Intracellular, Intercellular, and Stromal Invasion of Gastric Mucosa, Preneoplastic Lesions, and Cancer by Helicobacter pylori

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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,储 VITTORIO RICCI,§ and ENRICO SOLCIA‡

See editorial on page 1177. Background & Aims: It is not clear how Helicobacter pylori, an apparently extracellular pathogen colonizing the luminal side of the gastric epithelium, invariably causes an immune-inflammatory response on the stromal side of the mucosa. Penetration of H pylori into epithelial cell lines and its interaction with immuneinflammatory cells have been documented in vitro. Several investigations also showed in vivo bacterial penetration into the epithelium up to the lamina propria; however, the identification as H pylori of the bacterialike bodies observed in unchanged, metaplastic, or neoplastic mucosa remained sometimes questionable. Methods: To search for bacteria-like organisms, we used transmission electron microscopy on endoscopic biopsy specimens from 20 dyspeptic subjects and surgical specimens of neoplastic and nonneoplastic mucosa from 20 cancerous stomachs. To ascertain the H pylori nature of the organisms found, we used 6 different antibodies directed against bacterial lysates, purified vacuolating cytotoxin A, or purified cytotoxin-associated antigen A in immunogold tests. The results were compared with those of H pylori strains cultivated in vitro. Results: In nonmetaplastic gastric epithelium, cytochemically proven H pylori were detected, in the majority of cases, inside cytoplasm of epithelial cells, in intraepithelial intercellular spaces, and in underlying lamina propria, often in direct contact with immune-inflammatory cells and sometimes inside small blood vessels. Cytochemically proven H pylori were also observed inside 6 of 8 intestinal metaplasias and 9 of 20 cancers. Conclusions: H pylori penetrates normal, metaplastic, and neoplastic gastric epithelium in vivo, intracellularly, or interstitially to cause a strong immune-inflammatory response and promote gastric carcinogenesis.

H

elicobacter pylori is well-known to colonize gastric epithelium by floating in its mucinous coat or by adhering to its luminal surface. As a consequence, a

prominent immune-inflammatory response is invariably mounted in the underlying lamina propria.1–3 An intact epithelium should form a structural barrier preventing direct contact between the bacterium on the luminal side and reactive inflammatory cells on the stromal side. Therefore, to explain how a strong mucosal and systemic reaction may be elicited, H pylori-induced functional changes in the epithelium have been considered, with special reference to bacterial activation of the accessory immune competence inherent to gastric epithelium, which in turn may modulate underlying stroma cells.3–5 Among pertinent epithelial changes so far documented are de novo or increased expression of proinflammatory cytokines,4,6 proteases (such as cathepsins E, B, L, S, and D) known to be involved in antigen processing,3,5 or HLA-DR7 and costimulatory molecules such as B7-1 and B7-28 involved in antigen presentation. Such epithelial changes are likely to contribute to immune response and inflammation through a mechanism recalling in part that seen in M cells of Peyer’s patches and other lymphoid tissue-associated intestinal epithelial cells.9 –11 Several in vitro studies support a key role of direct interactions between H pylori or H pylori components/ products and immune cells such as T or B lymphocytes, dendritic cells, macrophages, monocytes, and mast cells.12–14 A number of light and electron microscopy studies— carefully reviewed by Petersen and Krogfelt,15 together with those reporting negative findings—suggested the presence of H pylori cells inside the gastric mucosa, either in epithelial cells and intraepithelial intercellular spaces or in the underlying lamina propria.16 –24 Despite these findings, H pylori is still commonly considered an essentially extracellular, noninvasive bacterium, an attitude unveiling some reluctance to accept published evidence. Among critical issues to be considered is some description of H pylori “intercellular space invasion,” where no distinction was made between bacterial Abbreviations used in this paper: CagA, cytotoxin-associated antigen A; IM, intestinal metaplasia; OMV, outer membrane vesicles; TEM, transmission electron microscopy; VacA, vacuolating cytotoxin A. © 2007 by the AGA Institute 0016-5085/07/$32.00 doi:10.1053/j.gastro.2007.01.049

