Ultrastructural observation on relation of H. pylori to gastric epithelia in chronic gastritis and peptic ulcer

Shan Min Yang,Bing Zhen Lin,Ying Fang,Yun Zheng

Subject headings gastritis; peptic ulcer; Helicobacter pylori

Yang SM,Lin BZ,Fang Y,Zheng Y.Ultrastructural observation on relation of H. pylori to gastric epithelia
in chronic gastritis and peptic ulcer.China Nati J New Gastroenterol, 1996;2(3):152-154

Abstract

 AIM  The relationship between Helicobacter pylori (Hp) and gastric epithelia in chronic gastritis and in petic ulcer was studied by transmission electron microscopy (TEM).

METHODS  Seventy-five gastric antral biopsy specimens from the patients examined by six other methods for Hp were fixed in glutaraldehyde and treated with tanin acid before OsOa-4 staining, then routinely processed for TEM studies (at least 4 semi- thin sections oriented for ultrathin sections in each sample).

RESULTS  The bacilli were detected by TEM within gastric mucosa in 53 of 55 patients infected with Hp. Ultrathin sections especially stained with tanin acid revealed clearly glycocalyx by which the bacillus was connected with the epithelium. As the bacilli grouped in colony and bred, the adjacent mucous cells degenerated and were characterized by erosion of the juxtaluminal cytoplasm, vacuolation or blebs, even desquamation of cell. Evidence was accumulated showing that the bacilli located in the lumen attracted neutrophils which intended to migrate into intercellular space of epithelia or into the lumen to exert the effect of Hp phagocytosis.

CONCLUSION  The sensitivity and specificity of  TEM diagnosis is 96% and 95% respectively. Tanin acid is suitable for the preservation of glycocalyx of cell. The colonized bacilli, usually with the wide periplasmic pools, contributed to the spectrum degeneration of epithelia, including mucous neck cells. If Hp infection persists, the degeneration and regeneration of mucous neck cells occur alternatively and ultimately the generative stem cells were damaged, as a result, chronic atrophic gastritis could occur.




INTRODUCTIONª¥
Ultrastructural studies have demonstrated that there was loss of apical microvilli and depletion of mucin granules where ª«H. pylori were seen^£Û1,2£Ý. Further investigation on Hp adherence, cell penetration, immune response of gastric epithelia is definitely needed to delineate the relationship between the bacilli and the epithelia or the monocytes.ª¤ª¤

MATERIALS AND METHODS
Biopsies of gastric antrum (4 biopsies) and body (1 biopsy) and serum from 75 patients with chronic gastritis or peptic ulcer were studied individually by culture, histopathology, cytology of smear, Hp rapid analytical chemistry urease kit (Hp-RACU), Hp enzyme linked immunosorbent analysis kit (Hp-EIA), and Hp polymerase chain reaction kit (Hp-PCR) £ÛMade by Cancer Research Center, Xiamen University£Ý. Positivity in more than three methods are considered as good standard for detection of ª«H. pyloriª« infection. One of antral biopsies was also examined by a JEM 100CX/¢ò transmission electron microscope. Biopsies were prefixed in 2.5% glutaraldehyde, treated for four hours in 1% tanin acid (except for 10 samples), then postfixed and stained in 1% osmium tetroxide and 1% K₃Fe(CN)₃ mixture. Ultrathin section (90nm), selected from the oriented semi-thin sections (at least 4 block/sample) were examined by TEM especially on the relationship between the bacilli and the gastric epithelia.ª¤ª¤

