Twelve weeks' gluten challenge with both moderate and low amounts of gluten sufficed to induce small-bowel mucosal morphological damage in the majority of volunteer treated celiac disease adults not, however, in all. An increase in the density of CD3+ IELs paralleled this phenomenon especially in the moderate-dose gluten challenge group. At the same time, moderate gluten doses triggered more gastrointestinal symptoms leading to premature withdrawals from the study. This suggests that in the 12-week time-frame smaller gluten doses of 1-3 g per day might be preferable to higher doses in order to obtain sufficient complete read-outs at the end of a challenge study.
In this study, small-bowel mucosal Vh/CrD measurement seemed to be the most sensitive parameter in both moderate and low gluten doses in assessing gluten-induced damage in celiac disease. This conception is also in line with previous findings, where the Vh/CrD index has been used to show harmful effects of gluten in both short-term gluten challenges and long-term follow-up studies [8, 13, 26, 27]. It is not surprising that the IEL count was a reliable marker for gluten toxicity only with moderate, but not with low gluten doses, as it has previously been shown that in celiac disease during a gluten-challenge, IELs accumulate in the surface and crypt epithelium in a dose-dependent manner . It is also important to note that even if the majority of celiac disease patients after gluten challenge yielded positive read-outs both in the Vh/CrD index and in the IEL count, there were some discrepant cases responding only with one or other parameter (Table 2). Marsh classification entailing six diagnostic categories (type 0-type 3c) has been widely used in the diagnostic work-up and follow-up of celiac disease . Marsh grades type 1-type 3c all indicate cursorily that small-bowel mucosal IEL density is increased (>40 IELs/100enterocytes), but different categories point more specifically to different morphological features (such as normal villous morphology, crypt hyperplasia or villous atrophy). Nonetheless the Marsh classification does not take into account minor gluten- induced mucosal changes (e.g. a significant decrease in Vh/CrD or increase in density of IELs within one Marsh grade) or discrepant results in Vh/CrD or IEL counts during a gluten challenge. It would thus be advisable in gluten challenge research projects, to use two separate continuous readout parameters in assessing gluten-dependent functional mucosal changes, i.e. the morphological Vh/CrD and the inflammatory IEL changes, as opposed to applying a simplified grouped classification.
Even if abdominal symptoms after the consumption of cereals do not generally constitute a specific marker of untreated celiac disease , gluten-induced gastrointestinal symptoms in our challenge study showed high positive predictive value (87%) for small-bowel mucosal injury in celiac disease patients. However, in 22% of the cases who developed significant small-intestinal damage, symptoms remained absent. It is widely known that many untreated celiac disease patients do not suffer from symptoms even if they have a manifest gluten-induced small-bowel mucosal lesion . Furthermore, in some treated celiac disease patients, symptoms may disappear on a strict gluten-free diet despite continued ongoing mucosal villous atrophy [30, 31]. Such persistent small-intestinal villous atrophy in symptom-free patients clearly carries a risk of subsequent severe complications such as osteoporosis and malignancies . This warrants caution in relying solely on symptoms or other patient-related outcomes without small-bowel biopsies in gluten challenge research projects.
In studies where a gluten challenge has been applied for diagnostic purposes in children on a normal daily gluten-containing diet (10-20 g daily), a small-bowel mucosal histological relapse has usually developed within three to twelve months in the majority of patients [8, 9]. Seroconversion of EMA and TG2 antibodies is known to be a reliable predictor of histological relapse and antibody testing has helped in the timing of the biopsy . In this study with low to moderate gluten doses, only 43% of the challenged adult celiac disease patients evinced a significant serological response. In line with earlier challenge studies, here all those who had increased serum autoantibodies during the challenge also developed small-bowel villous atrophy. However, at the same time serology missed nine cases with significant small-bowel mucosal changes (Tables 2 and 3). These cases might have called for a longer gluten challenge period or higher gluten amounts. However, it is well-known that serology may fail to detect occasional slight dietary transgressions and incomplete mucosal healing [30, 32, 33], and therefore celiac serology is not always a reliable means of assessing gluten-induced harm. Furthermore, it is important to note that after the gluten challenge all three antibody - EMA, TG2 and DPG antibody - tests performed similar way, and recently introduced DPG antibody test was not superior to conventional serological assays. As small-bowel mucosal TG2-specific autoantibody deposits have also been found in seronegative celiac patients and patients with early-stage celiac disease [23, 24, 34], it has been hypothesized that these mucosal autoantibodies might also appear early during a gluten challenge. In our study, mucosal autoantibodies followed the morphological damage, and they added only little to the normal morphometrical measurements. It might be that after a short-term acute gluten challenge the distribution of mucosal autoantibody depositions is patchy, and single mucosal biopsies may not reveal the positivity. It remains to be seen whether they will be of use at earlier time-points in the gluten challenge, but due to the invasive nature of small-bowel biopsies we were not able to take specimens on several occasions during the challenge.
There were some celiac disease patients who did not respond in terms of any of the measured outcome variables during the 12 weeks' gluten challenge. It is well-known that celiac patients show varying sensitivity to gluten and the process of mucosal deterioration may take years or even decades [35–37]. Furthermore, it should be remembered that the phenotype of the disease may vary in the same person over time; celiac patients suffering originally from abdominal symptoms at diagnosis may later experience only extraintestinal symptoms such as dermatitis herpetiformis when gluten is re-introduced into the diet . These issues need to be taken into account when sample sizes in study cohorts are determined.
At the baseline of the study, the majority of the treated celiac disease patients evinced increased densities of IELs and one patient even had villous atrophy with crypt hyperplasia despite adherent to a strict long-term gluten-free diet. According to the literature, such cases exist and it has been shown that even 80% of treated patients may have ongoing villous atrophy during a strict diet [38–40]. These persistent mucosal changes might be due to constant residual gluten contamination in the food, which again would indicate that alternative treatment options would be desirable.