JOURNAL OF PATHOLOGY, VOL.
161: 65-70 (1 990)
ULTRASTRUCTURAL STUDY OF HELICOBACTER PYLORI-ASSOCIATED GASTRITIS J. L. KAZI*, R. SINNIAHt, V. ZAMANt, M. L. N G t , N. A. JAFAREY*, S. M. ALAM*, S. J. ZUBERIS AND A. M. KAZIS
*Department of Pathology, Jinnah Postgraduate Medical Centre, Karachi, Pakistan: tDepartment of Pathology and Microbiology, National University of Singapore, Singapore; $Pakistan Medical Research Council, Pakistan; 6Department of Medicine, Jinnah Postgraduate Medical Centre, Karachi, Pakistan Received 17 July 1989 Accepted 22 January 1990
SUMMARY Endoscopic biopsies of antral mucosa from 26 patients with Helicobaeterpylori-associatedgastritis were studied by electron microscopy (EM). Scanning electron microscopy (SEM)showed clustering of H. pylori in the intercellular areas, being entrapped by the microvilli which were decreased at the sites where the bacilli were seen. The observations of SEM were confirmed by transmission electron microscopy (TEM),which showed adherence of the bacilli to the cell surface,producingcup-shaped depressions in the epithelial cells, and occasionally intracellularinfiltration by H.pylori. There were also depletion of mucus granules,degenerative changes, and disruption of intercellularjunction complexes of the epithelial cells. Post-treatment biopsies showed complete disappearance of the bacilli, and ultrastructural changes associated with H. pylori infection were resolved. KEY
pylori-associated gastritis, scanning electron microscopy, transmission electron microscopy, posttreatment changes.
INTRODUCTION Helicobacter pylori (H. pylori), first isolated by Marshall, has been associated with gastritis and peptic ulceration.' The neonatal gnotobiotic pig model of infection by H. pylori provides substantial evidence for its pathogenicity.' However, the mechanism by which H. pylori-associated gastritis develops is unknown, and the virulence determinant of H. pylori has not been established. Several potential mechanisms by which H . pylori induces mucosal -damage include disruption of the cytoskeleton,' degradation of gastric mucus,3 and cytotoxin production by the b a ~ i l l i .The ~ , ~ aim of
this study was to determine by electron microscopy (EM) the pathogenetic effect of H. pylori on the surface epithelial structures of the stomach mucosa. MATERIALS AND METHODS
Pre- and post-treatment biopsies were taken for EM studies from 26 cases of H . pylori-associated gastritis. These cases were treated with bismuth saiicylate for 3 weeks. The post-treatment biopsies were taken within 48h of completion of treatment. Biopsies for light microscopy were fixed in 10 per cent buffered formalin. Paraffin-embedded 4 pm sections were stained with haematoxylin and eosin (H&E) for diagnosis of gastritis, and modified Giemsa stain for detecting H. pylori. Biopsies for Addressee for correspondence: Professor Raja Sinniah, EM studies were fixed in 4 per cent cacodylate Department of Pathology, National University of Singapore, National University Hospital, Lower Kent Ridge Road, buffered glutaraldehyde. Pretreatment biopsies Singapore 05 1 I. fixed in glutaraldehyde were sliced into two pieces: 0022-341 7/90/050065-06$05.00 0 1990 by John Wiley & Sons, Ltd.
J. L. KAZI ET AL.
shows a decrease of microvilli in the intercellular areas where H.pyforiare Seen (arrows). x 12 OOO
RESULTS one for scanning and the other for transmission electron microscopy (TEM). Glutaraldehyde-fixed Scanning electron microscopy of the gastric post-treatment biopsies were processed for TEM studies. mucosal biopsies showed a striking concentration of H. pylori in the intercellular areas of epithelial cells. There was a marked overall increase in microvillus Scanning electron microscopy structures, more at the periphery of the cell than at The biopsies for scanning electron microscopy the apex. In many areas, the microvilli were seen to (SEM) were fixed in 4 per cent glutaraldehyde for be evenly distributed over the cell surface, and were 16 h at 4"C,post-fixed in 2 per cent osmium tetrox- decreased at places where the bacilli were present ide, and dehydrated in a graded series of acetone. (Fig. 1). They were in close contact with the bacilli, They were critical point-dried from acetone using which appeared to be entrapped between these proliquid carbon dioxide as the exchange medium. cesses (Fig. 2), an observation which was confirmed After mounting onto stubs, the specimens were by TEM (Fig. 3). coated with gold paladium to a thickness of Transmission electron microscopic studies conapproximately 25pm and examined in a SEM at firmed the observations of SEM. There was cluster25 kV. ing of bacilli in the intercellular areas and a decrease of microvilli where the bacilli were present. There Transmission electron microscopy were several morphological changes that were conBiopsy tissue fragments were fixed in glutaralde- sistent in all specimens and in all fields where the hyde at 4°C and sliced with a razor blade to facilitate bacilli were seen. H.pylori did not show any preferorientation. Specimenswere washed twice in cacody- ential site of adherence to cell membrane, but conlate buffer and post-fixed for 1 h in 1 per cent osmium tact between the two cell surfaces was always very tetroxide solution. Specimens were dehydrated and intimate (Fig. 4), producing cup-shaped depressions embedded in epoxy resin, and thin sections were on the surface epithelial cells at most points of conmounted on bare grids. Sections were stained by a tact. The epithelial cells showed depletion of mucus standard sequential method of saturated aqueous granules and degenerative changes. The intercelluuranyl acetate for 1 h followed by lead citrate. lar junctional complexes were disrupted and the
EM OF H . PYLORI ASSOCIATED GASTRITIS
Fig. 2-SEM shows H . pylori entrapped amongst the microvilli. x 70 500
Fig. G T E M shows H.pylori with a polar flagella in close contact with the cell membrane. x 39 510
bacilli were found to be insinuated deep in-between the epithelial cells (Fig. 5). In places, the surface epithelial cells were totally disrupted by the bacilli (Fig. 6). After extensive search H. pyIoriGere found infiltrating into the epithelial cells, and were seen within the cytoplasm and in cytoplasmic vacuoles (Fig. 7). After treatment disappearance of the bacilli and reversal of the surface mucosal degenerative changes were observed. The architecture of the surface epithelium, the population of mucus granules, microvilli of the epithelial cells, and intercellular junctional complexes were restored.
