Altogether 25 adult celiac disease patients fulfilling the inclusion criteria were enrolled for the study (Table ). At the time of the diagnosis, the majority (88%) had suffered from abdominal symptoms. When volunteering for this study, the patients had been a median of 11 years on a strict gluten-free diet, and all were in clinical remission.
Demographic data on the 25 celiac disease patients
At baseline in the current study, all patients except one had normal Vh/CrD ratios (median 2.9, range 1.3-4.2) on small-intestinal biopsy (Table ). The mean density of CD3+ IELs was 63 cells/mm (range 23-146). Altogether six patients had borderline positive celiac disease antibody levels (three had positive TG2 antibodies, two EMA and one DGP antibodies); in the rest all three antibodies were negative. When TG2-targeted autoantibodies were assessed at small-bowel mucosal level, moderate IgA-deposits were seen in two and faint in four patients. In the remaining 19 cases, mucosal TG2-autoantibody depositions were negative at the beginning of the challenge study.
Effect of moderate (3-5g) and low (1-3g) amounts of gluten on small-bowel mucosal villous height crypt depth ratio (Vh/CrD), densities of CD3+ intraepithelial lymphocytes (IELs) and gastrointestinal symptoms in treated celiac disease patients.
Altogether four celiac disease patients belonging to the moderate gluten dose group discontinued the study due to abdominal symptoms shortly after the challenge was initiated (after three, eight, ten and 23 days), and they were excluded from the final analyses. Celiac serology remained negative in all. The first two drop-outs with extremely brief gluten challenges underwent the follow-up endoscopy, and no small- bowel mucosal deterioration was evident.
The remaining 21 patients comprised the final study population (Tables and ), for whom the gluten challenge lasted a median of 84 days (range 29-103 days) with an average of 3.1 g daily gluten consumption (range 1.3-5.0 g/day). When the cohort was further divided according to the average daily gluten intake, ten patients belonged to the moderate-dose (3-5 g) and 11 to the low-dose (1-3 g) gluten challenge group (Tables and ). In the moderate-dose group, three patients withdrew prematurely (after 29, 38 and 61 days) by reason of abdominal symptoms such as diarrhea, vomiting and abdominal pain; one patient discontinued at day 45 for personal reasons. Intended end-point examinations were carried out in all four cases immediately after cessation of the challenge. All celiac patients in the low-dose gluten challenge group completed the study as planned after 12 weeks' challenge. Patients with a moderate gluten dose were thus more prone than those with a low dose to withdraw prematurely from the study (p = 0.035).
Effect of moderate (3-5g) and low (1-3g) amounts of gluten on serum transglutaminase 2 (TG2) antibodies and small bowel mucosal TG2-targeted autoantibody deposits in treated celiac disease patients.
During the gluten challenge, a clinically significant decrease in Vh/CrD was found in 14 (67%) out of the 21 celiac patients; seven were in the moderate and seven in the low gluten dose group (Table , Figures and ). The Vh/CrD in the moderate-dose group deteriorated from a mean 3.0 (95%CI 2.8-3.2) to 1.7 (95%CI 1.0-2.4) and in the low-dose group from 2.9 (95%CI 2.5-3.4) to 1.9 (95%CI 1.2-2.6), both changes being statistically significant (p = 0.002 and p = 0.016, respectively). The changes in Vh/CrD did not correlate with daily gluten intake (r = -0.138, p = 0.552).
Changes in small bowel mucosal villous height and crypt depth ratios in moderate-dose group at baseline and at completion of the study.
Changes in small bowel mucosal villous height and crypt depth ratios in low-dose group at baseline and at completion of the study.
In line with the morphological findings, a clinically significant change in the density of CD3+ IELs was seen in altogether 14 (67%) subjects; eight in the moderate-dose and six in the low-dose of (Table , Figures and ). In the moderate gluten dose group the IEL count increased significantly from a mean 49 cell/mm (95%CI 38-60) to 88 cell/mm (95%CI 70-106, p = 0.001). In the low-dose group the increase did not reach statistical significance (from a mean 78 cell/mm [95%CI 56-100] to 98 cell/mm [95%CI 61-135], p = 0.188). All in all, daily gluten intake did not correlate with increased inflammation (gluten intake vs. ΔCD3+ IEL count, r = 0.251, p = 0.273).
Changes in small bowel mucosal densities of CD3+ IELs in moderate-dose group at baseline and at completion of the study.
Changes in small bowel mucosal densities of CD3+ IELs in low-dose group at baseline and at completion of the study.
Altogether nine (43%) celiac disease patients, five in the moderate- and four in the low-dose gluten challenge group, showed significant increases in TG2 antibody titers (Table ). The subjects consuming moderate doses of gluten evinced clearly higher TG2 antibody titer responses than those consuming low doses; this was already seen after four weeks' challenge (Figure ). Interestingly, after the highest positive titer during the gluten challenge, the serum TG2 antibody levels tended to decrease even if gluten consumption continued (Figure ). Serum EMA and DGP antibody titers were in line with TG2 antibody results; furthermore, after the gluten challenge all these three antibodies appeared in the serum simultaneously (data not shown). Small-bowel mucosal TG2-targeted autoantibody deposits generally followed the gluten-induced serological response in sera and morphological changes seen in the biopsy specimens (Tables and ).
Changes in antibody levels. Transglutaminase 2 (TG2) antibody levels at baseline, at 4 and 8 weeks, and at the end of the study. Closed squares indicate moderate daily gluten dose 3-5 g and open squares low gluten doses (1-3 g per day).
At the outset, all subjects were free of gastrointestinal symptoms. Altogether 15 (71%) out of the 21 celiac disease patients experienced mild to moderate abdominal symptoms upon gluten challenge; eight of them (including the four withdrawing prematurely, see above) were from the moderate-dose and seven from the low-dose group (Table ). None developed severe symptoms. Thirteen (87%) out of the 15 symptomatic patients also developed small-intestinal mucosal changes (significant decrease in Vh/CrD and/or increase in the density of CD3+ IELs). Of note, symptoms did not reveal four celiac disease patients having clinically significant gluten-induced small-bowel mucosal damage.