Urinary NOx:creatinine ratios during gluten challenge in children with celiac disease

OBJECTIVES: Celiac disease is a gluten-induced small bowel enteropathy. Inflammation is known to be associated with enhanced nitric oxide (NO) production. An increase in urinary nitrate and nitrite (NOx) reflects increased NO production. The urinary NOx:creatinine ratio can be used as an indicator of the endogenous NO production. The aim of the study was to determine whether the urinary NOx:creatinine ratio of celiac disease patients increases during gluten challenge. METHODS: The authors studied 20 patients with unconfirmed celiac disease who had been following a gluten-free diet for at least 1 year. These patients underwent an 80-day gluten challenge. Urinary samples were obtained before and 10, 20, 40, and 80 days after starting the gluten challenge. The Griess reagent method was used for measuring urinary NOx. RESULTS: Gluten challenge confirmed the diagnosis of celiac disease in 15 of 20 patients. The NOx:creatinine ratios (mmol:mmol) of the biopsy-confirmed celiac disease patients were significantly higher than those of the unconfirmed celiac disease patients (0.67 vs. 0.17 on day 10; 0.78 vs. 0.15 on day 20; 0.85 vs. 0.25 on day 40; and 0.85 vs. 0.17 on day 80). CONCLUSIONS: Gluten challenge resulted in an increased urinary NOx:creatinine ratio in patients with biopsy-confirmed celiac disease. The NOx:creatinine ratio could be useful for the serial evaluation of disease activity.     

Antiendomysial antibody test reliability in children with frequent diarrhea and malnutrition: is it celiac disease?

BACKGROUND: The aim of this study was to evaluate the specificity of the immunoglobulin A (IgA) antiendomysial antibody test in the diagnosis of celiac disease in a group of malnourished children with acute diarrhea, chronic diarrhea, or parasitosis, because the reliability of this test has been questioned when applied to this specific group of patients. METHODS: Serum IgA level, IgA antiendomysial antibody (EMA) test, and stool examination were performed in 315 children, ranging in age 6 months to 13 years (range, 41 +/- 2.9 months), affected by malnutrition, isolated or in association with diarrhea or parasitosis. Independent of results, 33 children with a strong suspicion of celiac disease, also underwent IgA antitransglutaminase antibody test and jejunal biopsy. RESULTS: The EMA test was negative in 313 children, including the 43 with parasitosis, being positive in two patients in whom biopsy disclosed typical celiac mucosal abnormalities (1:157). The 31 children with negative EMA test who underwent biopsy also showed negative antitransglutaminase antibody results. Their biopsies disclosed normal mucosa in 1 patient, variable degree of jejunal atrophy (grade 1 and 2) in 27 patients, and grade 3 abnormalities in 3 patients. One of these three children, showing severe jejunal atrophy, died. The diagnosis of celiac disease was apparently not confirmed by a protracted gluten challenge in the other two children. CONCLUSIONS: The specificity of the EMA test seems to be high also in children with chronic malnutrition and diarrhea. However, the possibility of false-negative tests among immunologically compromised children cannot be excluded. In doubtful cases, the gluten challenge is required in malnourished children with clinical picture, biopsy finding, and evolution suggestive of celiac disease.     

Early prediction of relapse during gluten challenge in childhood celiac disease

Thirty-seven children, in whom celiac disease had been diagnosed because of flat mucosa on a gluten-containing diet and recovery on a gluten-free diet, were challenged with gluten powder, 10 g/day, in addition to an otherwise gluten-free diet. A small intestinal biopsy was performed before the challenge; clinical symptoms, a 1-h blood xylose test, and gliadin antibody measurement were used to establish the timing of the confirmatory biopsy. All but one case relapsed within 205 days (mode, 60 days). In no case was the relapse clinically evident. Raised levels of gliadin antibodies, a fall in xylose absorption, or both predicted the relapse in 37%, 7%, and 57% of cases, respectively. Evaluated individually, each test gave a considerable rate of false negative results. Discriminant coefficients produced for each test were used to compute a score that allowed the classification of patients into relapse/no relapse categories with a good degree of accuracy. The discriminant score rose sharply after only 15 days of challenge, indicating that it is possible to predict the relapse long before any clinical symptom appears.     

