factors like CagA, SHP2 and members of the MAPK/ERK signaling system, add special interest to the investigation of the biologic role and pathologic potential of PaCS. Materials and Methods Biopsy samples, retrieved from our archival histology collection, were taken in the period 1981–95 from the gastric antrum and corpus of 26 subjects (15 males and 11 females, aged between 26 and 79 years) undergoing routine endoscopic and histologic examination for dyspepsia as requested by the physician in charge of the patient and with the written consent of the patient. One of the specimens from both the antrum and the corpus was processed for TEM and the pertinent semithin resin section used for diagnostic purposes together with those of routine histologic material. No specimen specifically and/or exclusively devoted to the present study was taken. The study has been approved by the Ethics Committee of Fondazione IRCCS Policlinico San Matteo (Pavia, Italy) as a reinvestigation of archival material along the same line (i.e., diagnosis of H. pylori-dependent gastritis) as for the original consensus. The samples were fixed in 4% formaldehyde and embedded in paraffin for histologic investigation, or fixed for 4 hours with 2% formaldehyde and 2.5% glutaraldehyde in 0.1 M phosphate buffer (pH 7.3), followed by 1% osmium tetroxide for 1 hour, embedded in Epon-Araldite resin and processed for TEM. Paraffin sections were stained with hematoxylin-eosin, Giemsa or H. pylori immuno- peroxidase [2,17]. Semithin (0.5 mm) resin sections were stained with toluidine blue, while ultrathin sections were stained with uranyl-lead or the immunogold procedure [2,9], using antibodies against: a) H. pylori OMPs, urease, CagA and VacA, b) NOD1 receptor, c) E1A/B ligases, polyubiquitinated or mono/polyubiqui- tinated proteins, 20S proteasome core subunits, 19S proteasome S2 subunit, and 20S proteasome b5i subunit, d) SHP2 tyrosine phosphatase, ERK 1/2 kinases, ribosomal protein S16 and other proteins (detailed in Table S1). Anti-rabbit or anti-mouse IgG labeled with 5, 10, 15 or 20 nm gold particles (British Bio Cell, Cardiff, UK) were then used. Tests to evaluate the specificity of immunogold labeling were carried out using antibodies absorbed with excess antigen and omitting or substituting the specific antibodies in the first layer of the immunogold procedure. Positive and negative controls were obtained by parallel investigation of H. pylori cultures, epithelial cell cultures, and gastric mucosa specimens as in previous studies [2,9]. ESI analyses were performed by using a LEO 912AB electron microscope as described by Pezzati et al. [53]. Briefly, the net phosphorus distribution was obtained by computer processing of images collected at different energy loss values according to the three-window method. The final phosphorus map (coded in pseudocolors) was then superimposed on the ultrastructural organization of the same field obtained at 250 eV (i.e., at an energy loss where most of the elements contribute to the image). H. pylori BCF was produced as previously described [7] using the well-characterized urease+/CagA+/VacA+ wild-type H. pylori strain 60190 (ATCC 49503). Briefly, bacteria were grown in Brucella broth (BD Diagnostics, Sparks, MD) supplemented with References 1. Chan WY, Hui PK, Leung KM, Thomas TM (1992) Modes of Helicobacter colonization and gastric epithelial damage. Histopathology 21: 521–528. 2. Necchi V, Candusso ME, Tava F, Luinetti O, Ventura U, et al. (2007) Intracellular, intercellular, and stromal invasion of gastric mucosa, preneo- plastic lesions, and cancer by Helicobacter pylori. Gastroenterology 132: 1009–1023. 3. Amieva MR, Salama NR, Tompkins LS, Falkow S (2002) Helicobacter pylori enter and survive within multivesicular vacuoles of epithelial cells. Cell Microbiol 4: 677–690. H. pylori and PaCS 1% Vitox (Oxoid, Basingstoke, UK) and 5% fetal bovine serum (FBS; Gibco, Grand Island, NY) for 24–36 h at 37uC under microaerobic conditions and continuous shaking. Bacteria were then removed by centrifugation and the supernatant sterilized by passage through a 0.22 mm cellulose acetate filter. Cultured cells were incubated with H. pylori BCF diluted 1:3 in culture medium. VacA (with a s1a/m1 vacA genotype) was purified from BCF of H. pylori 60190 strain, grown in Brucella broth containing 0.2% b- cyclodextrins (Sigma, St Louis, MO) instead of FBS, by ammonium sulphate precipitation and gel filtration chromatogra- phy in accordance with Cover et al. [54]. Purified VacA was then labeled with Cy5 dye, stored in melting ice and, immediately before use, alkali activated by drop-wise addition of 0.4 N NaOH [8]. Cultured cells were incubated with 2 mg/ml activated Cy5- VacA. Human epithelial cell lines HeLa (ATCC CCL-2; from cervix adenocarcinoma) and AGS (ATCC CRL-1739; from gastric adenocarcinoma) were grown in DMEM supplemented with 10% FBS and 200 mM glutamine at 37uC in a humidified atmosphere of 5% CO2 in air. After washing, subconfluent cell monolayers were incubated at 37uC for 24 h under the different experimental conditions. Cells were then either fixed and processed for TEM as described above or fixed with 4% paraformaldehyde, permeabilized with 0.1% saponin, and pro- cessed for immunofluorescence as previously described [8,22] using Alexa 488-labeled anti-mouse IgG (Molecular Probes, Eugene, OR) or Texas Red- or Cy5-labeled anti-rabbit IgG (Jackson Immunoresearch, West Grove, PE) as secondary antibodies. Nuclear counterstaining was made with Hoechst 33258. TCS SP2 confocal laser scanning microscope (Leica, Heidelberg, Germany) equipped with 63x oil-immersion objective was used. Ubiquitinated protein-positive (i.e., FK2-reactive) cytoplasmic spots per cell and their colocalization with VacA or proteasome were quantified using the ImageJ software (NIH, Bethesda, MD). Supporting Information Table S1 Characteristics of antibodies used for TEM and confocal microscopy. Found at: doi:10.1371/journal.pone.0009716.s001 (0.02 MB PDF) Acknowledgments The authors thank Dr. A. Oldani (Department of Physiology, University of Pavia, Italy) and Dr. M.C. Panzeri (Advanced Light and Electron Microscopy BioImaging Center, San Raffaele University, Milan, Italy) for their invaluable help in confocal microscopy and electron spectroscopy imaging, respectively. Author Contributions Conceived and designed the experiments: VR ES. Performed the experiments: VN PS VR. Analyzed the data: VN PS VR ES. Wrote the paper: VR ES. 4. Odenbreit S, Pu¨ls J, Sedlmaier B, Gerland E, Fischer W, et al. (2000) Translocation of Helicobacter pylori CagA into gastric epithelial cells by type IV secretion. Science 287: 1497–1500. 5. Stein M, Rappuoli R, Covacci A (2000) Tyrosine phosphorylation on the Helicobacter pylori CagA antigen after cag-driven host cell translocation. Proc Natl Acad Sci USA 97: 1263–1268. 6. Viala J, Chaput C, Boneca IG, Cardona A, Girardin SE, et al. (2004) Nod1 responds to peptidoglycan delivered by the Helicobacter pylori cag pathogenicity island. Nat Immunol 5: 1166–1174. PLoS ONE | www.plosone.org 12 March 2010 | Volume 5 | Issue 3 | e9716