H. pylori and PaCS Figure 2. Characterization of PaCS in H. pylori-colonized human gastric epithelium in vivo. (A) and (A1) (both 4,000x) In two foveolar cells (A) three PaCSs (asterisks), one of which enlarged in the inset (upper left corner, 50,000x) to show their particles, lack phosphorus signal (red colour) when viewed under ESI analysis (A1). Note the intense signal of nucleoproteins in the nucleous, cytoplasmic ribosomes surrounding PaCSs and, in B and B1 (both 8,000x), a luminal H. pylori adhering to epithelial cell microvilli. (C, 6,300x) and (D, 6,300x) H. pylori bodies inside PaCSs, two of which fairly-well preserved (C; enlarged in c, 16,000x), the other (D; enlarged in d, 16,000x) heavily degenerated. Note a peribacterial clear space, the immunogold reactivity of both bacteria and PaCS for H. pylori OMPs (5 nm gold) and VacA (10 nm gold) in c and its inset (44,000x), while the PaCS only, but not bacterial remnants, reacts for human 20S-b5i subunit of the immunoproteasome (d). (E, 25,000x) OMP-reactive intracellular H. pylori enclosed in a supranuclear vacuole of a foveolar cell. (F, 6,300x) Several small, thin PaCSs are sparse inside the RER at the base of an epithelial cell, enlarged in f (32,000x) to show their particles clearly less dense than ribosomes, as well as their selective immunogold reactivity for CagA. g, Golgi area; Lp, lamina propria; n, nucleous; sg, secretory granule. doi:10.1371/journal.pone.0009716.g002 dence from VacA-storing vacuoles at ultrastructural level, despite sharing VacA content. It should be noted that, at variance with in vitro non-polarized cells, where PaCSs and VacA-storing vacuoles were usually admixed in the same cytoplasmic areas (Figure 4D), in superficial-foveolar cells of gastric biopsies they were topo- graphically separated, with PaCSs mainly located in the deep infra/perinuclear, ribosome-rich cytoplasm and VacA-storing endosomes, vacuoles or phagosomes in the ribosome-poor, apical/supranuclear cytoplasm. Concerning CagA, small, discrete immunogold deposits were found in the foveolar cell cytoplasm immediately below some adhering bacteria (Figure 4E and F), a finding which may be compatible with a non-endocytotic way of entry. NOD1 receptor in PaCS As NOD1 has been identified as a major intracellular H. pylori sensor of epithelial cells [6], we tested NOD1 antibodies on H. pylori-colonized human superficial-foveolar cells of gastric biopsies. PLoS ONE | www.plosone.org 4 March 2010 | Volume 5 | Issue 3 | e9716