The only effective treatment for coeliac disease is a lifelong gluten-free diet that excludes all food products containing wheat, rye, and barley. However, the necessity of avoiding oats remains controversial (1). Early small-scale reports on coeliac disease patients suggested intestinal malabsorption and exacerbating abdominal symptoms after ingestion of oats (2,3), but more recent in vitro and in vivo studies have questioned its toxicity (4–11). There is now a large body of evidence to support the safe consumption of oats in the vast majority of both children and adults having coeliac disease and dermatitis herpetiformis (4–11). Nonetheless, some concerns persist regarding recommending oats to all coeliac patients. The purity of oat products cannot always be guaranteed and contamination of oats with other gluten-containing cereals during harvesting and milling is known to occur (12,13). Furthermore, there would appear to be a small subset of coeliac patients who experience more abdominal symptoms while consuming an oat-containing diet as against the conventional gluten-free diet (14,15). There are studies showing that the symptoms induced by the consumption of oats are not associated with small-bowel mucosal damage (15,16). It was recently demonstrated that 3 of 9 oat-intolerant patients with coeliac disease had avenin-reactive T cells in the small-bowel mucosa (17). Oats have sequence similarities to wheat at the amino acid level (18), and in in vitro studies oat avenin has been shown to stimulate gliadin-reactive T cell lines (17,19). The clinical relevance of these somewhat discrepant findings remains obscure, and more investigations on the toxicity and immunogenicity of oats in coeliac disease are thus called for.
In addition to gluten-induced small-bowel mucosal inflammatory and morphological changes, humoral response to transglutaminase-2 (TG2) is highly pathognomic for active coeliac disease. These circulating coeliac disease-specific autoantibodies disappear during a gluten-free diet but reappear if gluten is reintroduced (20). Recent evidence shows that anti-TG2 autoantibodies are produced locally in the small-bowel mucosa (21,22), where they can be found deposited extracellularly already early in the disease process before manifest mucosal lesion with villus atrophy has set in and before autoantibodies are detectable in the circulation (23–25). Interestingly, in an earlier study by Shiner et al (26) it was suggested that in treated coeliac disease patients small-bowel mucosal subepithelial IgA deposits appear rapidly after gluten challenge—even hours before marked mucosal lymphocytosis is detectable. This prompted us to hypothesize that detection of TG2-targeted IgA-class autoantibody deposits in the small-bowel mucosa may offer a useful tool for revealing early gluten-induced minor mucosal changes in coeliac disease. We used this method to investigate the toxicity of oats in children with coeliac disease during a 2-year follow-up.
METHODS
Patients and Study Design
This study is part of a randomized controlled trial, whose details have been presented elsewhere (27). In brief, 23 consecutive children aged 7 years or older with previously diagnosed coeliac disease (median age 13 years, range 7–18 years, 7 female) were enrolled. At the time of diagnosis all evinced positive serum IgA-class endomysial antibodies and villous atrophy with crypt hyperplasia in the small-bowel mucosa. After maintaining a conventional gluten-free diet avoiding wheat, rye, barley, and oats for at least 2 years all had experienced a good clinical, serological, and histological response. At the baseline of the study, 13 children (median age 11 years, range 9–18 years, 6 female) in remission were randomized to undergo open oats challenge and 10 (median age 13 years, range 7–8 years, 1 female) a gluten challenge allowing the consumption of wheat, rye, and barley in addition to oats. When clear small-bowel mucosal relapse was verified during the gluten challenge, patients reverted to a gluten-free diet, avoiding wheat, rye, and barley but continuing oat consumption. Small-bowel mucosal biopsies and serum samples were taken at baseline and after 6 and 24 months from the patients ingesting only oats during the entire study period. In patients undergoing a gluten challenge, the first follow-up examination was carried out when clinical symptoms suggested a relapse or coeliac antibodies seroconverted positive. After the relapse and commencement of an oat-containing gluten-free diet, follow-up examinations were carried out in the same way at 6 and 24 months as in the oats challenge group. During the 2-year trial, oats had no detrimental effect on intestinal mucosal villous morphology, densities of CD3+, αβ+ and γδ+ intraepithelial lymphocytes (IEL) or HLA DR expression. In contrast, the gluten challenge group relapsed after 3 to 12 months, but complete recovery from the disease was accomplished in all on an oat-containing gluten-free diet during the 2-year follow-up (27).
