However, in many such cases TEM detected intracellular H. pylori bodies or their remnants, more frequently in the supranuclear cytoplasm and enveloped by a host membrane. Occasionally, they were also found in RER-rich basal cytoplasm, usually devoid of host enveloping membrane while directly contacting UPS-reactive 13-nm particles to form a PaCS. Such a pattern supports the involvement of H. pylori and/or its products in the genesis of at least a part of the PaCSs in vivo, while fitting with PaCS neogenesis and expansion in cultured cell lines incubated with H. pylori BCF. The colocalization of H. pylori products with NOD1 inside the PaCS is especially interesting in view of the demonstration that this receptor has a crucial role in sensing H. pylori PG to elicit NF-kB activation and chemokine-dependent proinflammatory response [6]. We also colocalized with NOD1 in PaCS both OMPs, known to be linked to PGs and LPS inside H. pylori outer membrane and related vesicles [9,10], and a moderately acidic polyanion conferring selective metachromatic basophilia of the type displayed by carboxylated glycoconjugates like PGs [23,24]. Of major interest is also the colocalization of bacterial products with ubiquitin-activating enzyme E1, polyubiquitinated proteins and proteasome components since interactions between bacterial toxins/virulence factors and the UPS of infected cells are well- known to occur [14,31,32] resulting in a modulated host cell response to bacteria. As shown for Salmonella typhimurium, when intracellular bacteria escape from their membrane-enclosed invasion vacuole to directly contact host cell cytosol, the ubiquitin system may also have a role in recognizing and degrading them through selective proteasome recruitment into peribacterial cytosol [13]. These findings are reminiscent of the H. pylori bodies showing various degenerative changes that we found lying free inside some PaCSs of human gastric mucosa, in direct contact with UPS components. Direct interaction of H. pylori or its products and UPS may underscore an attempt by colonized epithelial cells to dispose (or modulate activity) of bacteria and their toxins/virulence factors. Bacterial products/UPS colocalization was also observed inside minute collections of proteasome-like particles first appear- ing in the cytoplasm adjacent to RER-bound or free polyribo- somes, a likely site of origin of PaCSs. This finding may suggest that UPS and the PaCS particles are newly synthesized at a ribosomal level (or preferentially redistributed there) in response to bacterial products accumulation. Indeed, immunoproteasome has been shown to increase substantially at a RER/ribosomal level after microbial incubation [33,34], resulting in proteasome- mediated bacterial antigen release associated with stimulation of class I MHC peptide neosynthesis and activation of specific immune responses [35]. In bioptic specimens, we found UPS-positive PaCSs a) in superficial-foveolar gastric epithelium, especially when showing heavy luminal H. pylori colonization in the absence of cytotoxic changes, b) in mucin-poor goblet cells of colonic-type (type III) intestinal metaplasia, with or without dysplastic changes, showing intraepithelial (but not luminal) H. pylori, and c) albeit less frequently, in superficial-foveolar cells lacking luminal bacteria while showing sparse intracytoplasmic H. pylori. In control cultured cells, UPS-positive PaCSs were observed only occasionally and in the absence of any H. pylori product-related reactivity. Incubation of the same cell lines with H. pylori BCF induced formation, enlargement and quantitative enhancement of structures positive for H. pylori toxins/virulence factors, NOD1, ubiquitinated proteins and proteasome, which closely reproduced cytochemical and ultrastructural patterns of corresponding in vivo structures, thus confirming a significant role of H. pylori in their development. Other structures, unreactive for proteasome components, bacterial products or NOD1 and essentially formed by a H. pylori and PaCS meshwork of filamentous proteins resembling that reported by Simonsen et al. [36] and Kaganovitch et al. [37] in autophagy- related structures, were found occasionally in gastric foveolar epithelium and, more frequently, in cultured cell lines. A variety of discrete cytoplasmic structures potentially related to PaCS or filamentous structures has indeed been reported in epithelial and non-epithelial, cell lines, especially under conditions (including bacterial LPS treatment) altering the quality control of endogenous or exogenous, natural or mutated, misfolded proteins [38–42]. Among such previously described structures, only the ALIS (aggresome-like induced structure) caused by intracellular Legionella pneumophila in macrophages and dendritic cells [42] was induced by incubation with a bacterium. In that case, ALIS showed colocalization of ubiquitinated proteins and proteasome, thus resembling immunocytochemically the H. pylori-induced PaCSs, although no information on its high-resolution ultrastructure was given. The RER-associated DALIS (dendritic cell aggresome-like induced structure) [38] or ALIS [40,41] induced in vitro by bacterial LPS in dendritic cells, macrophages and epithelial cells, though rich in ubiquitinated proteins, lacked proteasome reactiv- ity. It remains to be ascertained, through further ultrastructural studies, whether PaCSs are akin to cytochemically similar structures arising in conditions of increased misfolded proteins production such as the UPS-positive JUNQ (juxtanuclear quality control compartment) [37], as infection may cause neosynthesis of misfolded proteins [43,44]. Much work remains to be done, especially at molecular level, to understand the biologic and pathologic role of PaCS. Although H. pylori infection appears to induce or enhance its formation, PaCS is not part of the severe cytotoxic changes arising in the epithelium at sites of more heavy colonization by tightly adhering bacteria. These more ‘‘acute’’ inflammatory and cytopathic effects affecting epithelium integrity and barrier function seem likely to have a direct role in the genesis of ulcerative disease [17,45]. On the other hand, epidemiologic, histopathologic and molecular studies suggest a relevant role of chronic H. pylori infection, with special reference to CagA-positive strains, in gastric carcinogenesis [2,46– 50] through a long-standing sequence of inflammatory, atrophic, metaplastic and dysplastic changes leading to intestinal-type cancer [48,51]. Recent molecular studies have clarified in part the role of CagA in this process by showing complex formation with the oncogenic protein SHP2 and activation of the ERK kinases pathway so as to elicit proliferative, morphogenetic and antiapoptotic effects of potential neoplastic relevance [19,21,28]. Our finding of selective colocalization of CagA, SHP2 and ERK kinases inside some PaCS may suggest this structure has a role in the regulation of cell growth and neoplastic transformation, also in view of its highly enriched UPS content and the mounting evidence for a role of UPS in cancer origin or progression [52]. PaCS detection, together with bacteria and their virulence factors, inside gastric precancerous lesions is in keeping with this suggestion and, more in general, with the hypothesis of a H. pylori/CagA-mediated carcinogenesis. In conclusion, it seems clear that PaCS, the novel intracellular structure where H. pylori products concentrate in vivo in colonized gastric epithelium as well as in vitro in human epithelial cell lines incubated with H. pylori products/virulence factors, is a distinctive cytoplasmic compartment where both NOD1, a selective H. pylori receptor, and UPS components are co-concentrated. This structure may have a role in bacterial recognition and handling, and may modulate the activity of toxins/virulence factors and induce pertinent immune responses, especially through immuno- proteasome. The presence of PaCSs in gastric preneoplastic lesions, and their selective colocalization with putative oncogenic PLoS ONE | www.plosone.org 11 March 2010 | Volume 5 | Issue 3 | e9716