Patients and controls
Patients were enrolled at the time of CD diagnosis, which was based upon clinical and serologic findings and subsequently confirmed by histology. After the commencement of a GFD, dietary compliance was established based on dietary history and negativization of EMA and atTG.
The control population consisted of non-coeliac subjects, randomly selected from the same geographical area of CD patients, with no family history of CD or other autoimmune diseases, undergoing an upper endoscopy to investigate dyspepsia and/or heartburn or for positive family history for gastric cancer and expressing their will to participate to the study before the exam. Controls were finally enrolled if their esophageal-gastro-duodenoscopies resulted negative for coeliac, neoplastic, or inflammatory diseases.
Patients and controls were recruited at the Endoscopy Unit of the Department of Internal Medicine, “A. Gemelli” Hospital, Catholic University of Rome. A written informed consent was obtained from all participants before entering the study protocol. The protocol and the consent form had been approved by the local Ethical Committee and conformed to the ethical guidelines of the Declaration of Helsinki (1975).
Collection of peripheral blood samples and flow cytometry
At baseline, 5 ml of peripheral blood were collected from eligible patients and controls. Blood samples from a subset of CD patients were also collected 7 days and 1, 3, 6, 12, and 24 months after the beginning of the GFD.
All the specimens were collected in heparinized tubes and immediately prepared for analysis. The count of CD133+ and CD34+ cells co-expressing CD45 was measured by conventional dual-color flow cytometry, using phycoerythrin (PE)-conjugated anti-CD133/2 antibodies (Miltenyi Biotec, Bergisch Gladbach, Germany) or PE-conjugated anti-CD34 antibodies [BD Biosciences (BD), Mountain View, CA, USA], in combination with fluorescein isothiocyanate (FITC)-conjugated anti-CD45 antibodies (BD). Fluorochrome-conjugated isotype-matched monoclonal antibodies were used to establish background fluorescence. Briefly, small aliquots of peripheral blood were incubated with the above-mentioned antibodies for 30 min at 4°C. After red cell lysis with the FACS lysing solution (BD), the samples were washed twice in PBS (Sigma, St Louis, MO, USA) and kept at 4°C until analysis. Samples were acquired on a FACS-Calibur® flow cytometer (BD): at least 5 × 104 events/determination were acquired in list mode and the results were analyzed using the Cell Quest® software (BD).
The percentage of positive cells among white blood cells (WBC) was calculated by subtracting the value of the appropriate isotype controls. All the data shown in the present report refer to the fraction of CD133+/CD45+ and CD34+/CD45+ cells that are representative of immature hematopoietic cells.
Upper gastrointestinal endoscopy, duodenal sample collection, atTG and EMA serology
Upper gastrointestinal endoscopy with duodenal biopsies (6 specimens from the distal duodenum) was performed on patients with positive serology for atTG IgA and EMA. Once CD diagnosis was histologically confirmed, the patients started a GFD; during the follow-up, they underwent repeated endoscopies with collection of duodenal biopsies at 6, 12, and 24 months following the beginning of the GFD. At the same time points, patients were clinically evaluated and serum levels of atTG and EMA were measured.
Control subjects underwent a single upper gastrointestinal endoscopy with duodenal biopsies (6 specimens from the distal duodenum); at enrollment, they were also clinically evaluated and serum levels of atTG and EMA were measured.
Histology and immunohistochemistry
Histological severity of duodenal damage was evaluated according to the Marsh classification modified by Oberhuber [5] on 4-µm paraffin sections stained with hematoxylin-eosin (H&E): infiltrative lesions with intraepithelial lymphocytosis were defined as Marsh I, infiltrative/hyperplastic lesions as Marsh II, and partial, subtotal, and total villous atrophy as Marsh III (a, b and c, respectively).
Lymphoid infiltrate (CD3) and putative ISC density (CD133/Lgr5) were assessed by immunohistochemistry on 4-µm paraffin sections. Immunohistochemistry was performed on deparaffinized sections using the avidin–biotin-peroxidase complex method (ABC-Elite Kit; Vector Laboratories, Burlingame, CA, USA). For antigen retrieval, paraffin sections were microwave-treated in 0.01 M citric acid buffer, pH 6.0 (2 cycles for 5 min each at 750 W), followed by inhibition of endogenous peroxidase with 3% H2O2 for 5 min. Then, the sections were incubated with the following primary antibodies: anti-CD3 (rabbit polyclonal; dilution 1:50; Dako; Glostrup, Denmark); CD133 (rabbit polyclonal; dilution 1:50; Biocare Medical, Concord, CA) and anti-Lgr5 (anti-G Protein-Coupled Receptor GPR49, rabbit polyclonal, dilution 1:1,000; MBL Int. Corp., Woburn, MA, USA). After a 1-h incubation at room temperature, immunodetection was performed using goat anti-rabbit secondary antibody (Vector Laboratories) and freshly made diaminobenzidine as a chromogen.
The number of intraepithelial lymphocytes (IEL) and epithelial cell nuclei in a randomly chosen, uninterrupted length of surface (villous) epithelium (>500 cells) was counted and the mean for the number of IEL/100 cells was calculated.
Quantification of ISC was obtained through the count of the number of Lgr5+CD133+ epithelial cells per crypt from five randomly chosen crypts and the computation of the mean value/crypt.
The entire area of the epithelium in each tissue section was measured on light microscopy using ×20 objective lenses and ×10 eyepiece.
Statistical analysis
Regarding the circulating CD133+ and CD34+ HSC counts, the results were expressed as mean ± SD. Student’s t test for unpaired data was used for the statistical analysis.
As for the assessment of villous atrophy, lymphoid infiltrate and intestinal CD133+ and Lgr5+ cells, statistical analysis was performed using the Mann–Whitney U test.
A p value <0.05 was considered statistically significant. The data analysis was generated using Microsoft Excel software (Microsoft Corporation, Redmond, WA, USA) and Real Statistics Resource Pack software (Release 3.5; Copyright 2013–2015 Charles Zaiontz; http://www.real-statistics.com).