Intracellular, Intercellular, and Stromal Invasion of Gastric Mucosa, Preneoplastic Lesions, and Cancer by Helicobacter pylori
2007; Elsevier BV; Volume: 132; Issue: 3 Linguagem: Inglês
10.1053/j.gastro.2007.01.049
ISSN1528-0012
AutoresVittorio Necchi, Maria Elena Candusso, Francesca Tava, Ombretta Luinetti, Ulderico Ventura, Roberto Fiocca, Vittorio Ricci, Enrico Solcia,
Tópico(s)Veterinary medicine and infectious diseases
ResumoBackground & 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 immune-inflammatory 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 bacteria-like 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. 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 immune-inflammatory 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 bacteria-like 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. See editorial on page 1177. 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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. 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 Epon-Araldite 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.3Fiocca R. Luinetti O. Villani L. Chiaravalli A.M. Capella C. Solcia E. Epithelial cytotoxicity, immune responses, and inflammatory components of Helicobacter pylori gastritis.Scand J Gastroenterol. 1994; 29: 11-21Crossref PubMed Scopus (97) Google Scholar, 34Andersen L.P. Holck S. Povlsen C.O. Campylobacter pylori detected by indirect immunohistochemical technique.APMIS. 1988; 96: 559-564Crossref PubMed Scopus (62) Google Scholar Fourteen cases were H pylori positive, and 6 were negative on light microscopy investigation. Semithin (0.5 μm) resin sections were stained with toluidine blue, whereas ultrathin sections were contrasted with uranyl-lead and the immunogold procedure as previously reported,35Sommi P. Ricci V. Fiocca R. Necchi V. Romano M. Telford J.L. Solcia E. Ventura U. Persistence of Helicobacter pylori VacA toxin and vacuolating potential in cultured gastric epithelial cells.Am J Physiol. 1998; 275: G681-G688PubMed Google Scholar, 36Fiocca R. Necchi V. Sommi P. Ricci V. Telford J. Cover T.L. Solcia E. Release of Helicobacter pylori vacuolating cytotoxin by both a specific secretion pathway and budding of outer membrane vesicles Uptake of released toxin and vesicles by gastric epithelium.J Pathol. 1999; 188: 220-226Crossref PubMed Scopus (202) Google Scholar 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 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.35Sommi P. Ricci V. Fiocca R. Necchi V. Romano M. Telford J.L. Solcia E. Ventura U. Persistence of Helicobacter pylori VacA toxin and vacuolating potential in cultured gastric epithelial cells.Am J Physiol. 1998; 275: G681-G688PubMed Google Scholar, 36Fiocca R. Necchi V. Sommi P. Ricci V. Telford J. Cover T.L. Solcia E. Release of Helicobacter pylori vacuolating cytotoxin by both a specific secretion pathway and budding of outer membrane vesicles Uptake of released toxin and vesicles by gastric epithelium.J Pathol. 1999; 188: 220-226Crossref PubMed Scopus (202) Google ScholarTable 1Characteristics of Antibodies UsedAntigenCodeAntibodiesSourceWorking dilutionATCC 43504 strain lysateV4074RP, 1.5 mg protein/mLaH pylori specific, H. heilmannii-negative in light microscopy tests.A1:200CH 20426 strain lysateB0471RP, 0.34 mg/mL IgbReactive with both H pylori and H heilmannii.B1:200CagA, recombinantHPP-5003-9RP, IgG 2 mg/mLC1:20CagA, 1–300, recombinantsc-25766RP, IgG 200 μg/mLD1:10VacA, recombinantHPP-5013-9RP, IgG 0.7 mg/mLC1:100VacA, purified123RP, whole serumE1:300NOTE. 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.a H pylori specific, H. heilmannii-negative in light microscopy tests.b Reactive with both H pylori and H heilmannii. Open table in a new tab 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. 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 described37Ricci V. Sommi P. Fiocca R. Romano M. Solcia E. Ventura U. Helicobacter pylori vacuolating toxin accumulates within the endosomal-vacuolar compartment of cultured gastric cells and potentiates the vacuolating activity of ammonia.J Pathol. 1997; 183: 453-459Crossref PubMed Scopus (77) Google Scholar 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, Vanderbilt University, Nashville, TN).38Cover T.L. Tummuru M.K.R. Cao P. Thompson S.A. Blaser M.J. Divergence of genetic sequences for the vacuolating cytotoxin among Helicobacter pylori strains.J Biol Chem. 1994; 269: 10566-10573Abstract Full Text PDF PubMed Google Scholar, 39Tummuru M.K.R. Cover T.L. Blaser M.J. Mutation of the cytotoxin-associated cagA gene does not affect the vacuolating cytotoxin activity of Helicobacter pylori.Infect Immun. 1994; 62: 2609-2613Crossref PubMed Google Scholar 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.40Fiocca R. Cornaggia M. Villani L. Capella C. Solcia E. Samloff I.M. 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Klersy C. Grillo F. Pandolfo N. Fiocca R. Identification of a lower grade muconodular subtype of gastric mucinous cancer.Virchows Arch. 2004; 445: 572-579Crossref PubMed Scopus (12) Google Scholar 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 anti-VacA 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.Figure 2Western 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 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-5003-9 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) With both anti-VacA antibodies, immunoreactivity was localized within the periplasmic space and the outer membrane of bacterial bodies, flagella, and outer membrane vesicles (OMVs) (Figures 1C and 1E and 3D), in accordance with previous findings.35Sommi P. Ricci V. Fiocca R. Necchi V. Romano M. Telford J.L. Solcia E. Ventura U. Persistence of Helicobacter pylori VacA toxin and vacuolating potential in cultured gastric epithelial cells.Am J Physiol. 1998; 275: G681-G688PubMed Google Scholar, 36Fiocca R. Necchi V. Sommi P. Ricci V. Telford J. Cover T.L. Solcia E. Release of Helicobacter pylori vacuolating cytotoxin by both a specif
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