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 immunoperoxidase [2] (
link), [17] (
link). Semithin (0.5 µm) resin sections were stained with toluidine blue, while ultrathin sections were stained with uranyl-lead or the immunogold procedure [2] (
link), [9] (
link), using antibodies against: a)
H. pylori OMPs, urease, CagA and VacA, b) NOD1 receptor, c) E1A/B ligases, polyubiquitinated or mono/polyubiquitinated proteins, 20S proteasome core subunits, 19S proteasome S2 subunit, and 20S proteasome β5i 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] (
link), [9] (
link).
ESI analyses were performed by using a LEO 912AB electron microscope as described by Pezzati
et al.[53] (
link). 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] (
link) 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 1% Vitox (Oxoid, Basingstoke, UK) and 5% fetal bovine serum (FBS; Gibco, Grand Island, NY) for 24–36 h at 37°C under microaerobic conditions and continuous shaking. Bacteria were then removed by centrifugation and the supernatant sterilized by passage through a 0.22 µm 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% β-cyclodextrins (Sigma, St Louis, MO) instead of FBS, by ammonium sulphate precipitation and gel filtration chromatography in accordance with Cover
et al.[54] (
link). 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] (
link). Cultured cells were incubated with 2 µg/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 37°C in a humidified atmosphere of 5% CO
2 in air. After washing, subconfluent cell monolayers were incubated at 37°C 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 processed for immunofluorescence as previously described [8] (
link), [22] (
link) 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).
Necchi V., Sommi P., Ricci V, & Solcia E. (2010). In Vivo Accumulation of Helicobacter pylori Products, NOD1, Ubiquitinated Proteins and Proteasome in a Novel Cytoplasmic Structure. PLoS ONE, 5(3), e9716.