Oat Product and Dietary Assessment
At baseline, children and their parents were given instructions by a trained dietician on the oat-containing gluten-free diet and gluten challenge (27). A detailed dietary analysis was assessed repeatedly by means of interview and 4-day record of food intake. The rolled, uncontaminated oats (1 single batch; Melia Ltd, Raisio, Finland; details of the cultivar were not provided by the manufacturer) were given to the patients free of charge. During the gluten challenge, patients bought their wheat-, rye-, barley-, and oats-containing products freely from grocery stores, and thus no specific cultivar was used. During a 2-year trial, the median daily consumption of oats was 45 g/day (range 13–81 g/day) in the oats challenge group. In the gluten challenge group, the median daily intake of gluten in the form of wheat-, rye-, barley-, and oat-containing normal bread was 14 g (range 7–19 g/day), and after adopting an oat-containing gluten-free diet the median intake of oats was 41 g/day, range 24 to 59 g/day. One patient in the oats challenge group had transient dietary lapses at the 6-month time point; all of the rest adhered to a strict gluten-free diet throughout the study (27).
Small-bowel Mucosal Morphology and TG2-specific IgA Deposits
All small-bowel intestinal biopsies were obtained by an adult Watson capsule in X-ray control next to the ligamentum of Treitz in jejunum. One part of the biopsy specimen was paraffin embedded and stained with hematoxylin and eosin to study small-bowel mucosal morphology and to determinate the villous height–crypt depth ratios (Vh/CrD) (28). From well-oriented biopsy sections Vh/CrD < 2 was considered characteristic for active coeliac disease.
The other part of the biopsy was snap-frozen and embedded in optimal cutting temperature compound (OCT, Tissue-Tec, Miles Inc, Elkhart, IN). In each case, altogether 6 unfixed, 5-μm-thick sections from the frozen small-bowel specimens were processed, 3 for investigation of IgA deposits and 3 for double-colour labelling for both IgA and TG2 by direct immunofluorescence. IgA was detected using fluorescein isothiocyanate-labelled rabbit antibody against human IgA (Dako AS, Glostrup, Denmark) at a dilution of 1:40 in phosphate-buffered saline (PBS), pH 7.4. For the double labelling, sections were stained for human IgA (as above, green) and for TG2 (red) using monoclonal mouse antibodies against TG2 (CUB7402, NeoMarkers, Fremont, CA), followed by rhodamine-conjugated anti-mouse immunoglobulin antibodies (Dako), both diluted 1:200 in PBS. In normal small-intestinal mucosal samples IgA is detected only inside the plasma and epithelial cells. In contrast, it has been shown that in active coeliac disease on a gluten-containing diet a clear TG2-targeted subepithelial IgA deposition can be found below the basement membrane along the villous and crypt epithelium and around mucosal vessels. In earlier studies it has been shown that such small-intestinal mucosal IgA deposition targets against TG2 (23,29). In short, when this IgA was eluted from the tissues, it targeted purified TG2 both in enzyme-linked immunosorbent assay (ELISA) and Western blot (23). In addition, when TG2 binding to fibronectin was disrupted by chloroacetic acid, disappearance of extracellular IgA deposits was demonstrated in coeliac small-bowel samples. Furthermore, we have shown that IgA deposits in the small bowel of active coeliac disease patients have the ability to bind external TG2 added to the tissue (29). The method used here was based on our previous experiments to detect TG2-specific antibodies in situ in tissue sections by their colocalization with TG2 when double-labelled by immunofluorescence. In this study the IgA deposits were graded semiquantitatively according to their intensity along the basement membrane in the villous-crypt area as follows: negative (−), weak positive (+), moderate positive (++), and strong positive (+++). In cases in which the intensity of the staining was patchy, the intensity of IgA deposits was graded from different areas and a mean value of the staining was given. All evaluations were carried out blindly without knowledge of disease history or laboratory findings. In our laboratory inter- and intraobserver variation have both been 98% in the detection of IgA deposits (29).
Serology
Serum IgA-class antibodies against TG2 were detected by ELISA using human recombinant TG2 as antigen, with a cutoff line of 5.0 U/mL (Celikey, Phadia, GmbH, Freiburg, Germany).
Statistics
Quantitative data were expressed as medians and ranges, and qualitative data as percentage of abnormal values. Statistical differences were evaluated using the Wilcoxon test, as appropriate. Correlations were tested by Spearman correlation test. All testing was 2-sided, and P values <0.05 were considered statistically significant. All calculations were performed with the Statistical Package for Social Sciences version 14.0 (SPSS, Inc, Chicago, IL).