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*Department of Human Pathology, University of Pavia, Pavia; ‡Pathologic Anatomy Service, IRCCS Policlinico S. Matteo, Pavia; §Department of Experimental Medicine, University of Pavia, Pavia; and 储Department of Pathology, University of Genova, Genova, Italy

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penetration of intercellular luminal clefts in between hypertrophic, luminally expanded epithelial cells (a frequently occurring noninvasive phenomenon) and true invasion of intercellular spaces delimited by basolateral membranes situated below intercellular tight junctions. Immunohistochemical studies have clearly documented bacteria in the lamina propria of 25%–30% of endoscopic biopsies also showing H pylori on the mucosal surface.20,25 Unfortunately, sometimes, insufficient specificity of the antibodies used prevented unequivocal conclusions as to the H pylori identity of observed bacteria,25,26 thus leaving still open the question as to whether H pylori itself or its products actually encounter in vivo lamina propria immune cells.27 Recently, H pylori has been shown to disrupt tight junction function in vitro, possibly through intraepithelial injection of cytotoxin-associated antigen A (CagA) protein.28 This change, also confirmed by in vivo experiments in mice,29 might open the paracellular route for bacterial penetration deep into the mucosa. In addition, H pylori has been found to be engulfed by epithelial cells in vitro,30,31 thus adding support to some ultrastructural studies showing putative bacterial bodies inside the epithelial cell cytoplasm of H pylori-colonized human gastric mucosa.18,19,22 With the exception of a single bacterial body,32 these essentially morphologic, ultrastructural observations still await direct cytochemical confirmation of bacterial identity. However, if confirmed, they may challenge the widely held belief that H pylori is an extracellular, noninvasive pathogen and may also indicate a possible intracellular route of bacterial transport to inflamed lamina propria. Of high interest are also recent investigations by Semino–Mora et al,33 suggesting the presence of intracellular and interstitial H pylori in preneoplastic as well as in neoplastic lesions of the gastric mucosa. These findings, which may substantially expand current concepts on the role of the bacterium in gastric carcinogenesis, were well supported by their light microscopy and molecular investigations, although being sometimes of difficult interpretation as to the exact nature of the bacteria-like bodies described in ultrastructural pictures.

Therefore, it seemed opportune to reinvestigate in vivo the issue of H pylori invasiveness by systematically analyzing H pylori-colonized gastric mucosa as well as its metaplastic or neoplastic changes to search for the presence of bacteria and their virulence factors or antigens in epithelial cells, intercellular spaces, and underlying stroma. In an attempt to provide new, more compelling evidence, we combined systematically transmission electron microscopy (TEM) with immunocytochemical tests using antibodies directed against H pylori and its virulence factors, vacuolating cytotoxin A (VacA) or CagA, to check directly the H pylori nature of putative bacterial bodies seen at an ultrastructural level.

Materials and Methods Biopsy samples were taken from the antrum and corpus of 20 subjects undergoing routine endoscopic and histologic examination for dyspeptic symptoms. From each biopsy site, part of the samples was fixed in 4% formaldehyde and embedded in paraffin for conventional histologic investigation, and part was fixed in a solution of 2.5% paraformaldehyde and 2% glutaraldehyde, followed by 1% osmium tetroxide, embedded in EponAraldite mixture, and processed for TEM. Alternatively, some aldehyde-fixed specimens were embedded in the hydrophilic resin LWR (London Resin Company, Basingstoke, United Kingdom). Paraffin sections were stained with H&E, alcian blue-periodic acid Schiff (AB-PAS), high iron diamine (HID), Giemsa, and the immunoperoxidase procedure using polyclonal rabbit antibodies (Dako, Glostrup, Denmark) directed against whole H pylori lysate.3,34 Fourteen cases were H pylori positive, and 6 were negative on light microscopy investigation. Semithin (0.5 ␮m) resin sections were stained with toluidine blue, whereas ultrathin sections were contrasted with uranyl-lead and the immunogold procedure as previously reported,35,36 using 6 different antibodies directed against whole bacterial lysates or highly purified VacA or CagA antigens (Table 1) and gold-labelled goat anti-rabbit IgG (British Bio Cell, Cardiff, United Kingdom). Specificity tests were carried out using antibodies absorbed with excess antigen or

