Gliadin

All α-gliadin DNA sequences obtained in this study were translated to protein sequences and converted into FASTA format. In addition, public domain gliadin and glutenin sequences from bread wheat were extracted in FASTA-format from the Uniprot database with the following conditions: Triticum aestivum and (gliadin or glutenin). The program PeptideSearch [28 (link)] was used for matching the predicted epitopes from α-gliadin with the databases described above. Only perfect matches were considered in the scoring.

Each distinct protein spot was excised from at least three 2-DE gels in order to digest them separately with trypsin, thermolysin and chymotrypsin. MS/MS analysis of enzymatic digests of protein spots was carried out with a QSTAR Pulsar i quadrupole time-of-flight mass spectrometer (Applied Biosystems/MDS Sciex, Toronto, Canada) that was equipped with a nano-electrospray source and nano-flow liquid chromatograph [27 (link)]. Automatic determination of the appropriate collision energy (relative to m/z) was carried out by the Analyst QS 1.1 software. When analyzing samples digested either with chymotrypsin or thermolysin the intercept of the collision energy values was decreased by eight units relative to that used with trypsin. The spectra from each digest were used to interrogate a "SuperWheat" database (Version # 100211) as described in [59 (link)]. For this study, the "SuperWheat" database was constructed by concatenating the following publicly available databases; NCBI non-redundant green plant protein sequences (download date: 2/11/2010) [84 ], nucleotide sequences translated in all six reading frames of contigs from TaGI Releases 10.0 and 11.0 [63 ], US Wheat Genome Project [85 ], HarvEST 1.14 (WI all NSF "stringent" assembly from 05/08/04) [86 ], NCBI Unigene Build #55 [84 ], and all ESTs from Butte 86 developing grain, as well as translated sequences (reading frame only) of 94 Butte 86 contigs (Additional file 2), including those for alpha-gliadins and gamma-gliadins [12 (link),13 (link)]. Additionally the database contained a list of proteins known to be common laboratory contaminants [87 ] and sequences for thermolysin. The "SuperWheat" database contained 2,094,746 protein sequences. Two search engines, X!Tandem [57 (link),87 ] and Mascot version 2.1 (Matrix Science, London, UK) [88 ,89 (link)] were used to match the peptide mass spectra to spectra generated in silico from database peptides. It has been demonstrated that Mascot and X!tandem yield somewhat different results [59 (link),70 (link)]. Mascot identified significantly more tryptic peptides than did X!tandem, while X!tandem identified more peptides from proteins digested with thermolysin or chymotrypsin. Scaffold Version 2_02_04 [62 ] was used to assemble and visualize MS/MS derived peptide and protein identifications. A "subset" database was generated from the initial search of the SuperWheat database by exporting from Scaffold all protein sequences that had a 20% or greater probability of being a match. Appended to these 2,134 sequences was an equal number of decoy protein sequences from the archaeobacter Jannaschia sp, translated sequences from the set of Butte 86 contigs not already included in the subset database, and the set of common protein contaminants. A "second pass" search [90 (link),91 (link)] was conducted with both search engines and the results assembled and validated with Scaffold. Identifications of proteins were required to meet the following criteria: at least two peptides having a parent mass tolerance threshold of less than or equal to 100 ppm and a greater than 90% peptide probability as specified by the Peptide Prophet algorithm [92 ]. Scaffold Version 3.00.03 was used to compile the final set of MS/MS based peptide and protein identifications, using the MUDPIT algorithm to independently analyze the data for each spot. The false discovery rate was generally found to be 0.0% under the filter settings used. The data associated with this manuscript may be downloaded from ProteomeCommons.org Tranche using the following hash:
hCc5INiKGH0m4DEfxLbShm1F+us+JyZ/HENjkOTlGcni8NmnyoEwU5i7Onf/Po2kNtnP10SCdgODD6Swo0hgF69d3dIAAAAAAAB6hg==

