(2 mg/0.02 mM acetic acid) c) non-sensitized mice (CFA only) subsequently gavaged with indomethacin (3.5 mg/kg). In vitro intestinal permeability Two sections of jejunum from each mouse were used for Ussing chamber studies. Briefly, 5 cm of jejunum samples were collected and divided into 2 segments. Each segment was opened along the mesenteric border, flattened and mounted in an Ussing chamber 2 with an opening of 0.6 cm . Tissues were bathed in oxygenated Krebs buffer containing 10 mM glucose (serosal side) or 10 mM mannitol (luminal side) at 37uC. After a 20-minute equilibration period, conductance (G: mS/cm2) were recorded. Mucosal to serosal transport of macromolecules was assessed by adding horseradish peroxidase (HRP; type II, Sigma-Aldrich, Ontario), a commonly used macromolecular marker, in the luminal side. Serosal samples (500 ml) were obtained at 30 minutes intervals for 2 hours. Intact HRP was assessed using a modified Worthington method with o-dianosidine dihydrochloride (Sigma-Aldrich, On- tario) as the substrate, and mucosal to serosal fluxes were calculated according to standard formulae and expressed as pmol/cm2/hr. Examination of epithelial cell injury Jejunal sections were obtained and immediately fixed in 2.5% glutaraldehyde in 0.1 mol/L sodium cacodylate buffer (pH 7.4) for 2 hours, transferred to sodium cacodylate buffer and stored at 4uC overnight. Tissues were subsequently processed for electron microscopy, and photomicrographs were prepared. Ultra-struc- tural epithelial damage was evaluated in enterocytes by the presence of alterations in brush border, mitochondrial edema and tight junction (TJ) morphology. Epithelial damage was determined by transmission electron microscope (JEOL, Tokyo) of enterocytes in sections from 4–6 animals in each of the 4 study groups. The number of mitochondria with disrupted cristae within the apical region of enterocytes were counted on coded 5000x magnification photomicrographs measuring a total of 300 mm2 (125–250 mitochondria/photomicrographs, 5 photomicrographs/mouse, 6 mice/group) using Adobe CS3 Extended (Adobe Systems Incorporated, California). The fraction of altered mitochondria, defined as number of mitochondria with disrupted cristae divided by total number of mitochondria in a view, was calculated. Mitochondria on the edge of micrographs were excluded for evaluation, since neither their boundaries nor area could be accurately determined. The fraction of disrupted TJ structure was calculated as total altered TJ divided by total number of TJ evaluated in 20 fields per mouse in a blinded manner (3–20 tight junctions/field, 60–400 tight junctions/mouse, 4 mice/group). A field is defined as one square in the EM grid, measuring 8100 mm2. Apical junctional analysis by quantitative real-time PCR Total RNA from a 30–60 mg proximal small intestine section was isolated using RNeasy mini kit (Qiagen, Ontario). cDNA was synthesized from 2 mg of purified total RNA using M-MLV reverse transcriptase (Invitrogen, Ontario). Quantitative real-time PCR was performed with 1:20 dilutions of cDNA. The reaction consisted of iQTM SYBR Green Supermix (Bio-rad, Ontario) for quantitative PCR, primers at 0.5 mM, and 1 ml of cDNA. Amplification was performed using iQ5 Real-Time Detection System at 95uC for 3 min followed by 37 cycles at 94uC for 15 s, 55uC (ZO-1) or 58uC (E-Cadherin) or 59uC (GAPDH) for 20 s, and 72uC for 25 s. Q Real-time PCR was performed using the following primers: ZO-1 59-AGGACACCAAAGCATGTGT- GAG-39/and 39-GGCATTCCTGCTGGTTACA-59; NSAID and Gluten Sensitivity E-Cadherin 59-GCACATATGTAGCTCTCATC-39 and 59C- CTTCACAGTCACACACATG-39. GAPDH 59-CCATGGAGA- AGGCTGGGG-39 and 59-CAAAGTTGTCATGGATGACC-39 was used as housekeeping gene. CT values reported by iQ5 software were used in the study. PCR efficiencies for each amplicon were determined by making 10-fold serial dilutions of cDNA and then amplifying the cDNA using primers to both the gene of interest and housekeeping gene. Relative expression levels were calculated using the Pfaffl method [52], with efficiency correction for each primer set, using REST software [52]. A melting curve analysis was performed by heating the reactions from 50u to 99uC in 0.2uC intervals while monitoring fluorescence. Splenocyte proliferation, cell cultures and cytokine analysis Peptic-tryptic digests of gliadin (PT-gliadin) was prepared as described previously [54]. Spleen cells were harvested and cultured (46105 cells/well) in 96-well tissue culture plates at 37uC, 5% CO2 for 72 h in the presence or absence of 500 mg/ml PT-gliadin and/or 5 mg/ml indomethacin. The cultures were 3 pulsed with 1 mCi/well [ H]-thymidine for 18 h. The cultured cells were harvested onto glass fibre filters using Filtermate harvester (Cambridge Technology, Massachusetts). The radioac- tivity incorporated was determined with a Beta Scintillation Counter (Beckman, California). Results are expressed as stimula- tion index (SI) and calculated as: SI = (mean cpm of triplicate cultures containing antigen)/(mean cpm of cells cultured with medium alone). Splenocyte supernatants were collected 48 h after incubation with or without PT-gliadin and/or indomethacin. The presence of pro-inflammatory cytokines in the supernatant was measured using pro-inflammatory CBA kit (BD Bioscience, California) and analyzed using BD FACSarray Bioanalyzer System (BD Biosci- ence, California). Fluorescent in situ hybridisation (FISH) Oligonucleotide probes are summarized in Table S1. The group-specific probes were labeled at the 59-end with fluorescein isothiocyanate (FITC), showing green fluorescence. EUB 338 probe, targeting conserved sequences within the bacterial domain, was used as positive control [55]. NON EUB 338 probe was used as negative control to eliminate the background fluorescence [56]. Both control probes were labelled at the 59end with either the indocyanine dye Cy3, showing red fluorescence, or with FITC. Aliquots of 36 ml fixed samples were incubated with 4 ml of each fluorescent probe (50 ng/ml) in hybridization solution (10 mM Tris–HCl, 0.9 M NaCl, pH 8.0, and 10% [w/v] sodium dodecyl sulphate) at appropriate temperature (45–50uC) overnight. After- wards, bacterial cells were incubated with 400 ml washing solution (10 mM Tris–HCl, 0.9 M NaCl, pH 8.0) at 50 uC for 30 min to remove non-specific binding of the probes. Hybridized cells were finally pelleted by centrifugation (12 000 g for 5 min) and resuspended in 400 ml of PBS for flow cytometry detection. Bacterial groups were enumerated by combining each FITC- labelled group-specific probe with the EUB 338-Cy3 probe, and expressed as a ratio of cells hybridizing with the FITC-labelled specific probe to cells hybridizing with the EUB 338-Cy3 probe. This proportion was corrected by subtracting the background fluorescence obtained with the negative control probe NON EUB 338 [57,58]. Flow cytometry detections were performed using anEPICSH XL-MCL flow cytometer (Beckman Coulter, Florida, USA) as previously described [58]. This instrument is equipped with two light scatter detectors that measure forward (FSC) and side scatter (SSC) and fluorescence detectors that detect appro- PLoS ONE | www.plosone.org 9 July 2009 | Volume 4 | Issue 7 | e6472