Table 1. Characteristics of Antibodies Used Antigen ATCC 43504 strain lysate CH 20426 strain lysate CagA, recombinant CagA, 1–300, recombinant VacA, recombinant VacA, purified

Code V4074 B0471 HPP-5003-9 sc-25766 HPP-5013-9 123

Antibodies RP, RP, RP, RP, RP, RP,

protein/mLa

1.5 mg 0.34 mg/mL Igb IgG 2 mg/mL IgG 200 ␮g/mL IgG 0.7 mg/mL whole serum

Source

Working dilution

A B C D C E

1:200 1:200 1:20 1:10 1:100 1:300

NOTE. A: Biømeda, Foster City, CA. B: Dako A/S, Glostrup, Denmark. C: Austral Biologicals, San Ramon, CA. D: Santa Cruz Biotechnology, Santa Cruz, CA. E: Dr. T.L. Cover, Nashville, TN. RP, rabbit polyclonal. aH pylori specific, H. heilmannii-negative in light microscopy tests. bReactive with both H pylori and H heilmannii.

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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 17874 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 17874 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).

omitting the specific antibodies in the first layer of the immunogold procedure. Positive controls were obtained by parallel investigation of H pylori cultures (2 wild-type CagA⫹/VacA⫹ strains: 60190 [ATCC 49503] and CCUG 17874 [from Culture Collection, University of Göteborg]) and H pylori-colonized mucosa from previous studies.35,36 We also assessed the specificity of each antibody used by means of SDS-PAGE (under reducing conditions), followed by Western blotting, of bacterial lysates or broth culture filtrates. These were prepared as previously described37 from the CCUG 17874 and 60190 strains as well as from isogenic mutants of the 60190 strain in which vacA (60190:v1 strain) or cagA (60190:M22 strain) genes were disrupted by insertional mutagenesis (kindly supplied by T. L. Cover, Vander-

bilt University, Nashville, TN).38,39 The enhanced chemiluminescence revelation system (ECL Advance; Amersham Biosciences, Amersham, United Kingdom) was used. Samples of neoplastic tissue and nonneoplastic mucosa taken during surgery from 20 cancerous stomachs were also processed for light and electron microscopy using the same procedures as for bioptic specimens. In nonneoplastic mucosa, intestinal metaplasia (IM) was detected in light microscopy specimens from 12 of the cases and in TEM specimens from 7 cases. Histologic types and cellular phenotypes (including reactivity for MUC2, MUC5, MUC6, pepsinogen II, and CAR5 antigen) of preneoplastic and neoplastic lesions were characterized according to previously reported histologic, ultrastructural, and cytochemical criteria.40 – 44

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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 60190 H pylori strain. Lane 1: control. Lane 2: OMVs; molecular size markers on the left. A main 50- to 54kilodalton 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 HPP5003-9 anti-CagA antibodies on whole bacterial lysates from different H pylori strains: 60190 (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.