Fig. 3-TEM shows a cross-section of H. p$or; entrapped between two microvilli. x 108 300
Before H . pylori was isolated, Fallah ef af. in 19766and Fung et al. in 1979’ had described scanning electron microscopic changes in chronic nonspecific gastritis. Both groups described an increase in mucosal projections which wereevenly distributed throughout the surface of the cell and many of them were much larger (and microvillus-like) than the bulbous mucosal projections with peripheral concentrations as found in normal gastric biopsies.6 Although Fallah et aL6did not observe any bacteria,
J. L. KAZI ET AL.
Fig. 5-TEM shows two bacilli insinuated between the epithelial cells (arrows). The epithelial cells show depletion of mucus granules. loss of microvilli, and degenerationofcytoplasm. x 16 300
Fig. G T E M showing disruptionof epithelial cells at the site of bacterial clustering. x 10 200
EM OF H. PYLORI ASSOCIATED GASTFUTIS
Fig. 7-TEM shows H. pylori on epithelial cell membrane and infiltrating the cell to lie in the cytoplasm and within a cytoplasmic vacuole (arrows). x 38 600
Fung et aL7 reported the presence of bacteria by TEM. Fung et aL7 graded the electron microscopic changes in chronic gastritis; grade I showed an increase in microvilli in the periphery of the cells. In grade I1 changes, microvilli were also seen in the central area and the peripheral zones, and in grade 111, microvilli were found to be evenly distributed throughout the cell surface. The increase in the number of microvilli in H . pylori-associated gastritis indicates that the cells are reacting to a stimulus, and is part of a protective responseagainst thebacilli, whichissupported by the heightened cellular and humoral immune responses seen in H . pylori-associated gastritis.* Microvilli were possibly developed as a barrier, and the close adherence of the bacilli to the cell membrane could only occur after the microvilli were damaged by the bacilli, probably by its toxins. Cantey ef al. in 19819 described the attachment of Escherichia coli strain RDEC-1 to rabbit intestinal epithelial cells in two stages. In the initial stage, bacteria attached to intact microvilli, and in the late stage, they attached to mucosal epithelial cells that had lost their microvillous border as a result of bacterial toxic products.
The segmented filamentous micro-organisms commonly found in rat ileum attach directly to epithelial cells and produce conspicuous alterations in the microvillous border of absorptive cells. These alterations include the disruption or even complete removal of microvilli from broad areas of the cell surface." In giardial infection of rat intestine, the ventral surface of giardia serves as an organ of attachment to the microvillous border. Circular dome-shaped lesions or alterations of the microvillous border of villous epithelial cells were observed interspersed among attached trophozoites of giardia." The intimate adherence of H . p-vlori to the cell membrane producing cup-shaped depressions on the surface is similar to the adherence pedestals produced by an enterocyte adherent Escherichia coli strain, which was neither enterotoxigenic nor entero-invasive and was found to be the cause of protracted diarrhoea in infants." Our TEM findings are consistent with the obserLoss of microvilli at vations of other the sites of H . pylori adhesion. depletion of mucus granules, disruption of intercellular junctional complexes, and the appearance of intracytoplasmic
J. L. KAZI ETAL.
degenerative vacuoles are possibly produced by the toxin liberated by H. pylori. Leunk et u I . showed ~ that the broth culture filtrates of H. pylori induced non-lethal cytopathic effects in vitro in seven of nine mammalian cell lines tested. TEM revealed that the response consisted of intracellular vacuolization. H. p-vlori toxin production could be involved in the development of peptic ~ l c e r a t i o n . ~ There is controversy in the literature regarding epithelial infiltration by H. pylori. Only a few workers have claimed such an invasion. MeyrickThomas et d . I 9 described the presence of bacilli within the mucus vacuoles of the epithelial cells. Buck et al.I3 also found them rarely inside the epithelial cells, and Lee et aL2*found the bacilli within the lysosome of an epithelial cell. Bode et d.I6have described the infiltration of H . pylori in 10 per cent of their patients with active duodenal ulceration. However, the invasion was seen only in metaplastic surface mucus cells of the duodenum. The bacilli were found between mucus granules, attached to vacuoles, and close to lysosome-like structures. Our ultrastructural study was limited to the gastric biopsies infected by H. pylori and did not include duodenal biopsies. In the gastric biopsies examined by us, we searched extensively for epithelial invasion by H. pylori by observing sections at several levels of each biopsy, and did see evidence of intra-epithelial invasion by the bacilli. Restoration of the normal architecture of the gastric mucosa, with the disappearance of H. pylori in post-treatment biopsies, is further evidence for the pathogenic role of H. pylori in gastric mucosal damage and gastritis. Our observations also point to the possible role of microvillus structures in H. pvlori-associated gastritis. ACKNOWLEDGEMENTS
This study was supported by grants from the National University of Singapore and Jinnah Postgraduate Medical Centre, Karachi, Pakistan. We thank Mrs Josephine, Miss Azima, and Mr John Chia of the National University of Singapore for technical assistance, and Mr Zaigham Ali and Ms Veronica Mok for secretarial assistance.
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