Participants provided written informed consent. The Human Research Ethics Committee Melbourne Health (2003.009) and The Walter and Eliza Hall Institute Human Research Ethics Committee (03/04) approved the study. Male and female patients aged 18 to 70 years with biopsy-confirmed CeD following GFD for at least 8 weeks were eligible. Patients were excluded if their score was over 12 in the Celiac Dietary Adherence Test (33), serum TG2 immunoglobulin A (IgA) and deamidated gliadin peptide IgG were both elevated, immunomodulators or immunosuppressive medications were used during the previous 2 months, oral or parenteral corticosteroids were used within the previous 6 weeks, or females were pregnant, lactating, or breastfeeding. A double-blind food challenge consisting of either 5 g of vital wheat gluten flour (Bob’s Red Mill Natural Foods Inc., Milwaukie, OR), which was estimated to contain 3 g of gluten protein according to the Osborne equation, or gluten-free fine-ground white rice flour (“McKenzie’s Rice Flour,” Ward McKenzie’s Pty Ltd., Australia) was added to one serve of (7 g) gluten-free “Vitafresh Low Calorie Lime” or “Vitafresh Low Calorie Sweet Navel Orange” (Hansells Food Group, New Zealand) mixed together in 100 ml of water consumed over 2 min. Adverse events were recorded hourly and graded according to “The Common Terminology Criteria for Adverse Events (CTCAE) version 4.0” (U.S. Food and Drugs Administration Guidance for Industry: Toxicity Grading Scale). Each hour, a modified version of the CeD patient reported outcome questionnaire (CeD PRO) was completed by participants to grade their worst experience for 11 symptoms over the previous 1-hour graded on a whole-number scale from 0 (none) to 10 (worst possible) (34). Blood was collected before and hourly up to 8 hours in the first six patients, and for later patients, only at baseline and at 4 and 6 hours. Blood for plasma was collected and processed as described for phase 1 clinical studies. Blood for serum was collected into Vacutainer Plus plastic serum tubes (BD 367986). Serum was separated from blood after 2 hours at room temperature by centrifuging at 2000g for 20 min.

The clinical procedures, outcomes, and associated laboratory methods for a second, separate unmasked gluten food challenge have been reported in detail elsewhere (5). Participants were on GFD and in histological and serological remission. Quantitative histology was determined in baseline duodenal biopsies. Participants consumed one 50-g muesli bar daily that contained 7.6 g of gluten flour (5.7 g of gluten protein) and was free of fermentable oligosaccharides, disaccharides, monosaccharides, and polyols. Blood samples were collected at baseline and at 2, 4, and 6 hours after consuming the muesli bar on day 1. Plasma from these blood samples was kept frozen at −80°C and later analyzed for cytokines using a magnetic bead assay, and results have been previously reported (5). For the present study, the ECL assay was used to reassess IL-2, IL-8, and IL-10 in frozen plasma samples from the first study day. Symptoms were recorded by patients on a visual analog scale at baseline and then every 2 hours up to 6 hours after consuming gluten on the first study day. The frequency of effector-memory gut-homing CD4+ cells specific for DQ2.5:glia-α1a, DQ2.5:glia-α2, DQ2.5:glia-ω1 and DQ2.5:glia-ω2, and DQ8:glia-α1 and DQ8:glia-γ1b epitopes was assessed in blood collected at baseline according to previously described methods (35). These data have been previously published (5).

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