IgA anti-gliadin antibodies in the monitoring of gluten challenge in celiac disease

The aim of this study was to evaluate the reliability of serum IgA anti-gliadin antibodies (IgA-AGA) in the monitoring of gluten challenge and in the prediction of mucosal relapse in children with celiac disease (CD) in order to reduce the challenging procedure to a minimum. Serial evaluations of serum IgA-AGA titers and 1-h blood xylose levels were performed in 17 children with celiac disease during gluten challenge. Jejunal biopsy was generally done after two consecutive measurements of positive IgA-AGA. The morphological appearance of the mucosa and intraepithelial lymphocyte infiltration were also evaluated. A serum positive for IgA-AGA was observed in 16 of 17 patients between the 15th and 35th day of challenge. The challenge was concluded in all children after 20-45 days from the introduction of a gluten-containing diet after histological confirmation of CD. Plasma xylose test was less reliable in this respect. We conclude that IgA-AGA measurement by gluten challenge is likely to simplify and allow earlier diagnostic confirmation of celiac disease in children, without intestinal biopsy.     

R1 reticulin antibodies: markers of celiac disease in children on a normal diet and on gluten challenge

R1 reticulin antibodies were found in sera from patients with childhood celiac disease (CCD). Although the overall sensitivity of R1 in the diagnosis of CCD was relatively low (16/43 = 37%), when only those cases in an active phase of the disease were considered, the sensitivity increased (16/24 = 67%). In spite of its low sensitivity, the R1 assay did show a high degree of specificity, as this antibody was not found in children with post-enteritis syndrome or in healthy controls. R1 antibodies, when found in active CCD, always turned out to be positive when tested on human liver as substrate. While this fact did not enhance the sensitivity of the test, it strengthened its specificity, since R1, found in pathological conditions other than CCD, was nearly always negative on human tissue. Although the R1 reticulin antibody test cannot replace jejunal biopsy in the diagnosis of CCD, its assay, particularly on human liver as substrate, can be considered a useful tool in the screening of celiac patients.     

Serum IgG and IgA anti-gliadin antibodies as markers of mucosal damage in children with suspected celiac disease upon gluten challenge

Serum anti-gliadin antibodies (AGAs) of the IgG and IgA isotypes were determined in 17 children (mean age of 5.6 years) by means of an enzyme-linked immunosorbent assay (ELISA). All children were suspected of celiac disease. They had been on dietary treatment for at least 10 months before they were challenged with gluten. Based on jejunal biopsy findings, 10 of the 17 children had to be considered positive. The sensitivity of the measurement of AGA at 6 weeks after gluten challenge was found to be 90% for IgG, 100% for IgA, and 100% for IgG/IgA combined. The specificity for IgG, IgA, and the IgG/IgA combination was 100, 71, and 100%, respectively. Twelve weeks after gluten challenge, the sensitivity as well as the specificity of AGA determination for IgG, IgA, and IgG/IgA were 100%. It is concluded that testing both IgG AGA and IgA AGA in children suspected of celiac disease is valuable in monitoring the course of the diagnostic provocation protocol and that jejunal biopsies can be abolished. This inexpensive tool can be useful in reducing the number of intestinal biopsies.     

Immunofluorescent antigluten antibody test. Titer and profile of gluten antibodies in celiac disease

Circulating antibodies to gluten fractions have been detected in most patients with celiac disease during gluten ingestion. The various detection techniques, however, are rather complex and inadequate for routine clinical use. Recently, a new indirect immunofluorescent method, named the antigluten antibody (AGA) test, has been developed. To establish the reliability of the test we compared six groups of patients: (1) 15 patients with biopsy-proved celiac disease of whom 13 had positive test results (the other two had already been receiving a gluten-free diet for two to three weeks); (2) 13 malnourished patients without celiac disease who had damaged mucosa, of whom only two had positive test results; (3) 21 patients with celiac symptomatology but normal mucosa, of whom four had positive test results; (4) 42 patients with other intestinal tract diseases, of whom seven had positive test results; (5) 28 patients with extraintestinal diseases, of whom only two had positive test results; and (6) 26 patients with autoimmune diseases, of whom five had positive test results. All patients with celiac disease who had positive test results and who were receiving a gluten-containing diet had titers of 1:40 to 1:80 in the IgG class, while all other patients with positive AGA test results had a low titer of 1:10 to 1:40, in the IgM class mainly. We conclude that the AGA test is an effective screening tool for patients with celiac disease and may serve as a practical index of dietary gluten avoidance.     