Blood for testing was taken on empty stomach and the following routine tests were performed: blood cell count, C-reactive protein, count, glucose, bilirubin, urea, creatinine, profile of lipids, thyroid-stimulating hormone, free thyroxine, free triiodothyronine, vit. D3, vit. B12, alanine and asparagine aminotransferase, gamma-glutamyltranspeptidase, alkaline phosphatase, amylase, lipase, antibodies for tissue transglutaminase, and deaminated gliadin peptide. Fecal calprotectin (FC) was evaluated by sandwich ELISA test in Quantum Blue Reader (Buhlmann Diagnostics, Amherst, NH, USA). In addition, the ¹³C urea breath test was performed to exclude Helicobacter pylori infection and the lactulose hydrogen breath test was applied to eliminate patients with small intestinal bacterial overgrowth.
Urine samples for the analysis of TRP metabolites were collected in the morning on an empty stomach into a container with a solution of 0.1% hydrochloric acid as a stabilizer. L-tryptophan and its metabolites 5-HIAA, KYN, KYNA, and QA were determined in urine using liquid chromatography–tandem mass spectrometry (LC–MS/MS—Ganzimmun Diagnostics AG, Mainz, Germany; D-ML-13147–01-01). The levels of these metabolites were expressed in mg/g creatinine. The proportions between the levels of 5-HIAA acid TRP, as well as between KYN and TRP, were also calculated. The 5-HIAA/TRP and KYN/TRP ratios were considered as markers of the tryptophan hydrolase 1 and 2,3-dioxygenase activity, respectively.

Gliadin (Sigma, USA) was dissolved in 1% Dimethyl sulfoxide (DMSO). In order to determine the appropriate concentration of gliadin with the most inflammatory effect on MQs, three different concentrations of gliadin (100, 200, 400 μg/ml) were used (although previous studies considered the concentration of 200 μg/ml as the best concentration for gliadin in this regard)^{6 (link)}. Based on our findings, the concentration of 200 μg/ml of gliadin was used as the optimal concentration too.
A. muciniphila were added to MQ’s culture at a multiplicity of infection (MOI) of 1, 10 and 100 respectively^{25 (link)}.
To investigate the effect of A. muciniphila on the MQ’s response to gliadin peptide, A. muciniphila was added to the MQ cells before stimulation with gliadin as pre-treat (preventive effect) and after stimulation with gliadin as post-treat (protective effect).
To evaluate the preventive effect of A. muciniphila on the inflammatory effect of gliadin, 1 × 10⁶ of THP-1 cells were seeded in 6 well cell culture treated plate. After 48 h of PMA treatment, the culture media with PMA was changed with penicillin and streptomycin-free medium (to prevent bacterial death) followed by rest for 24 h prior to experiments. Cells were treated with A. muciniphila in three concentrations (MOI = 1, 10, 100) for 24 h and then the treatment with gliadin was done for the next 24 h (pre-treat). Furthermore, to evaluate the protective effect of A.muciniphila, after 24 h stimulation with gliadin, cells were treated with different concentrations (MOI = 1, 10, 100) of A. muciniphila (post-treat). A well was treated with 20 ng/ml LPS (Santa Cruz Biotechnology Cat No. sc-3535), as positive control, to compare its induction pattern with gliadin and bacteria. Two wells that were treated with gliadin alone and A. muciniphila alone and a well full of PMA-treated THP-1 cells without any treatment was considered as the negative control group (Fig. 6). Experiments were performed in triplicate (technical replicates).Figure 6

Schematic illustration of the study design.

The following autoantibodies of class immunoglobulin G (IgG) were analyzed in the sera of WD patients and controls with the use of an indirect immunofluorescence technique: antinuclear antibodies (ANA), anti-smooth muscle antibodies (SMA), anti-mitochondrial antibodies (AMA), anti-parietal cell antibodies (APCA), anti-liver/kidney microsomal antibodies type 1 (LKM-1), and anti-neutrophil cytoplasmic antibodies (ANCA). According to ESPGHAN recommendations [23 (link)], the presence of ANA was detected using commercial testing slides with Hep-2 cell line (EuroImmun, Lubeck, Germany) at 1:20 screening dilution. Positive results for the ANA tests were confirmed by the immunoblot method (ANA3b test, EuroImmun, Germany). SMA, AMA, APCA, and LKM-1 were evaluated on a commercially-available rat tissue substrate (liver, kidney and stomach; BioSystems, Barcelona, Spain) at a 1:20 screening dilution.
For ANCA detection, commercial slides with Hep-2 cell and neutrophils fix with formalin and ethanol were used (EuroImmun, Germany) at a 1:10 screening dilution.
All of the specimens positive at 1:10 or 1:20 dilution were retested with the use of two-fold dilutions to determine the final antibody titer. Microscopic assessment was done by two independent diagnosticians.
For detection of celiac-specific antibodies, the presence of anti-tissue transglutaminase 2 (anty-tTG2 IgA) autoantibodies in class IgA and anti-deamidated gliadin peptide (DPG) antibodies in class IgG were analyzed with the use of an automated Thermo Scientific Phadia 100 system (Phadia, Sweden). According to the manufacturer’s protocols, an antibody concentration >10 and <7 U/mL was considered as positive and negative, respectively. Positive results were confirmed by testing anti-endomysial (EuroImmun, Germany) antibodies (EMA) in both IgA and IgG classes.