RESULTS
On the ultrastructure, ª«H. pyloriª« appeared as dense opaque. Curved or spiral bodies were usually surrounded by electron-lucent zone in mucin pool. They were also located at the sites close to the microvilli of epithelial cells usually in the gastric neck region or abutted onto the depression of plasma membrane which resulted from microvilli lost (Fig. 1) or apocrine secretion. Sometimes, the organisms drilled into the nike of discharged mucous, of disrupted cellular junction, of rolled microvilli, of fragmentated cells, and were found to be insinuated deep inª²between the epithelial cells (Fig. 2). Hp infected patients were confirmed in 55 (30 chronic gastritis and 25 pepticulcer) of 75 objects (by at least three diagnostic standards) from which curved or spiral organisms were detected in 53 patients (with 1 false positive) by TEM in the gastric mucosa (96%). Specificity of TEM was 95%, with 19 true negative and 1 false negative. Stained with tanin acid, glycocalyx was observed at the surface of organisms,  especially  on the tip of microvilli which possessed glycocalyx material (Fig. 3). Where the bacilli grouped in colony and bred, the adjacent mucous cells degenerated and were characterized by the vacuolation (Fig. 4), accumulation of lysosome and appearance of mucous apocrine or blebs displaying of myeline figure, even drop-out of epithelium. Evidences were accumulated that the bacilli located in the lumen attracted polymorphoneuclear leukocytes (PMNL) which intended to migrate into intercellular space of the epithelia (Fig. 5) or into the lumen to exert the effect of Hp phagocytosis (Fig. 6).ª¤ª¤

DISCUSSION
In order to preserve fine structure from destroying effect of osmium tetroxide. We tried to fix biospies in tannic acid solution and found that some fragile glycocalyx on the bacilli and the epithelia remained intact (Fig. 3). Based on our observation, where the bacilli grouped in colony, there were the dividing figures of organism and usually there were vacuolation of adjacent epithelial cells. Similar observation was noted by Caselli^£Û3£Ý. As for the bacilli firmly adhering to the epithelium or hiding in the niche of injured cells, it was presumed that the microhabitats of niche permitted the bacilli to directly release cytotoxin or vacuotoxin and to impair the cells^£Û4£Ý. As organisms grouped in colony, ammonia was released directly to their harbouring environment where it was suitable for the bacilli to escape microbicidal effect of Hp for a long time.ª¥
         Much evidence has been accumulated to show that Hp can be cleared by neutrophils from the site of infection most effectively through the opsonophagocytotic process ª«in vivoª« and ª«in vitroª« in which that specific IgG antibody promoted complementª²dependent phagocytosis and killing of Hp by PMNL was demonstrated by Caselli^£Û5£Ý. Until now, with the limited literatures, the evidence of phagocytosis of the organisms by PMNL in antrum ª«in vivoª« was scant. And evidence was accumulated to show that the bacilli were located in the lumen, a certain protein or chemotactic factor of Hp^£Û6£Ý attracted neutrophils migrating into the epithelia, occasionally even into the gland to exert the activity of phagocytosis. Degranulation of proteolytic enzymes from neutrophils may also contribute to gastric mucosa damage, especially in the cells of generative region mucous neck region. As a result of persistent Hp infection, the degeneration and regeneration in mucous neck region alternatively occur and give rise to the atrophic gastritis. Regeneration of the stem cells of mucous neck region would ultimately promote the gene instability which accounts for genetic mutation in neoplastic transformation in gastric stem cells.ª¤

Fig. 1 The bacillus abuts upon the depression of plasma membrane (*) of the vacuolated cell. (¡Á21000)ª¤

Fig. 2 The bacilli (*) penetrating deep inª²between the mucous cells, the tight junction was broken and the
intercellular space was dilated (¡ü). (¡Á21000)ª¤

Fig. 3 The bacilus shows the first step of adherence initiated by the direct contact (¡ü) of the organism to
the microvilla glycocalyx. (¡Á28500)ª¤

Fig. 4 A great number of bacteria grouped in a colony shown with periplasmic pools(s). Note the bacilli abutting
upon the depression of the plasma membrane of mucous neck cells lost microvilli (¡ü) and vacuolated. (¡Á8700)ª¤

Fig. 5 Neutrophil (*) penetrated into the epithelia and migrated to the bacilli located in the apical region of
mucous neck cells. (¡Á10800)ª¤

Fig. 6 The bacillus (¡ü) is phagocytized by neutrophillocated in the lumen. In the phagosome, two lysosomes
approach to the bacillus, of which the cell wall was lysed and discontinuous (¡ü). (¡Á43500)

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Department of Cell Biology, Cancer Research Center, Xiamen 361005 Fujian Province, China.ª¤
*Supported by Science Foundation of Xiamen. No.95801.ª¤
Correspondence to Dr Yan Shan Ming,Department of Cell Biology, Cancer Research Center,
Xiamen 361005 Fujian Province, China.ª¤
Tel.+86¡¤529¡¤2017309.
Received 4th April, 1996, revised 25th July 1996.ª¤