Patients and controls
In this blind, cross-sectional study we included 15 consecutive untreated patients with active CD and 9 individuals with NCSRWS who attended from 2014 to 2016 to the outpatient gastroenterology clinic at the Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, a tertiary referral medical facility in Mexico City. CD was diagnosed when patients met the following criteria: 1) CD compatible clinical data: chronic diarrhea, weight loss, bloating, abdominal discomfort, fatigue or nutrient deficiencies, 2) positive IgA anti endomysium antibodies (EMA IgA; IF, Inova Diagnostics, San Diego, CA, USA. Normal < 1:5), IgA tissue Transglutaminase antibodies (anti-tTg IgA; ELISA, Orgentec; Mainz, Germany. Normal < 10 U/mL) and IgA/IgG anti deaminated gliadin peptide antibodies (IgA and IgG-DGP; ELISA, Orgentec; Mainz,Germany. Normal < 10 U/mL) and 3) duodenal mucosa VA according to Marsh-Oberhuber criteria (24).
The diagnosis of NCSRWS 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 were on a gluten-free diet, 3) relapse of symptoms with the ingestion of gluten-containing food, 4) negative CD serological markers (EMA IgA, anti-tTG IgA, anti IgA/IgG -DGP) 5) no evidence of wheat allergy (IgE serological test) and, 6) normal duodenal mucosa.
A qualified nutritionist with expertise in CD evaluated all patients. Symptom severity (abdominal discomfort or pain, bloating, diarrhea, and constipation) was assessed at baseline, while on an unrestricted diet, 6 weeks after following a gluten-free diet and after completing a 6 weeks challenge with 10 grams of gluten per day using a visual analog scale (VAS; 0-10). Only NCSRWS patients underwent a gluten free/gluten containing diet challenge. We did not perform a double-blind gluten/placebo-controlled trial challenge in any case.
Diet compliance was evaluated during bi-weekly out-patient visits. All serological tests (EMA IgA, anti-tTG IgA, anti - DGP) were performed at baseline visit and after completion of a gluten free diet or gluten challenge.
Headache, tingling or numbness in feet or hands, fatigue, musculoskeletal pain, brain fog (mild transient cognitive impairment), rash and oral ulcers were considered extra-intestinal symptoms and they were specifically evaluated using a visual analog scale (VAS; 0-10). A good clinical response to the gluten-free diet was considered when there was a decrease in the intensity of symptoms of at least 50% compared to the baseline VAS score.
We excluded patients with other gastrointestinal diseases, history of gastrointestinal surgery, active or previous infection diseases, clotting disorders, renal insufficiency, pregnancy or breast feeding, active use of antimicrobial, probiotics, immunosuppressive drugs, non-steroidal anti-inflammatory drugs or corticosteroids. The 10 subjects included in the control group had undergone an upper endoscopy, fulfilled the functional dyspepsia ROMA III criteria and had both: negative CD serology and normal duodenal histology (25).
All endoscopic duodenal biopsies were obtained while the patient was on a gluten containing diet. 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 slices.
Isolation of Mucosal Lymphocytes (mLs) (IELs) from Duodenal Tissue
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 40 μm 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 1μL of Brefeldin A (BD Golgi Plug) for 1 h at 37°C with 5% CO2. Live-dead assay and cellular count from cellular samples was performed (>90%) on a Neubauer chamber (trypan blue) as previously reported. (26)
Tissues placed on positively charged slides were incubated with mouse monoclonal anti-human IL-1, 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-beta1 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 immunohistochemistry 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. As we have done before, cytokine-expressing cells were reported as the percentage of positive cells in three fields (X320) taken from the epithelium and lamina propria (27). Results are expressed as the median, mean and 5th/95th percentiles.
Peripheral Blood Mononuclear Cells (PBMCs) Isolation
Using a sample of venous blood, we isolated 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 were performed on cellular samples (>90%).
1X10 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 cyto10x/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-γcells, 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 autofluorescence 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) control were stained in parallel.
As in prior reports from our group samples were analyzed with an Attune Acoustic Focusing Cytometer Blue/Red (Life Technologies) (26,28). We recorded more than 10,000 events for each sample, and they were analyzed with Attune® Cytometric Software v2.1
This work was performed according to the principles expressed in the Declaration of Helsinki. The study was reviewed and approved by the institutional ethics and research committee (GAS-1298-14/15-1; August 11, 2014). Each patient gave and signed a written informed consent.
Due to the exploratory nature of the study and prevalence of CD, NCSWS and FD, a convenience sampling method was used. Statistical analysis was performed using GraphPad Prism for Windows (version 6.01 GraphPad software Inc. USA).
Immunohistochemical data are expressed as the median, mean and 5 /95 percentiles. We used Kruskall Wallis test for non-parametric variables. We performed one-way analysis of variance on ranks by Holm-Sidak method and
Dunn´s test for all pairwise multiple comparison procedures and comparisons versus a control group. P<0.05 was considered statistically significant.