1. Volume 132, Issue 3, Pages 1009-1023 (March 2007)
Intracellular, Intercellular, and Stromal Invasion of Gastric Mucosa, Preneoplastic Lesions, and Cancer by Helicobacter pylori 
Vittorio Necchi, Maria Elena Candusso, Francesca Tava, Ombretta Luinetti, Ulderico Ventura, Roberto Fiocca, Vittorio Ricci, Enrico Solcia 
Gastroenterology 
Volume 132, Issue 3, Pages (March 2007)
DOI: /j.gastro
Copyright © 2007 AGA Institute Terms and Conditions

2. Figure 1
Aldehyde-osmium fixed bacterial culture (A, D, and E) or gastric biopsy (B, C, F, and G); immunogold procedure; uranyl-lead counterstain, with 10 (B, C, D, and G)-, 15 (C and F)-, or 20-nm (A, E, and G) gold particles. Immunogold labeling with H pylori lysate V4074 antibodies of H pylori cultured in vitro (A, CCUG strain; original magnification, 26,000×) or colonizing gastric mucosa (B; luminal side of the epithelium; original magnification, 70,000×) shows intense reactivity of the outer membrane, its periplasmic, and, especially, its external leaflet with related appendages (pili?). Note also reactivity of membranaceous remnants from lysed bacteria (top right in A) and of flagella (top in B). Only flagella of H pylori are labeled by H pylori lysate B0471 antibodies (D; gastric lumen; original magnification, 70,000×). VacA immunoreactivity is localized within outer membrane and periplasmic space in cultured H pylori cells (E; CCUG strain, 123 serum; original magnification, 56,000×). No reactivity is found in H heilmannii by using H pylori V4074 antibodies, apart from some labeling of flagella (F; gastric lumen; original magnification, 28,000×; compare with A, B, and C). (C and G) Double immunogold procedure, performed sequentially on the 2 sides of the same ultramicrotomic section, showing colocalization of H pylori outer membrane proteins (V4074 antibodies, 10-nm gold particles) and VacA (123 antibodies, 15-nm [C] and 20-nm [G] gold particles) in a well-preserved, typically spiral H pylori (C; original magnification, 60,000×) contacting the luminal membrane of a superficial epithelial cell (bottom right) and in a lysed bacterial ghost with preserved membranes (note detached outer membrane on the right) adhering to lateral membrane plications (left and upper) in an intercellular space (G; original magnification, 63,000×; sparse V4074 reactivity marked with arrowheads).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

3. Figure 2
Western blotting analysis of H pylori antibodies. (A) Immunoblotting pattern of H pylori lysate V4074 antibodies on purified outer membrane vesicles (OMVs) from H pylori strain. Lane 1: control. Lane 2: OMVs; molecular size markers on the left. A main 50- to 54-kilodalton band and 3 other bands (of approximately 24, 28, and 75 kilodaltons) are recognized by the antibodies. All of the 4 bands are apparently H pylori-specific because they all are absent in the paired control (lane 1: uninoculated Brucella broth supplemented with 5% fetal calf serum and processed in the same manner as OMV-containing broth culture filtrate for purifying OMVs). (B) Immunoreactivity of HPP anti-CagA antibodies on whole bacterial lysates from different H pylori strains: (CagA+/VacA+; lane 1) and its isogenic mutants 60190:M22 (CagA−/VacA+; lane 2) and 60190:v1 (CagA+/VacA−; lane 3). Molecular size markers on the left. The antibodies detect an immunoblotting band at 120–130 kilodaltons, specific for CagA because it is absent when the CagA-defective strain is used.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

4. Figure 3
Immunogold staining with HPP anti-CagA (A–C) or 123 anti-VacA (D) antibodies and 20-nm gold particles. (A) CCUG H pylori culture showing reactivity inside bacterial bodies (original magnification, 60,000×). (B) An H pylori organism adhering to the lumenal surface of gastric epithelium shows 2 CagA reactive spots, one inside its body (bottom left) and the other near to an adhesion site (middle right) (original magnification, 60,000×). (C) CagA-reactive spot immediately adjacent to a tight junction between 2 epithelial cells in an endoscopic biopsy sample colonized by H pylori (not shown). Note slight dilation of interjunctional space at both tight (above) and adherens (below) junctional sites (original magnification, 117,000×). (D) Luminal cleft (l, lumen) showing 3 H pylori with VacA-reactive outer membranes, a flagellum (f), and 3 OMVs (v), one of which is contacting the luminal membrane overlying a tight junction (tj). Note also a microvillus (m) and several intracytoplasmic mucin granules (original magnification, 25,000×).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

5. Figure 4
Ultrastructure of intercellular junctions and spaces in H pylori-colonized gastric epithelium from endoscopic biopsies of the same patient. (A) Three H pylori organisms (enlarged in a; original magnification, 16,800×) lying in the deep intercellular intraepithelial space, just above the basal membrane. Note also luminal bacteria (top) overlying an apparently preserved tight junction, dilation of the underlying intercellular space, filled with lateral membrane plications, and an intraepithelial granulocyte (middle right) (original magnification, 6300×). (B) Fully detached intercellular junction (arrowhead, enlarged in b; original magnification, 25,000×) overlying a dilated intercellular space; compare with normal intercellular junctions (arrows) and spaces (original magnification, 15,000×). Note in b the mucus-filled intercellular space below (and luminal microvilli above) the detached junction (asterisk). (C and D) Dense H pylori bodies immunostained with V4074 antibodies and 15-nm gold particles in intercellular spaces. Note contact with lateral epithelial surfaces (C; original magnification, 7800×; enlarged in c; original magnification, 31,000×) and with lateral membrane (left) plications filling a dilated space (D; original magnification, 31,000×), where membranaceous remnants of lysed bacteria (bottom right) are also reactive.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

