Patients and controls
In this blind and cross-sectional study, we included 15 consecutive untreated patients with active CD and 9 individuals with NCGS who attended to the Department of Gastroenterology outpatient clinic at the Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, a tertiary care medical facility in Mexico City.
CD was diagnosed when patients met the following criteria: 1) Compatible clinical data: chronic diarrhea, weight loss, bloating, abdominal disconfort, fatigue or, nutrient deficiencies, 2) positive serum autoantibodies: anti-endomysium (EmA IgA), anti-transglutaminase (anti-tTg IgA) and anti-deamidated gliadin peptide antibodies (AGA-DGP IgA and IgG) and, 3) VA according to Marsh-Oberhuber criteria.16
Diagnosis of NCGS was considered when patients presented with: 1) intestinal and extra intestinal symptoms associated with the ingestion of gluten-containing food, 2) a clear clinical response while they followed a gluten-free diet (GFD), 3) relapse of symptoms with the ingestion of gluten-containing foods, 4) negative CD (EmA IgA, anti-tTG IgA, AGA-DGP) and wheat allergy (IgE serological test) serological markers and, 5) normal duodenal biopsies.
All patients were evaluated by a qualified nutritionist with expertise in CD. Symptoms severity (abdominal discomfort or pain, bloating, diarrhea, and constipation) was assessed using a visual analog scale (VAS: 0-10) at baseline, while on an unrestricted diet, 6 weeks after they followed a GFD and after concluding a 6 week challenge based on a diet adjusted to contain 10 grams of gluten par day. Diet compliance was evaluated at biweekly out-patient visits during the gluten-challenge. All serological tests (EmA IgA, anti-tTG IgA, AGA-DGP) were performed at baseline visit and at conclusion of the 6 weeks gluten challenge. The presence of headache, tingling or numbness on feet or hands, fatigue, musculoskeletal pain, brain fog, rash and oral ulcers were considered extra-intestinal symptoms and were specifically evaluated.
We considered a good clinical response to the GFD when there was a decrease in the intensity of symptoms of at least 50% compared to the basal VAS. We did not perform a double-blind gluten/placebo-controlled trial challenge in any case.
We excluded patients with other gastrointestinal diseases, history of gastrointestinal surgery, active or previous infectious diseases, clotting disorders, renal insufficiency, pregnancy or breast feeding, active use of antimicrobial, probiotics, immunosuppressive drugs, non-steroidal anti- inflammatory drugs or corticosteroids.
As a control group we included 10 subjects that fulfilled ROME III criteria for functional dyspepsia (FD) with negative CD serology and who had undergone an upper endoscopy with normal duodenal histology (GT-FD).
Biopsy Sampling
During upper endoscopy four tissue samples from the second portion of the duodenum were obtained, two of them were placed immediately in ice-chilled Hank buffer solution (HBSS) /5% fetal bovine serum (SFB, GIBCO). The others were fixed in 10% formaldehyde and subsequently embedded in paraffin wax and cut into 4 μm thick sections.
Intraepithelial lymphocytes (IELs) Isolation from Duodenal Tissue
Duodenal mucosa samples (epithelium and lamina propria) were cut with a scalpel blade and incubated in phosphate buffer 1x (PBS) / ethylenediamine tetra acetic acid (EDTA) 2mM at 34ºC for 30 min while being agitated. After that, samples were treated with Collagenase IV (Sigma) at 60 U/ml for 1 h at 34°C while being agitated. The cell suspension was then passed through a 40μm cell strainer (Cell Strainer BD Falcon), washed with 2 ml of PBS, and centrifuged at 800 g for 10 min at 25°C. The resulting pellet was homogenized in 1 mL of PBS and incubated with 1 μL of Brefeldin A (BD Golgi Plug) for 1 h at 37°C with 5% CO2. Live-dead assay and cellular count was realized on cellular samples (>90%) on Neubauer chamber (trypan blue).
