vitro and in vivo with CagA, or ERK kinases (Figure 6C) involved in a MAP kinase signaling pathway activated by CagA [28]. Tests on consecutive ultramicrotomic sections through the same PaCS or on the same section using multiple antibodies linked to gold particles of different size confirmed colocalization of each bacterial protein with NOD1, polyubiquitinated proteins and proteasomal components, sometimes with gold particles tightly adherent to each other so as to suggest possible interaction of respectively linked molecules (Figure 6A, a1 and a2). Non-particulate, UPS-negative structures In addition to PaCS, other types of cytoplasmic structures were observed by TEM, less commonly in gastric epithelium and more frequently in epithelial cell lines. They showed a loose filamentous meshwork often forming an irregular honeycomb-like scaffold, or with foci of more compact fibrillar structure, in the absence of 13- nm-thick particles (Figure 6D and d). These particle-free, fibrillar to honeycomb-like structures were sometimes found to coexist with PaCS, adjacent to each other inside the same cytoplasmic area, occasionally with patterns suggesting that they may originate from PaCS by dissolution of the 13-nm particles with retention of the interparticle scaffold. PaCS distribution Both PaCS under TEM and metachromatic areas of toluidine blue-stained resin sections under light microscopy were mainly found in superficial-foveolar cells of H. pylori-infected gastric mucosa, with progressively increasing number and size from the renewal zone to the surface epithelium. Less numerous or smaller PaCSs were seen in superficial-foveolar cells devoid of luminal bacteria, though showing sparse intracellular H. pylori and moderate mononuclear cell inflammation in the underlying lamina propria. PaCSs were not observed in acidopeptic, pyloric or cardial gland cells, nor in complete, small intestine-type (type I) metaplastic epithelium or lamina propria cells of inflamed gastric mucosa. In three cases, we found areas of colonic-type (type III) intestinal metaplasia whose sparcely granulated goblet cells showed intracellular H. pylori, especially in the supranuclear cytoplasm [2], as well as typical PaCSs in the infranuclear cytoplasm. PaCSs were also found in dysplastic foci observed in two such cases (Figure 6E and e). In H. pylori-positive mucosa, PaCSs were significantly less represented in epithelial cells showing cytotoxic changes like increased cell desquamation with minute erosions, severe irregu- larity of the luminal surface alternating cell bulging with intercellular luminal clefts, altered cell polarity, focal micropapil- lary hyperplasia of mucin-poor elongated cells, cytoplasmic vacuoles and autophagosomes (Figures 1C and 4A, B and C), compared with well-polarized cells lacking signs of severe epithelial damage (Figures 1A, B, B1 and 3A) and often coexisting in the same case or even in the same mucosal specimen (Figure 1C). Confocal microscopy of BCF-treated epithelial cells in culture Parallel confocal microscopy tests on BCF-treated HeLa or AGS cells confirmed ultrastructural findings by showing VacA in PaCSs, characterized by their concentration of ubiquitinated (i.e., FK2-reactive) proteins (Figure 6F). Indeed, in agreement with previous findings [7,22], a large amount of VacA was associated to cytoplasmic vacuoles which, on the contrary, lacked ubiquitinated proteins (Figure 6F). PaCS identification at confocal microscopy level was further confirmed by additional colocalization of H. pylori and PaCS proteasome (Figure 7A), which, like VacA colocalization, was essentially due to BCF treatment (Figure 7B). Interestingly, after 24 h incubation with H. pylori BCF, HeLa cells still showed a proteasome-negative 45% fraction of FK2-reactive spots (Figure 7B). This fact, which is paralleled by a similar behavior of VacA/FK2 colocalization, may be explained by the time- dependence development of both FK2- (Figure 7C) and protea- some-reactive (not shown) spots. It seems likely that the whole process of PaCS formation is time-dependent, with final recruitment of the proteasome to the aggregate of bacterial products and ubiquitinated proteins. The role of H. pylori and its products in PaCS formation is further stressed by the enhanced colocalization between ubiquitinated proteins and proteasome in BCF-treated cells compared to untreated control cells (Figure 7B) and by the observation that colocalization between ubiquitinated proteins and NOD1 receptor was present only in BCF-treated cells (Figure 7D). Discussion In this study a novel cytoplasmic structure is described in H. pylori- colonized human gastric epithelium which is essentially character- ized by accumulation of regularly spaced, 13-nm-thick, elongated particles inside a relatively clear, amorphous to filamentous background, to form a distinct cytoplasmic area surrounded by RER. Such a particle-rich cytoplasmic structure (PaCS) showed selective reactivity for a) bacterial products like CagA, VacA, urease and OMPs, sometimes coupled with H. pylori bodies or remnants, b) NOD1 receptor for bacterial peptidoglycans, c) E1 ubiquitin- activating enzyme and polyubiquitinated proteins, and d) several proteasome components, including the 20S core subunits, 19S-S2 subunit, and 20S-b5i subunit (also known as LMP7, characterizing the INFc-stimulated immunoproteasome). PaCSs apparently arise in the juxtaribosomal cytoplasm interposed between RER cisternae, at sites of bacterial products accumulation, and progressively grow by accumulation of newly formed particles, while pushing aside cytoplasmic organelles. In thin sections analysed at high resolution, some of the PaCS particles showed ultrastructural resemblance to the structure reported in vitro by X-ray analysis of the proteasome complex [25–27], with which they are likely to be identified, as also suggested by their direct immunoreactivity for proteasomal components. Previous immunogold studies documenting a close topographic correlation between proteasome immunoreactivity and RER [29] may also support this conclusion, given present separation of phosphorous-positive, proteasome-negative, RER- associated ribosomes from the phosphorous-negative, proteasome- immunoreactive 13-nm particles collected inside PaCS. PaCSs were more prominent in the basal cytoplasm of superficial-foveolar cells showing H. pylori colonization on their luminal side. Vesicular endosomes, often storing VacA, were seen to accumulate in the apical cytoplasm of colonized epithelium and to fuse each other to form VacA-storing vacuoles, as already shown in vitro [7,30] and in vivo [9,17]. Both electron and confocal microscopy of human gastric biopsies or cultured epithelial cells clearly separated PaCS from VacA-storing vacuoles and phago- somes, despite their common toxin content. In addition, minute, free CagA deposits were seen in the cytoplasm just underlying H. pylori adhesion sites. How both cytosolic (CagA) and endocytosed (VacA and OMPs) bacterial products reach deeply situated PaCS (an essentially cytosolic compartment) after entering the apical part of the cell, remains to be clarified. PaCSs were also observed in biopses from cases showing no H. pylori at routine light microscopy investigation of paraffin sections. PLoS ONE | www.plosone.org 9 March 2010 | Volume 5 | Issue 3 | e9716