6. Figure 5
Three bacteria found in the stroma (lower part of A, B, and C) underlying gastric epithelium (upper part), immunostained with V4074 anti-H pylori (A; original magnification, 4700×; enlarged in a; original magnification, 30,200×; 15-nm gold particles), 123 anti-VacA (B; original magnification, 5600×; enlarged in b; original magnification, 44,400×; 20-nm gold particles), or HPP anti-CagA antibodies (C; original magnification, 7500×; enlarged in c; original magnification, 38,400×; 15-nm gold particles). Note the inner lamellar structure of the bacterium in a and the close relationship with stroma cells of bacteria in b and c.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

7. Figure 6
Sections of gastric mucosa immunostained with V4074 anti-H pylori antibodies using 10-nm gold particles. (A) Three bacteria are found in the deep stroma (bottom) in between a blood vessel and a stroma cell, 2 of which are enlarged in a1 and a2 to show immunogold reactivity (original magnification, 58,000×). Note the unusual shape (a product of altered bacterial segmentation?) of the a1 bacterium, which closely resembles the luminal bacterium in a (original magnification, 58,600×) also immunoreactive for H pylori antibodies. Note also the marked dilation of intercellular spaces, filled with extensively developed lateral membrane plications, in H pylori-colonized epithelium (original magnification, 5400×). (B) A bacterium, enlarged in b (52,500×) to show H pylori immunogold reactivity, is found inside a stromal vessel. Note the basal epithelium on top right (original magnification, 7500×).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

8. Figure 7
Intracellular H pylori in gastric epithelium. (A) Well-preserved H pylori organism in a cytoplasmic vacuole, enlarged in a (original magnification, 55,200×; 15-nm gold particles) to show immunoreactivity with 123 anti-VacA antibodies. Note the 2 H pylori in a luminal cleft (top left) (original magnification, 13,500×). (B) Dense, laminated bacterium in a cytoplasmic vacuole of tangentially cut epithelium, enlarged in b (original magnification, 44,500×; 20-nm gold particles) to show immunostaining with V4074 anti-H pylori antibodies. Note the dilated intercellular spaces filled with membrane plications (original magnification, 8500×).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

9. Figure 8
Structurally altered (partly lysed?) intracellular bacteria found in the subapical cytoplasm of intestinal metaplasia from nonneoplastic mucosa of cancerous stomachs, immunostained with V4074 anti-H pylori antibodies using 15-nm gold particles. (A) Small intestine-type metaplastic cell with long, regular microvilli (original magnification, 42,000×). (B) Large intestine-type metaplastic cell with short, irregular microvilli and goblet-type secretory vesicles (original magnification, 21,000×).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

10. Figure 9
Mixed gastric (foveolar cell, f) and intestinal (goblet cell) metaplasia immunostained with V4074 anti-H pylori antibodies in nonneoplastic mucosa of a cancerous stomach (original magnification, 6700×; 15-nm gold particles). Note the 4 reactive bacteria inside cytoplasmic vesicles of 2 goblet cells, enlarged in (a; original magnification, 55,200×), (b; original magnification, 53,400×), and (c; original magnification, 52,200×) to show heavy immunogold deposition over outer membranes and their appendages and flagella.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

11. Figure 10
Dysplastic (A; original magnification, 3200×; B; original magnification, 8200×) and neoplastic (C; original magnification, 10,800×; D; original magnification, 3400×) lesions from the same cancer case, immunostained with V4074 anti-H pylori antibodies and 15-nm gold particles. Note a coccoid bacterium inside the cytoplasm of a stromal cell (A; enlarged in a; original magnification, 38,400×; see also a dysplastic epithelial cell on the top) and 3 bacteria, 1 intracellular and 2 intercellular, inside dysplastic epithelium (B; the 2 intercellular bacteria enlarged in b; original magnification, 33,000×). Four intercellular (C; enlarged in c; original magnification, 41,400×) and 5 intracellular (D; partly enlarged in d; original magnification, 25,000×) bacteria are shown inside cancer tissue, all positive for the H pylori immunogold test; see also in (d) the positive membranaceous remnants of other bacteria.
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions

12. Figure 11
(A and B) Mucinous cancer showing a small group of dense bacteria inside intercellular mucus (A; original magnification, 8400×) and a spiral bacterium adhering to the luminal surface of a neoplastic microgland (B; original magnification, 9100×), enlarged in (b) (original magnification, 42,000×, 15-nm gold particles) to show immunogold reactivity with V4074 H pylori antibodies. (C) Tubular cancer with colonic-type tumor cells (see luminal glycocaliceal bodies, small arrowhead and enlarged in (c), and goblet-type secretory vesicles; original magnification, 24,000×) colonized by a dense bacterium (large arrowhead) reactive with HPP anti-CagA antibodies, enlarged in (c) (original magnification, 42,000×; 15-nm gold particles).
Gastroenterology  , DOI: ( /j.gastro )
Copyright © 2007 AGA Institute Terms and Conditions