevention study from allergy-prone families, totally abstained from cow's milk and egg from gestational week 28 to delivery. This article presents the development of allergic disease at 5 years of age in their children, compared with the development of allergic disease in the children of the control mothers who took normal food throughout pregnancy. The prevalence of allergic disease could be evaluated in 198 children (95%). Allergic disease was monitored with questionnaires, skin prick testing, serum-IgE determinations, and physical examination. Eczema, allergic rhinoconjunctivitis, and asthma was equally common in the groups. Persistent food intolerance to egg was significantly more common in children of the mothers receiving the diet. This long-term follow-up confirms our previous findings that maternal elimination diet during late pregnancy does not prevent the development of allergic disease in the genetically predisposed child.

Development of atopic disease in babies whose mothers were receiving exclusion diet during pregnancy--a randomized study.

In a prospective, randomized study, we have monitored the effect of maternal abstention from cow's milk and egg on the development of atopy in babies. Two hundred twelve women were followed from midpregnancy. We report the occurrences of allergies in their babies up to 18 months of age, as assessed by skin prick testing, determination of serum IgE, questionnaires, and blinded physical examination by a pediatric allergist. Whatever the method that was used, there was no statistically significant difference between babies whose mothers received the "diet" or the "nondiet." Other factors known to influence the risk of atopy like heredity, sex, month of birth, breast-feeding, and exposure to tobacco smoke, animal dandruff, and solid food did not differ between the groups. The mothers receiving the exclusion diet, by their own choice, had diminished their intake of milk and egg during lactation also, and therefore, their babies were significantly less exposed to cow's milk before 6 months. Still, atopy was equally abundant among their children. Thus, maternal elimination diet during late pregnancy did not protect the baby against allergy.

Skin prick test in duplicate: is it necessary?

BACKGROUND: Duplicate skin prick testing has previously been recommended because of reports that accidental negative tests are common. However, duplicate tests also mean an extra allergen load, which may increase the risk of inducing a generalized reaction at the test situation, at least in the youngest infants. OBJECTIVE: To investigate whether the occurrence of both a positive and negative test result is a common feature when performing duplicate skin prick tests and can therefore justify the duplicate method. METHODS: A retrospective analysis of all skin prick tests performed in duplicate at the pediatric clinic at University Hospital in Linköping, Sweden, in 1997. RESULTS: Of 1,087 skin prick tests, 14 resulted in one positive and one negative test, or 1.3%. The corresponding figure in the youngest age group, (ie, <2 years of age) was 3 of 340 (0.9%). CONCLUSIONS: Considering the risk of inducing a summation of the reactions, and thereby a generalized allergic reaction, when applying an extra allergen load on the limited surface of the small arm, we conclude that the results of this study justify using single prick test, at least in the youngest age group and probably when testing children of all ages.

Maternal abstention from cow milk and egg in allergy risk pregnancies. Effect on antibody production in the mother and the newborn.

To study the possibility of intrauterine sensitization, 212 women were enrolled on a voluntary basis into a prospective, randomized study, comparing the effects of an elimination diet and normal food during late pregnancy. The diet group took no cow milk and no egg from week 28 to delivery, and extra calcium and casein hydrolysate (Nutramigen) supplement was given to fill the nutritional needs of mother and child. The control group took normal food, including approximately 1/2 liter of milk/day and 3-5 eggs/week. All families had a history of allergy in mother, father, or sibling. Maternal weight gain during pregnancy was significantly lower in the diet group. Birth weights showed no significant difference between the two main groups, but smokers in the elimination diet group had significantly smaller babies. IgE antibodies to cow milk and egg were significantly higher in atopic than in non-atopic women before the trial. The diet caused a significant fall in IgG-antibodies to cow milk and egg in both atopic and non-atopic subjects. Cord blood IgE determination revealed no significant difference between the groups. No IgE antibodies to cow milk or egg were detected in any of the cord sera. The participating babies are being followed up until 18 months of age.

Antibodies IgG, IgA, and IgM to food antigens during the first 18 months of life in relation to feeding and development of atopic disease.

Serum levels of IgG, IgA, and IgM antibodies against ovalbumin, beta-lactoglobulin, and gliadin were measured in a series of 210 "allergy-risk" infants and their mothers. The antibody levels were determined with ELISA in sera obtained from mothers at delivery, and from their babies at birth, 6 weeks, 6 months, and 18 months. High levels of maternal IgG, IgA, or IgM antibodies to food at delivery did not appear to protect the baby against development of atopic disease. Maternal avoidance of cow's milk and egg during pregnancy had no significant influence on the level of food antibodies in cord blood, but the mother's intake of these foods during lactation affected the immunologic response of the baby, not only to these antigens but also to gliadin as well. Babies with minimal cow's milk exposure before 6 months had significantly lower levels of IgG to beta-lactoglobulin than babies regularly exposed to cow's milk. We conclude that maternal elimination diet during lactation influences the immunologic response of the baby, but if this prevents the development of atopic disease or just delays the immunologic maturation remains to be evaluated.

Allergy screening including a comparison of prick test results with allergen-coated lancets (Phazet) and liquid extracts.

During an allergy screening of families with a history of allergy in one or more subjects, skin prick tests (SPT) were performed in duplicate in 314 adults. The tests were performed with a new type of lancet with a 0.9 mm long point, loaded either with allergen (Phazet) or used together with standardized extracts (10,000 and 100,000 BU/ml). Wheals obtained with both methods were compared, and related to total IgE concentrations and history. A wheal area of 3 and 7 mm2 was used as cut-off limit. Results with Phazet were close to those with Pharmalgen 100,000 BU/ml. With both methods precision was good for allergens, but less for Phazet histamine than for the corresponding wet test. SPT results obtained with Phazet and 100,000 BU/ml correlated better with history than the results with 10,000 BU/ml. Using a wheal size of 7 mm2 as cut-off limit the efficiency for five tested allergens was 91%. "False positive" tests, possibly indicating an unobserved or latent type of allergy, were found more often with higher serum IgE concentrations. The safety of SPT was confirmed and Phazet was well accepted by nurses and tested subjects.

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.