Ethical Considerations
The study protocol was approved by the Ethics Committee of Tampere University Hospital. All children and their parents gave their written informed consent.
RESULTS
At baseline, all treated children with coeliac disease were in clinical remission and had normal villous architecture at histology (27). Small-bowel mucosal median Vh/CrD was 4.1 (range 3.0–5.1) in the oat challenge group and 4.4 (range 3.2–5.9) in the gluten challenge group. In addition, serum IgA-class anti-TG2 antibodies were negative in all. When autoantibodies were sought at the small-bowel mucosal level, weak-to-moderate TG2-specific IgA deposits were present in 4 of 13 (31%) in the oat challenge group and in 3 of 10 (30%) in the gluten challenge group (Fig. 1A and B).
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FIG. 1: Intensity of small-bowel mucosal transglutaminase 2-specific IgA deposits in patients randomized to oats (A) or gluten challenge (B) groups. Two patients in the oats challenge group (A) who prematurely withdrew from the study were rebiopsied immediately after symptoms occurred (within 1 month after oat challenge was started), and these cases are indicated with open circles.
In the oat challenge group, there was no significant change in the intensity of mucosal IgA deposits during the 2-year trial (Fig. 1A). In contrast, in the gluten challenge group the intensity of IgA deposits increased parallel with the development of small-bowel mucosal villous atrophy with crypt hyperplasia, and at the time of relapse all had moderate-to-severe IgA depositions present in the mucosa (Figs. 1B and 2). During the gluten challenge, 2 patients were also biopsied before they developed small-bowel mucosal villous atrophy, and interestingly, both already evinced clear TG2-specific IgA deposits even if the villous morphology was still intact (Fig. 2B). When an oat-containing gluten-free diet was adopted after relapse of the disease, the intensity of small-bowel mucosal IgA deposits again decreased significantly within 6 months, and after 2 years with oats only 1 of 7 (14%) had minor depositions left in the mucosa (Figs. 1B and 2). At the end of the 2-year trial with oats all patients again had negative serum anti-TG2 antibodies and Vh/CrD was normal in all (median Vh/CrD 4.2, range 2.9–5.2).
FIG. 2: At baseline, this 13-year old boy had been on a conventional gluten-free diet avoiding wheat, rye, barley, and oats for 2 years and evinced normal small-bowel mucosa villous morphology and negative mucosal IgA deposits (A). After 6 months' gluten (wheat, rye, barley, and oats) challenge he still had normal villi, but clear IgA deposits were already present (B). The small-bowel mucosa relapsed after 12 months with gluten, and strong IgA deposits can be seen (C). After adopting an oat-containing gluten-free diet the mucosal IgA deposits slowly disappeared following villous recovery (D), after 6 months with oats; (E), after 2 years with oats. IgA is stained in green and transglutaminase 2 (TG2) in red; yellow (arrow) in composite figures indicates colocalization of coeliac-type IgA deposits and TG2.
When data from both study groups and all time points were collated, it was noted that the intensity of small-bowel mucosal IgA deposits correlated well with serum TG2-antibody levels (Spearman test r = 0.694, P < 0.001) (Fig. 3A), and the severity of small-bowel mucosal villous damage (r = −0.550, P < 0.001) (Fig. 3B).
FIG. 3: Correlation between the intensity of small-bowel mucosal transglutaminase 2-specific IgA deposits and serum IgA-class TG2 antibodies (A), (Spearman correlation coefficient was r = 0.694, P < 0.001), and between small-bowel mucosal IgA deposits and villous height-crypt depth ratios (B), (Spearman correlation coefficient was r = −0.550, P < 0.001). Dotted line indicates the cutoff value for positivity in TG2 antibody measurements from serum. Cases having standard gluten-free diet omitting wheat, rye, barley, and oats (n = 23) are shown in black circles, cases having gluten challenge (n = 10) in black squares, cases with oat containing diet (n = 39) in open circles, and 2 patients in oat group with premature withdrawals in open squares.
One patient in the oat challenge group admitted passing dietary transgressions at 6 months. At that time, his serum anti-TG2 antibody titre increased from 0.4 to 3.1 U/mL, still, however, remaining below the cutoff level, and the intensity of small-bowel mucosal IgA deposits was slightly increased. After continuing with a strict oat-containing gluten-free diet both serum and mucosal autoantibodies again decreased (Fig. 1A).
