Fig. 2.

Adhesion of H. pylori to guinea pig gastric epithelial cells mediated by MUC5AC mucin and Lewis (Le)X/LeY determinants. Binding of H. pylori to guinea pig primary gastric epithelial cells and gastric tissue specimens was evaluated by imaging H. pylori stained with anti-H. pylori antibodies (Ab) conjugated with fluorescein isothiocyanate (FITC) in a fluorescence reader or confocal microscope, counterstained with DAPI or phalloidine. a – guinea pig primary gastric epithelial cells non treated (in culture medium alone) or treated with selected H. pylori components: for 24 h with glycine acid extract – GE (10 μg/ml), urease subunite A – UreA (5 μg/ml), cytotoxin associated gene A (CagA) protein (1 μg/ml), H. pylori lipopolysaccharide (LPS Hp) or E.coli LPS –LPS Ec (25 ng/ml) or for 2 h with live H. pylori – Hp (2 × 10⁷ colony forming units - CFU/ml) were prepared. Further, cells non-treated (control of adhesion) or blocked with anti-MUC5AC or with anti-LeX/LeY antibodies were used in adhesion assay. a i – the intensity of fluorescence measured in a fluorescence reader at 495 nm (excitation) and 519 nm (emission), mean values ±SD. * Statistical significance for cells treated with an individual component vs untreated cells or ^# treated with individual antibody vs untreated cells (control cells), p < 0.05 in non parametric U Mann-Whitney test. Representative images of primary gastric epithelial cells untreated (a ii) or treated (a iii) with anti-MUC5AC antibodies before H. pylori binding experiments, stained with anti-H. pylori FITC antibodies in confocal microscope (Leica TCS SPE), at 495 nm excitation and 519 emission for FITC, 345 excitation/455 emission for DAPI and 591 nm excitation/608 nm emission for phalloidine (640 magnification). Gastric tissue specimens from non infected guinea pigs untreated or treated with anti-MUC5AC (b) or anti-LeX (c) blocking antibodies before 2 h exposure to live H. pylori (2 × 10⁷ colony forming units (CFU)/ml). bi and Ci – intensity of fluorescence measured using the software ImageJ version 1.48v (National Institute of Health, United States) at 495 excitation/519 emission, mean values ±SD. *Statistically significant values for gastric tissue blocked with anti-MUC5AC or anti-LeX antibodies vs unblocked specimens. Representative images of H. pylori adhesion to the guinea pig gastric tissue non treated with anti-MUC5AC (bii) or anti-LeX (cii) antibodies vs gastric tissue treated with such antibodies (b iii, iii) from fluorescence microscope (Axio Scope A1, Zeiss, Germany), at 495 nm excitation/519 nm emission for FITC, 345 nm excitation/455 nm emission for DAPI and 591 nm excitation/608 nm emission for phalloidine (100 × magnification)