Intestinal mast cells and neutrophil chemotactic activity of serum following a single challenge with gluten in celiac children on a gluten-free diet

The number of one subtype of mast cells (formalin fixation, toluidine blue staining), cells of the lamina propria, and intraepithelial lymphocytes were counted in the intestinal biopsy specimens of 14 children with treated celiac disease following a single challenge with gluten. The serum neutrophil chemotactic activity was measured at 0, 1, 3, 5, and 24 h after challenge. There was no significant change in the number of intraepithelial lymphocytes, but the biopsy samples obtained at 5 h showed a marked increase in the inflammatory cells of the lamina propria and a significant decrease in the number of mast cells. A pronounced decrease was present at 3-5 h in the number of eosinophil cells in the blood. The neutrophil chemotactic activity of sera showed a significant increment in 10 of 14 patients. The intestinal permeability of patients became abnormal, as detected by the increased absorption of lactulose. These findings suggest that degranulation of mast cells may be involved in the pathogenesis of the small intestinal mucosal injury in children with celiac disease.     

Even small amounts of gluten cause relapse in children with celiac disease.

BACKGROUND: Previously, a gluten challenge was customary to establish the diagnosis of celiac disease in children. There are no clear recommendations on how to perform this challenge or what markers to rely on for timing the biopsy after the challenge. The authors' aim was to monitor gluten intake, clinical symptoms, and antibody kinetics to evaluate the influence of gluten exposure during the challenge. METHODS: Twenty-five children under investigation for suspected celiac disease were challenged. One child was excluded because blood samples, food records, or biopsy was lacking. Median age at the postchallenge biopsy was 3.8 (2.7-8.8) years. The families kept daily records of the children's gluten intake and of symptoms that occurred. Blood samples were taken monthly for analysis of antigliadin and endomysium antibodies and total immunoglobulin A (IgA). A third biopsy was performed when clinical symptoms suggested a relapse. RESULTS: All 24 children showed deterioration of the mucosa or elevated antibodies during gluten challenge. Median duration of the challenge was 13 (5-51) weeks, and mean gluten intake was 1.7 (0.2-4.3) g/d and 0.1 (0.02-0.26) g/kg daily. CONCLUSIONS: Gluten intake during the challenge varied widely, and the parents were unable to give their children the recommended amount. Despite the small amounts given, all children showed signs of relapse at a clinical, laboratory, or histologic level. Much smaller amounts of gluten than previously suggested seem sufficient to cause relapse during gluten challenge in children.     

Complement studies in children with treated coeliac disease after gluten challenge

Changes of the complement components in the sera of 13 children with treated coeliac disease were studied after gluten challenge. The levels of C 1 and C 3-activator (factor B) were significantly decreased at 4 h after the challenge, as were the levels of total complement (CH 50) and the components C 1, C 4 and C 1-inactivator at 8 h. After 24 h most values returned to normal but there was another significant decrease in serum C 4 after 24 h, and for CH 50, C 1, C 2 and C 4 after 48 h.This study was supported by a grant from the Deutsche Forschungsgemeinschaft (Az. Op 12/11)     

Postpubertal gluten challenge in coeliac disease.

Altogether 38 postpubertal children with coeliac disease were rebiopsied. Mucosal abnormality in nine (24%) of them indicated poor adherence to the diet. Gluten challenge with a diet containing a normal amount of gluten was performed in those 29 patients with a normal mucosa. During challenge, rebiopsy was done when reticulin antibodies turned positive (mean 0.6 years, range 0.2-2.0) or at the end of the two year study. Histologically a clear relapse into coeliac disease was seen in all 23 patients who were positive for reticulin antibodies. At this time gliadin antibodies were positive in all but two. Sixteen (70%) of those who relapsed were completely asymptomatic. Three girls and one boy did not relapse within two years, indicating the possible recovery from coeliac disease to be 11%. All four had undergone gluten challenge earlier in childhood, after initial diagnosis and mucosal recovery, and this had resulted in mucosal relapse. To establish definite postpubertal recovery from coeliac disease in cases with normal mucosa at two years from challenge, further follow up studies of reticulin antibodies and later rebiopsy are needed. The reticulin antibody test seems to be suitable for prediction of mucosal relapse in coeliac disease.     