Two children with coeliac disease experienced abdominal pain and vomiting immediately after intake of oats, and within 1 month both withdrew prematurely from the study. The follow-up biopsies were taken upon withdrawal immediately after symptoms occurred, and they showed that the small-bowel mucosa villous morphology was normal and the densities of IELs were even lower than at the beginning of the study (27). Furthermore, at follow-up serum anti-TG2 antibodies and small-bowel mucosal autoantibody deposits remained negative in both patients (Fig. 4A and B).
FIG. 4: A 16-year-old boy developed abdominal symptoms immediately after ingestion of oats; small-bowel mucosal villous morphology remained normal and densities of CD3+ intraepithelial lymphocytes even decreased (from 86 cells/mm to 51 cells/mm). Mucosal IgA deposits were negative both before (A) and immediately after (B) oats challenge. IgA is labelled green, transglutaminase 2 in red; IgA deposition or co-localization of IgA and TG2 (yellow) was not detected.
DISCUSSION
We demonstrated by measuring the intensities of small-bowel mucosal TG2-specific autoantibody deposits that the ingestion of oats during a gluten-free diet for 2 years does not result in humoral immune activation in children with coeliac disease. In contrast, when wheat, rye, and barley are consumed in addition to oats, a marked small-bowel mucosal antibody response occurs parallel with small-bowel mucosal damage within 3 to 12 months. If oats were disease inducing per se, then the small-bowel mucosa would not have recovered after the relapse on a diet excluding wheat, rye, and barley but containing oats. The present findings are in accord with those in earlier studies in children (Table 1) and adults with coeliac disease or dermatitis herpetiformis, showing that oats have neither toxic nor immunogenic effects on the small-bowel mucosa and are tolerated by the majority of patients with coeliac disease (4–8,16).
TABLE 1: Previous oat trials in children with coeliac disease
Although several studies have suggested that oats can be safely added to the diet of patients with coeliac disease, reports that some develop more abdominal symptoms or may even have avenin-reactive T cells in the small-bowel mucosa while consuming oats raise some concern (15,17). Furthermore, some studies have reported somewhat higher withdrawal frequencies from oats challenge groups when compared with standard gluten-free diet groups also omitting oats (11,15). Most patients who dropped out of these studies were not properly followed up to study whether oat intolerance was due to relapse of the disease or whether it was related to something else in oats, for example, high fibre content (16). In the present study, both children with coeliac disease who developed dramatic abdominal symptoms immediately after ingesting oats were biopsied, but no signs of immune activation or relapse of coeliac disease were found. This notwithstanding, the possibility that some patients with coeliac disease may be truly oat intolerant and should thus avoid oats to remain in remission should be kept in mind.
In our challenge study, small-bowel mucosal TG2-specific IgA deposits proved to be clearly gluten-sensitive and they were, in fact, present in the mucosa even before the onset of villous atrophy (Fig. 2). Furthermore, in 1 case, the intensity of the IgA deposition transiently increased when the patient had advertent dietary lapses even if serum anti-TG2 antibody levels remained normal (Fig. 1A). The specificity for TG2 of IgA deposits have been shown in earlier studies by ELISA, Western blot, and in situ (23,29). Gluten sensitivity of mucosal IgA deposits has been also shown in studies concerning early-stage coeliac disease; mucosal IgA deposits have been detected in patients still evincing normal small-bowel mucosal villous architecture but who subsequently developed mucosal damage when gluten consumption was continued (23–25). Interestingly, IgA deposits can also be detected in the small-bowel mucosa of such patients with coeliac disease who do not have the autoantibodies in their serum (23,29). Because the coeliac disease–specific autoantibodies against TG2 are produced in the mucosa of the small intestine, it would appear that after gluten ingestion the autoantibodies are first sequestered in the bowel and that only later “spilling over” from the gut leads to their appearance in the serum.
In conclusion, this study showed that consumption of oats is safe for the majority of children with coeliac disease. Lifelong follow-up is recommended for children wishing to consume a diet containing oats. The detection of mucosal IgA-deposits is a potent means of monitoring treatment of coeliac disease, although not needed in routine surveillance. However, new treatment options in coeliac disease are under study, meaning that reliable and sensitive diagnostic tools to detect minor gluten-induced small-bowel mucosal changes are warranted. The detection of mucosal IgA deposits could provide such a tool and be useful also in the follow-up of clinically problematic cases.
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