A microbiological etiology for gastritis and peptic ulceration.
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Biomedical subjects
Publications and source records attributed to D G Newell.
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The mucosal antibody response, in female guinea-pigs vaccinated with the Skinner herpes simplex virus vaccine, has been investigated. The HSV-specific secretory IgA response was assessed using the cross-reactivity of an antiserum raised against human secretory component. Animals vaccinated subcutaneously at a distant site were shown to respond to subsequent infection with HSV by the production of HSV-specific vaginal IgG and secretory IgA. No vaginal HSV-specific antibodies were found in infected, non-vaccinated animals.
Campylobacter-like organisms, isolated from the gastric antrum of Rhesus monkeys, were compared with Campylobacter jejuni and C. pylori. They were similar to C. pylori by light microscopy, in ultrastructural morphology, in enzymic, fatty-acid-methyl-ester, and protein-profile analysis, and in antigenic reactivity with rabbit antisera to C. jejuni and C. pylori and with C. pylori-specific monoclonal antibody. Because this natural infection of the Rhesus monkey is associated with chronic gastritis, resembling the disease in humans colonised with C. pylori, we recommend the animal as a model for the investigation of human gastritis.
Histological examination of the stomachs of Rhesus monkeys at autopsy showed chronic gastritis in a high proportion of all ages. Lesions consisted of mild to heavy infiltration of the lamina propria by lymphocytes, plasma cells, and histiocytes. The antrum was most consistently affected, but lesions were also present in the fundus and pylorus. Gastric Campylobacter-like organisms (GCLO) apparently identical to human C pylori were cultured and/or detected immunohistologically in several animals. Electron microscopy showed the spiral bacteria on the epithelial surface and in gastric pits. They did not penetrate the cells but were intimately attached to the apical plasma membrane and caused loss of microvilli. Antibodies to C pylori were detected in serum of the monkeys by ELISA. The immunospecificity of this antibody response was confirmed by Western blotting techniques. A small number of cynomolgus monkeys examined had gastritis, which may also be associated with the presence of C pylori. Baboons did not have gastritis, nor was C pylori cultured from their stomachs. The study indicates that the Rhesus monkey has a naturally occurring gastritis associated with C pylori infection and may therefore be a suitable experimental animal for the human disease.
An ELISA system, using an acid extractable cell surface C. pylori antigen, has been used to detect anti-C. pylori antibodies in 176 endoscoped patients. The antibody concentration was related to histological diagnosis and colonisation with Campylobacter-like organisms. The criteria for sero-positivity was determined from the median antibody concentration in a group of 368 non-endoscoped control patients. The specificity and sensitivity of the sero-diagnostic assay for histological gastritis in antral biopsies was 83.0% and 95.6% respectively. The positive (79.3%) and negative predictive values (94.3%) indicate that such a sero-diagnostic assay could be used to screen patients prior to endoscopy.
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The outer membrane and surface exposed proteins of four strains of the gastric Campylobacter-like organism Campylobacter pyloridis were identified by SDS-PAGE of Sarkosyl-insoluble membranous material and 125I-surface-labelled whole bacteria. Although constant outer membrane proteins (molecular mass 61, 54 and 31 kDa) were observed in these strains, several variable 125I-labelled surface proteins were detected. C. pyloridis does not appear to express a single surface-exposed major outer membrane protein like that of C. jejuni and C. coli. Putative flagella proteins were identified from isolated flagella and acid-extractable surface material and by immunoblotting with anti-flagella antibodies. Several major protein antigens were observed by immunoblotting with anti-C. pyloridis antisera. At least two of these antigens cross-reacted with anti-C. jejuni antiserum. This cross-reaction appears to be caused primarily by flagellar antigens. However, one major protein antigen (61 kDa) was not cross-reactive with C. jejuni and may, therefore, be useful in serological tests for the specific diagnosis of C. pyloridis infections.
Eight monoclonal antibodies have been derived from Balb/c mice hyperimmunized with the purified flagella from Campylobacter jejuni strain 81116. These monoclonal antibodies are directed against flagella as demonstrated by reaction in ELISA against flagellate and aflagellate antigens, radio-immunoprecipitation and electro-immunoblotting techniques. Some of the antibodies react with a 60K minor protein as well as the 62K flagella protein. This protein may be related to an antigen expressed on the surface of the organism and detectable by immunogold labelling with one of the monoclonal antibodies. None of the antibodies causes the aggregation of bacteria or inhibits bacterial motility, unlike polyclonal anti-flagella antiserum. Moreover, none of the antibodies tested protected infant mice from colonization with C. jejuni strain 81116 even though partial protection (28%) was observed with syngeneic anti-flagella anti-serum. Absence of protection is probably due to the cryptic nature of the flagella epitopes investigated or lack of antibody activity in the gastrointestinal tract.
Intraperitoneal vaccination of female mice, before mating, with a whole cell, heat-killed (62 degrees C) vaccine of Campylobacter jejuni allowed the mother to confer immunity to her young, challenged orally 4-6 days after birth with the homologous strain. There was no protection against a strain of another serotype. Heating the vaccine to 100 degrees C destroyed its protective properties. A vaccine prepared from an aflagellate variant of the original strain was as protective as the original vaccine against challenge with the flagellated strain. Anti-flagellar serum antibody titres of the dams did not correlate with protection of their young.
