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. 2012 Jun 25;7(6):e39199. doi: 10.1371/journal.pone.0039199

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Serelli-Lee et al.

This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

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(A) The percentage of CD3⁺CD8⁻CCR6⁺IL-17A⁺ cells as a function of CD3⁺CD8⁻ PBMCs was assessed by flow cytometry. PBMCs were activated with PMA and ionomycin for 5 hours in the presence of GolgiStop. Cells were stained for cell surface CD3, CD8, and CCR6, fixed, permeabilised, and stained for intracellular IL-17A. Th17 cells were defined as CCR6⁺IL-17A⁺ events within the CD3⁺CD8⁻ compartment. Representative flow cytometry plots of individuals from group A, P, and N have been depicted. (B) Scatter plot of CD3⁺CD8⁻CCR6⁺IL-17A⁺ cells as a percentage of CD3⁺CD8⁻ cells among PBMCs that had been stimulated with PMA and ionomycin. Group A (n = 44), group P (n = 47), and group N (n = 48). The median and interquartile ranges have been represented on the scatter plot as horizontal bars. (C) The frequency of CD3⁺CD8⁻CCR6⁺IL-17A⁺ events within the CD3⁺CD8⁻ compartment for individuals from group P divided according to years since HP treatment. 1 year (n = 13), 2–3 years (n = 9), 4–9 years (n = 7), and ≥10 years (n = 3). (D) Number of CD4⁺IL-17A⁺ cells per high powered field (HPF) in gastric biopsy samples. Immunofluorescence microscopy was performed on gastric biopsies obtained from 8 patients in group A, 17 patients in group P, 12 patients in group N. For each patient sample, ten HPFs were evaluated and the average number of CD4⁺IL-17A⁺ cells per HPF was represented on the scatter plot. (E) Number of CD4⁺IL-17A⁺ cells per HPF in samples from group P stratified according to years since HP treatment. 1 year (n = 3), 2–3 years (n = 5), 4–9 years (n = 1), and ≥10 years (n = 3). (F − I) Cytokine concentrations in clarified homogenate obtained from mechanically disrupted gastric biopsy samples were measured using the MILLIPLEX® xMAP® bead-based cytokine quantification assay. (F) IL-17A concentration in gastric biopsy samples obtained from patients in group A (n = 7), group P (n = 15), and group N (n = 9). (G) IL-17A concentration in gastric biopsy samples from group P individuals depicted in (F) who were further sub-grouped based on the presence (PC+) or absence (PC−) of histological evidence of pre-cancerous lesions (chronic atrophic gastritis or intestinal metaplasia) in the gastric mucosa. PC+ (n = 12), PC− (n = 3). (H) IFNγ concentration in gastric biopsy samples obtained from patients in group A (n = 7), group P (n = 15), and group N (n = 9). (I) IL-8 concentration in gastric biopsy samples obtained from patients in group A (n = 7), group P (n = 15), and group N (n = 9). NS: not significant, *p<0.05, **p<0.001, ***p<0.0001.