Results Characterization of Antibodies Used and Reactive Bacterial Bodies Observed In aldehyde-osmium-fixed bacteria, either cultured or colonizing gastric mucosa, both antibodies directed against H pylori lysates reacted with flagella, whereas only the V4074 antibody reacted with the bacterial body by nicely depicting its outer membrane (Figure 1A–D). Only in aldehyde-fixed, LWR resin-embedded specimens did the B0471 antibody react with bacterial bodies, with a preference for their inner part. Western blotting analysis of bacterial lysates with the 2 antibodies showed a main reactive band at 50 –54 kilodaltons. In addition, B0471 antibodies detected bands at 37, 70, and 110 kilodaltons that were not shown by V4074 antibodies. On the other hand, with V4074 antibodies, enriched outer membrane vesicle preparations showed, in addition to the 50- to 54-kilodalton band, other bands of 24, 28, and 75 kilodaltons that were not labelled by B0471 antibodies (Figure 2A). None of these bands reacted with specific antiVacA or anti-CagA antibodies. It is clear from both immunocytochemical and immunoblotting investigations that the 2 antibodies against bacterial lysates recognize partly different antigens or antigenic epitopes, which are, however, different from VacA or CagA. With both anti-VacA antibodies, immunoreactivity was localized within the periplasmic space and the outer membrane of bacterial bodies, flagella, and outer mem-

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brane vesicles (OMVs) (Figures 1C and 1E and 3D), in accordance with previous findings.35,36 By immunoblotting of both H pylori lysates and broth culture filtrates, the 2 antibodies showed a clear-cut 90-kilodalton band that was specific for VacA because it was absent when the VacA-defective strain was tested and clearly different from the bands labelled by V4074 or B0471 anti-H pylori antibodies (not shown). CagA immunoreactivity was localized ultrastructurally to discrete bacterial body spots by both antibodies, including those corresponding to bacterial/cell adhesion sites (Figure 3A and B). Heavier immunogold labellings were obtained with HPP-5003-9 antibodies. The 2 antiCagA antibodies detected an identical immunoblotting band at 120 –130 kilodaltons that was specific for CagA because it was absent when the CagA-defective strain was used (Figure 2B) and clearly different from the bands recognized by all other antibodies tested. In addition to well-preserved bacteria, we observed H pylori degenerative forms in bacterial cultures as well as in mucosal specimens. In both conditions, together with some ultrastructural peculiarity of the bacterium, they retained at least some H pylori-associated immunoreactivities. This allowed us to identify correctly the bacteria, even when their morphology was greatly altered compared with typical spiral forms: densified, fibrillar, laminated, vacuolated, conglutinated, fragmented, or lysated so as to retain empty membranes only (Figure 1G and Figures 4 – 6). Compared with cultured or luminal bacteria, individual immunogold reactivites of invasive bacteria found in deeper mucosal strata or inside tumor tissue often showed changes in distribution and intensity (Figures 1 and 4 – 6). These included loss of peripheral membrane distribution of VacA and V4074 antibody reactivity or reduced density of CagA and VacA immunogold deposition. However, simultaneous or sequential application of the various antibodies to the same (Figure 1C and 1G) or adjacent sections, through the same bacterium, allowed a confident assessment of the H pylori nature of most invasive bacteria. The H pylori specificity of our immunogold tests, using anti-VacA, anti-CagA, or V4074 H pylori antibodies, is also supported by their lack of cross-reactivity (apart from sparse flagellar labelling by the V4074 antibodies) with Helicobacter heilmannii (Figure 1F), despite its known immunologic similarity to H pylori, which was confirmed by its reactivity with B0471 anti-H pylori antibodies on light microscopy.26

Endoscopic Biopsies The presence of H pylori adhering to foveolar epithelium or floating in its mucinous coat was confirmed by TEM investigation of all 14 endoscopic cases in which the bacterium was also detected by light microscopy. TEM did not detect luminal bacteria in 5

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Figure 3. Immunogold staining with HPP-5003-9 anti-CagA (A–C) or 123 anti-VacA (D) antibodies and 20-nm gold particles. (A) CCUG 17874 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⫻).