Nine monoclonal antibodies directed against the flagella of Campylobacter jejuni strain 81116 have been investigated for serotypic and cross-reacting activity using a panel of 17 Penner serotype strains of C. jejuni. Four monoclonal antibodies were exclusively specific for serotype-6 strains, which was the serotype of C. jejuni strain 81116. Two monoclonal antibodies cross-reacted with all the flagellated strains of C. jejuni tested. One of these cross-reacting monoclonal antibodies, CF5, was found to react with all other Campylobacter species except C. sputorum bulbulus but it did not react with other bacterial enteropathogens. An antigen-capture ELISA technique was established, using this monoclonal antibody, which could detect flagellar antigen in human faecal material. These anti-flagella monoclonal antibodies therefore may be valuable in the diagnosis and serotyping of C. jejuni in clinical material.
We studied the antigenicity of a wild-type flagellate and motile (F+M+) Campylobacter jejuni strain (81116) and two daughter mutants, one flagellate and immotile (F+M-) and one aflagellate and immotile (F-M-). By sodium dodecyl sulfate-polyacrylamide gel electrophoresis of acid-extracted surface proteins, a 63-kilodalton (kDa) band identified from sheared flagella as the flagellar protein was present in the F+M+ and F+M- strains but not in the F-M- strain. No other differences in protein profile among the three strains were noted. By Western blotting, serum from rabbits immunized with either the F+M+ or F-M- strain detected a 63-kDa protein in the F+M+ and F+M- strains but not in the F-M- strain. That the F-M- antiserum recognized the 63-kDa band suggests that small amounts of this protein or a cross-reacting antigen is present on the F-M- strain. By counterimmunoelectrophoresis of the acid-extracted preparations with immune sera, all three strains were found to share three major antigens, but a fourth antigen with a net positive charge was present only in the F+M+ and F+M- strains. Antisera to five C. jejuni and two Campylobacter fetus strains recognized the 63-kDa protein of purified F+M+ flagella in Western blots, demonstrating a common antigen is present, but enzyme-linked immunosorbent assay results suggest that the sharing of this antigen among Campylobacter strains is variable.
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The virulence of Campylobacter jejuni and C. coli isolated from various water sources was compared with that of clinical strains by in vitro assays of adhesion, invasion and cytotoxicity to HeLa cells. Variation in degree of attachment was observed, but this did not appear to be related to strain source, However, water strains were less invasive and less cytotoxic to HeLa cells than clinical strains as shown by immunofluorescence and electron microscopy. These differences were particularly evident between clinical and water isolates of the same serotype and biotype implicated in an outbreak of campylobacter enteritis in a school. The enhanced virulence of the clinical isolates, possibly induced by passage, was confirmed by colonization tests on infant mice.
The biochemical and biological properties of the flagella of Campylobacter jejuni have been investigated using two variants selected from a flagellate, motile clinical isolate (strain 81116): a flagellate, non-motile variant (SF-1) and an aflagellate variant (SF-2). Phenotypic and biochemical analysis of the strains and amino acid analysis of the isolated flagella suggest that the variants differed from the wild-type strain only in the absence of flagella and/or motility. The aflagellate variant poorly colonized the gastrointestinal tract of infant mice but the flagellate, non-motile variant colonized the mice as successfully as the wild-type strain. 35S-labelled organisms were used to investigate the attachment of the variants to human epithelial cell monolayers in vitro. The flagellate, non-motile strain attached more efficiently to the cells than the wild-type strain or the aflagellate strain. Differences in attachment suggest that an adhesin is intimately associated with flagella of C. jejuni and that active flagella mediate only a tenuous association with host cells. This adhesin attached most efficiently to cells of intestinal epithelial origin and was not specifically inhibited by various sugars.
Outer membrane proteins of Campylobacter jejuni and other campylobacter species were analyzed for their antigenic potentials by immunoblotting. Polypeptides were resolved by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred electrophoretically, and reacted with rabbit antisera to C. jejuni. Each Campylobacter species analyzed demonstrated a unique outer membrane protein antigenic profile; interspecies antigen sharing was observed to be compatible with the degree of DNA relatedness between the species. The most highly conserved outer membrane protein antigen was the flagellum (molecular weight, 62,000). An aflagellate mutant was found to be untypable with the heat-labile system, in contrast to its parental isolate. The immunogenic potentials of C. jejuni proteins were examined by immunoblot analysis of sera from infected humans. Sera of convalescent patients, reacted with their homologous C. jejuni isolates, recognized a variety of campylobacter proteins. The most consistent immunogen in human infection was the flagellar protein. Patient sera assayed by the immunoblot technique were easily distinguished from control sera, which did not recognize specific campylobacter antigens. These findings suggest that the campylobacter flagellar protein is an essential determinant of the heat-labile antigen typing scheme and is the dominant immunogen recognized during C. jejuni infections in humans.
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The outer membrane proteins of five clinical isolates of Campylobacter jejuni were identified by 125I-surface labelling and SDS-PAGE of outer membrane preparations. All isolates expressed a major outer membrane protein of variable molecular weight (43 000-46 000: 43K-46K). Several constant surface proteins were also identified including a 27K protein which was surface-exposed and acid-extractable but was not present in the outer membrane preparations. Isolated flagella comprised a major 62K protein and a minor 87K protein. Both proteins were absent in an aflagellate variant. The 62K protein was immunoblotted and immunoprecipitated by rabbit anti-flagella antisera.
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