Short-term gluten challenge in children with coeliac disease does not impair spontaneous growth hormone secretion

BACKGROUND: Growth retardation in children with coeliac disease has been attributed to impaired growth hormone (GH) secretion observed in stimulation tests. OBJECTIVE: This study aimed at investigating the possible change in spontaneous GH secretion during a standardised gluten challenge. PATIENTS: Twelve children with previous enteropathy suggesting coeliac disease and a normal pre-challenge biopsy on a gluten-free diet were included; eight of them completed all parts of the study, including repeated 24-h GH sampling. METHODS: At the start and the end of a 5-6 weeks standardised gluten challenge, blood was drawn at a constant rate for 24 h and collected for GH analysis at 20-min intervals. The graph of plotted GH values was analysed by means of a computer program (PULSAR). RESULTS: No significant changes were seen in the measures of maximum GH peak, baseline GH values, area under the curve over the baseline (AUCb), the number of GH peaks or mean GH concentration. GH secretion rate (GHt) increased slightly. None of the characteristics of the 24-h profile was significantly correlated to the change of IGF-I. CONCLUSION: No impaired GH secretion was found. Thus, we speculate that decreased growth rate in celiac disease may not be primarily caused by changes in GH secretion. Instead it may be caused by changed peripheral sensitivity to GH.     

Two different doses of gluten show a dose-dependent response of enteropathy but not of serological markers during gluten challenge in children with coeliac disease

In order to study dose-dependency in histopathological reactions and in changes of serological markers of mucosal relapse, gluten challenge was performed with two defined amounts of gluten in 54 children with earlier enteropathy. Gluten was provided in the form of powder and the patients were randomly allotted to either 0.2 (group A, n = 27) or 0.5 (group B, n = 27) grams per kg body weight per day. At the start and after 4 wk of challenge a small intestinal biopsy was performed. Biopsy specimens were evaluated, in accordance with defined criteria, graded and summarized in an enteropathy score. Blood was sampled at the start and after 2 and 4 wk of challenge. Serum levels of anti-gliadin antibodies (AGA) and anti-endomysium antibodies (EmA) were measured. Within 4 wk of challenge, 24 out of 27 patients in group A and all patients in group B had relapsed. After increasing the gluten dose to 0.5 g/kg/d during the subsequent 4 wk, the three non-relapsing patients also relapsed. Conclusion: The severity of mucosal inflammation was significantly higher for group B (p = 0.04) indicating a dose-related severity of the enteropathy. No significant difference was found for maximum AGA level, or in the proportion of patients that converted to pathological values for AGA or EmA.     

Screening by anti-endomysium antibody for celiac disease in diabetic children and adolescents in Austria

BACKGROUND: Unrecognized celiac disease (CD) may be found in a substantial proportion of patients with type I diabetes mellitus. METHODS: A cohort of 403 Austrian children and adolescents with type I diabetes mellitus (210 males and 193 females; age range, 1-22 years) was screened for celiac disease using the IgA anti-endomysium antibody test (EMA) and the immunoglobulin (Ig)G anti-gliadin (AGA-IgG) and IgA anti-gliadin (AGA-IgA) antibody test. RESULTS: Twelve patients' sera (2.98%) yielded positive EMA results and two patients' sera (0.49%) with IgA deficiency had high AGA-IgG values. All but one of these patients underwent intestinal biopsy. Six (1.49%) had clear histologic evidence of CD (flat mucosa), whereas three (0.74%) showed minor histologic changes (increase in intraepithelial lymphocytes) and four (0.99%), including the EMA-negative patients with IgA deficiency, had a normal mucosa. When the cases with silent and potential CD were combined, the overall prevalence in the current cohort was 2.98%. There was no difference in the hemoglobin (Hb)A1c level between antibody-positive and -negative patients, and subsequent gluten-free diet did not change this metabolic parameter. CONCLUSION: The prevalence of clinically unrecognized CD, found by EMA screening, is much higher in Austrian children with diabetes than in a comparable population without diabetes. The prevalence of CD in diabetic children in Austria is distinctly lower, however, than in several other countries.