of the 6 cases negative on light microscopy, whereas occasional bacteria were observed in the remaining case. Intraepithelial intercellular bacteria were found in 11 cases, all of which also showed luminal bacteria. Often, bacteria-filled spaces were in fact luminal clefts delimited by luminal-type cell membranes with sparse, short microvilli and closed at the bottom by tight junctions (Figures 3D, 4A, and 7A). Cytoplasmic extrusion into the lumen caused by cellular edema might explain such a condition. However, a prominent depth asymmetry was frequently observed between clefts on opposite sides of the same cell, a finding hardly explained by cellular edema, and possibly accounted for by local tight junction displacement/detachment and remodelling under bacterial influence. In addition, H pylori were also detected in true intraepithelial intercellular (lateral) spaces situated below a tight junction, from just below the junction to

just above the basal lamina (Figure 4A, C, and D). Although the bacteria were often in direct contact with lateral membranes of epithelial cells, no special adhesion structures, such as pedestals or apparent membrane fusions, were observed. Junctional systems overlying intercellular spaces penetrated by bacteria were sometimes altered ultrastructurally because of the detachment of juxtaposed membranes or reduced adhesion area. However, the observed patterns suggest that reassembly of junctional membranes may occur after bacterial passage. Only rarely were the junctions completely detached, thus leaving open communication between the lumen and intercellular space (Figure 4B). Luminal mucus was occasionally found to penetrate deeply into the intercellular space (Figure 4C). Immunoreactivity over tight junctions or nearby cytoplasm was more often detected with CagA than VacA antibodies; on the other hand, VacA-positive bacteria and OMVs were

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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.

often found close to the luminal end of tight junctions (Figure 3C and D). Spiral, coccoid, or irregularly shaped bacteria, often densified or with various kinds of degenerative patterns (lysed, lamellated, fused to each other, or even fragmented), were found in the lamina propria of 8 cases, all also showing H pylori in the lumen as well as in intercellular spaces (Figure 5). Correct identification of bacteria and their degenerative forms was based on immunocytochemical reactivity to anti-H pylori, anti-VacA, and/or anti-CagA antibodies, in addition to ultrastructural homology with corresponding luminal or cultured bacteria and related degenerative bodies (Figure 6A). H pylori and its remnants were observed in the interstitial stroma, often in close contact with immunocompetent and inflammatory cells (lymphocytes, plasma cells, dendritic

cells, macrophages, granulocytes) and sometimes inside such cells. In 4 cases, a few H pylori immunoreactive, spiral, or coccoid bacteria were also detected inside the lumen of blood capillaries, either adhering to the luminal surface of endothelial cells or lying free in the vessel lumen (Figure 6B). Intracellular H pylori bacteria were detected in 9 cases, all also showing luminal bacteria. Intracellular H pylori were scattered mainly in the supranuclear cytoplasm, inside endosomal vacuoles or multivesicular bodies, or enveloped by a closely apposed membrane, or even lying apparently free in the cytoplasm (Figure 7). Their shape ranged from well-preserved spiral forms (occasionally with flagella) to round coccoid bodies and irregular dense bodies whose bacterial origin was sometimes recognized only by their reactivity to immunocytochemical tests.

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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-5003-9 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.

Only a single biopsy from a dyspeptic patient revealed an area of IM on TEM. This was composed of goblet cells interspersed between columnar cells with well-developed, regular microvilli, thus closely resembling small intestine epithelium. Only 2, nonadhering H pylori were found in the lumen of such a “complete type” metaplastic area, which also showed rare bacteria in intercellular spaces and none in the underlying stroma. Bacteria and their remnants were found more often in the supranuclear cytoplasm of columnar cells. In nonmetaplastic mucosa from the same patient, we found scarce luminal or intercellular and no intracellular or stromal bacteria (data not shown).

Cancerous Stomachs Specimens of nonmetaplastic mucosa were taken from 11 cancerous stomachs, 9 of which showed patterns of H pylori distribution in the lumen, intercellular space, stroma, and cell cytoplasm, resembling those of biopsy specimens from dyspeptic patients. Metaplastic mucosa was observed in 7 cases, which was mainly of a “complete” small intestine type in 3 cases (Figure 8A) and of an