Immunohistochemistry
Tissues placed on positively charged slides were incubated with mouse monoclonal anti-human IL-1β, IL-6, IL-8, IL-10, IL-15, IL-22, IL-23, IFN-γ, TNF-α, and with rabbit polyclonal anti-human IL-2, IL-12p40, IL-17A, IL-21, or TGF-β1 antibody (Abcam, Cambridge, MA, USA) or anti-human IL-4 antibody (Bio Legend Inc., San Diego, CA, USA) at 10 µg/mL during 30 min. Binding was detected with Universal Dako labelled streptavidin biotin reagent+peroxidase for primary antibodies from rabbit, mouse and goat (Dako, Glostrup, Denmark). Spleen and ganglion samples were used as a positive control. Negative controls were carried out with normal human serum (1:100) and with the IHC universal negative control reagent (Enzo Life Sciences, Inc., Farmingdale, NY, USA), while phosphate buffer saline-egg albumin (SIGMA-Aldrich) was use in the reactive blank. Controls excluded nonspecific staining or endogenous enzymatic activities. We examined three different sections of each biopsy. Cytokine-expressing cells were reported as the percentage of positive cells in three fields (X320) taken from the epithelium and lamina propria. Results are expressed as the median, mean and 5th/95th percentiles.
Peripheral Blood Mononuclear Cells (PBMCs) Isolation.
We collected a sample of venous blood to isolate PBMCs by gradient centrifugation on Ficoll-Paque (Merck-Millipore). The bottom was resuspended in 1 mL of PBS 1x /Brefeldin A (BD GolgiPlug) and incubated at 37ºC in 5% CO2 during 1h. Live-dead assay (trypan blue) and cellular count was realized on cellular samples (>90%).
Flow Cytometry.
1X105 PBMCs or mLs were labeled with 5 𝜇L of antihuman CD4-FITC-labeled, monoclonal antibody (BioLegend San Diego, CA). Cells were permeabilized with 200 𝜇L of cytofix/cytoperm solution (BD Biosciences). Intracellular staining was performed with an anti-human Foxp3-PE-, IFN-γ-APC-Cy7-, IL-17A-PE-Cy7- (BioLegend), T-bet-PerCP-Cy5.5- (BD Pharmingen, San Jose, CA), and ROR-γt-APC-labeled (R&D Systems, Minneapolis, MN) mouse monoclonal antibodies. From the electronic bi-parametric gate of the singlets and living cells, we performed an analysis in the CD4+ lymphocytes population to identify CD4+/Foxp3+ cells, CD4+/T-bet cells, CD4+/INF-γ cells, CD4+/ROR-γt+ cells, CD4+/IL-17A cells. Results are expressed as the relative percentage of CD4+/IL-17A+−, CD4+/IFN-γ+−, CD4+/Foxp3+−, CD4+/T-bet+−, and CD4+/ROR-γt+−expressing cells in each gate. For an auto fluorescence control, we ran an unstained and permeabilized cell sample. An AbC anti-mouse bead kit (Invitrogen, UK) was used to adjust instrument settings, to set fluorescence compensation, and to check instrument sensitivity. Fluorescence minus one (FMO) controls were stained in parallel. Samples were analyzed with an Attune Acoustic Focusing Cytometer Blue/Red (Life Technologies). We recorded more than 10,000 events for each sample, and they were analyzed with Attune® Cytometric Software v2.1.
Ethical Considerations. This work was performed according to the principles expressed in the Declaration of Helsinki. Our Institutional research and ethics committees approved the protocol (GAS-1298-14/15-1; August 11, 2014). Each patient signed a written informed consent.
Statistical analysis
Data were analyzed using GraphPad Prism for Windows (version 6.01 GraphPad software Inc. USA) and Kruskall Wallis non-parametric test. Immunohistochemical data are expressed as the median, mean and 5th/95th percentiles. We performed one-way ANOVA on ranks by Holm-Sidak method and Dunn’s test for all pairwise multiple comparison procedures. A p value <0.05 was considered statistically significant.