“incomplete” type with goblet cells interspersed between cells showing foveolar cell-type granules (ie, a mixed, gastric/intestinal epithelium) in 2 cases (Figure 9). In the remaining 2 cases, we found mainly “colonic-type” metaplasia (Figure 8B), with abundant sulfomucin-positive goblet cells separated by columnar cells; in addition to short, sparse microvilli with long cytoplasmic roots of filaments, both cell types showed luminal glycocaliceal bodies and the apical dense or vesicular bodies described in previous papers as distinctive of colorectal epithelium and related growths45 and characterized by their reactivity with CAR-5 antigen.41 H pylori or its degenerative remnants were observed in 5 of the 7 IM cases, more frequently inside the supranuclear cytoplasm of goblet or columnar cells and in lateral intercellular spaces but rarely on the luminal side. In general, bacteria were found inside all types of IM, with special reference to incomplete, mixed, or colonic-type metaplasia (Figures 8 and 9). Purely morphologic, ultrastructural screening of the 20 cancers (3 specimens each) revealed bacteria-like bod-

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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⫻).

ies in 11 cases. Reinvestigation of pertinent tissue blocks with immunogold procedures and antibodies directed against whole H pylori lysates or specific virulence factors confirmed the H pylori identity of putative bodies in 9 cases. Associated dysplastic changes were found in 3 cases, 2 of which with bacteria (Figure 10A and 10B). Bacteria and related degenerative remnants were detected in both diffuse and glandular cancers, more frequently inside tumor cell cytoplasm, intracellular vacuoles, or intercellular spaces and, less frequently, in the lumen of glandular structures or in tumor stroma (Figures 10C and 10D and 11). Among tumor cell phenotypes affected by H pylori colonization were those reminiscent of immature goblet cells (ie, columnar, MUC2-positive cells with sparse secretory vesicles) and colorectal epithelium (ie, characterized by irregular, short microvilli with long cytoplasmic roots, glycocaliceal bodies, CAR5-positive apical dense or vesicular bodies). Bacterial coloniza-

tion also involved foveolar-type tumor cells, showing collections of secretory granules with a cerebroid-punctate ultrastructure, and mucopeptic cells (ie, pepsinogenreactive cells with double structure, often targetoid granules) tumors. Cancerous tubules mimicking small intestine epithelium were more rarely colonized by H pylori.

Discussion In this study, H pylori was unequivocally detected in intraepithelial, intercellular, and stromal sites of the majority of gastric biopsy specimens also showing bacteria on their luminal side. Ultrastructural similarity with well-characterized spiral, coccoid, or degenerative forms found in H pylori cultures or mucosal lumen, and specific immunogold reactivity with different antibodies directed against H pylori or its main

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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⫻).

virulence factors, confirmed the identity of observed bacteria and related degenerative forms or fragments. In keeping with recent in vitro demonstration that H pylori can functionally disrupt tight junctions, possibly through intraepithelial delivery of CagA,28,46 we found CagA accumulation over and around tight junctions of colonized gastric epithelium as well as ultrastructural alterations of the tight junctions covering intercellular spaces penetrated by H pylori cells. VacA toxin, found to increase paracellular permeability to small molecules (molecular weight, ⬍400) when added to the apical side of polarized epithelial cell monolayers,47 was not consistently detected over tight junctions or nearby cytoplasm; however, VacA-positive bac-

teria and related OMVs were frequently found in close contact with the epithelial luminal membrane covering the junctions. Our in vivo findings support the hypothesis that functional and structural alterations of the tight junctions may open the way to bacterial penetration into deep intercellular spaces, up to the underlying lamina propria. In fact, we observed a correlation between the presence of H pylori in the lamina propria and in the overlying intercellular intraepithelial spaces. Consistent transepithelial penetration of H pylori into infected gastric mucosa may help to understand how the colonized mucosa invariably mounts a prominent local immune-inflammatory response,1–3 as well as a systemic

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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 goblettype secretory vesicles (original magnification, 21,000⫻).

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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.

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.

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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-5003-9 anti-CagA antibodies, enlarged in (c) (original magnification, 42,000⫻; 15-nm gold particles).

immune reaction,48 and, under special genetic conditions, even extragastric autoimmune pathologies.49 –51 In fact, direct contact (including intracellular penetration in some cases) between H pylori or its remnants and stroma cells such as lymphocytes, plasma cells, dendritic cells, macrophages, and granulocytes has been observed. Occasionally, H pylori-immunoreactive bacteria have also been detected inside blood capillaries, a finding that may explain the H pylori bacteremia detected occasionally.52 Indirect interaction between luminal H pylori and mucosal immune-inflammatory cells also remains possible through bacterial activation of the accessory immune competence inherent to gastric epithelium. Bacteria or bacterial antigens uptake and processing by pertinent cathepsins, followed by presentation, in combination with class II HLA and B7 costimulatory molecules, to professional immunocompetent cells recruited in the lamina propria3,5,7,8 as well as de novo or enhanced expression of proinflammatory cytokines4,6 are among documented epithelial changes. A suggestive analogy with the function of Peyer’s patch M cells9 –11 is further stressed by current confirmation that H pylori cells may enter epithelial cell cytoplasm. However, we found that

bacteria, as well as potential antigen transport vesicles, are mostly restricted to the supranuclear cytoplasm in gastric epithelium, unlike in M cells. This finding also makes an intracellular route of H pylori cell/antigen transport to the lamina propria less likely. On the other hand, the capacity of dendritic cells of traversing intestinal epithelium to sample luminal bacteria or their antigens and transport them to the lamina propria or lymph nodes53 remains to be proven to also work in human gastric mucosa. Unequivocal, morphologic as well as cytochemical detection of H pylori (often well preserved, VacA and CagA storing and apparently vital) inside intact epithelial cells of human gastric mucosa confirms the intracellular pathogen potential of the bacterium. This is in keeping with the expression of Nod1, a known intraepithelial pathogen-recognition molecule, by H pylori-colonized epithelial cells54 as well as with the predominantly T-helper 1 type of immune response elicited.5,13 In addition, confirmation of the intracellular occurrence of H pylori is crucial when choosing appropriate antibiotics for bacterial eradication as well as when monitoring their in vivo effectiveness after completion of treatment.24,55,56 That

the bacterium can retain at least in part its vitality and bioactivity is supported by the demonstration of persistent intracellular expression of H pylori virulence genes.33 The presence of H pylori in IM is particularly interesting and has already been suggested by a few light and electron microscopy investigations.33,57,58 This finding substantially expands the field of a possible interaction between H pylori and IM in gastric carcinogenesis, from an early preneoplastic step corresponding to epithelial progenitors59 and IM genesis to the whole process of its progression to neoplasia. Indeed, it supports a persistent in vivo activity of several H pylori-activated molecular mechanisms of gastric carcinogenesis recently proposed. These include CagA-mediated intracellular disruption of growth regulation,60,61 inflammation-elicited nuclear factor-␬B transcription factor activation,62 and silencing of DNA mismatch repair genes.63 H pylori occurrence in dysplastic as well as in neoplastic growths, as recently proposed33 and confirmed by the present ultrastructural immunocytochemical investigation, may further substantiate and expand H pylori contribution to gastric carcinogenesis and help explain some beneficial effects of bacterial eradication on IM-related cancer risk64,65 as well as on cancer recurrence.66

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Received September 28, 2006. Accepted November 30, 2006. Address requests for reprints to: Vittorio Ricci, MD, PhD, Department of Experimental Medicine, Section of Human Physiology, University of Pavia Medical School, Via Forlanini 6, 27100 Pavia, Italy. e-mail: [email protected]; fax: (39) 0382 987664. Supported in part by grants from the Italian Ministry of Health to IRCCS Policlinico S. Matteo, from the University of Pavia (Fondo d’Ateneo per la Ricerca; to E.S. and U.V.), and from the Italian Ministry for University and Research (to V.R. and U.V.) (Progetti di Ricerca di Interesse Nazionale 2004065448_002 and 2004064334_003, respectively). V.R. and E.S. share last authorship.

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