Unless treated, Helicobacter pylori persists as a chronic lifelong infection in the gastric mucosa of almost half the human population. Peptic ulcer disease or gastric cancer result in a small proportion of cases, thus it is important to develop effective methods to identify those patients at high risk of developing disease. The studies in this thesis aimed firstly to develop more accurate serological tests for non-invasive diagnosis of a H. pylori infection; secondly, to develop tests to identify virulence markers of disease-associated H. pylori strains; and thirdly, to determine implications of these tests for disease pathogenesis. Methods: Gastric biopsies and blood samples were collected from patients undergoing a routine upper gastrointestinal endoscopy at the Nottingham University Hospital NHS Trust- Queen's Medical Centre Campus, Nottingham. In addition, a panel of H. pylori isolates from Belgian population, including patients with duodenal ulcer, gastric cancer and, an age and gender matched gastric cancer control group with non-ulcer dyspepsia, was also characterised. ELISA assays were developed using serial dilution methods to accurately and quantitatively measure anti-H. pylori and anti-CagA IgG titres. PCR genotyping was carried out on H. pylori isolates for vacA region polymorphisms, cagA status and determination of the number of cagA EPIY A-C motifs. cagA mRNA levels in gastric biopsy tissues and cultured H. pylori strains were assessed using real- time RT-PCR. Naturally occurring polymorphic differences in the cagA promoter regions were identified by sequence analysis. Results and conclusions: The ELISA assays developed in this study allowed reliable detection of H. pylori infection and its virulence factor CagA. These assays performed well to overcome previously reported limitations of serological methods. Antibody titres closely associated with the severity of inflammation in the gastric mucosa but not with gastric atrophy. The relationship between anti-CagA IgG responses and numbers of EPIY A-C motifs was investigated and surprisingly patients infected with less virulent cagA types were found to have significantly higher titres. These strains were also found to express significantly higher cagA transcript levels both in vivo and in vitro. Sequence analysis of the cagA promoter region identified few potential naturally occurring polymorphisms. Polymorphic differences at position -54 within the inverted repeat of the cagA promoter region were found to be important in determining cagA transcription and site directed mutagenesis confirmed this. Genotyping analysis of the Belgian samples showed that vacA s l and il-type H pylori strains were closely associated with DU and ,GC and there was also a good concordance with cagA status. However, these strains were not significantly associated with mononuclear cell infiltration and neutrophil infiltration. In conclusion, serological detection of H pylori and its virulence factor CagA is a useful diagnostic tool and the intensity of the immune response in patients infected with these virulent strains is a good indicator of inflammatory process in the gastric mucosa. It was observed that a complex interaction between H pylori virulence and host exists, and that the cagA+ strains are likely to modulate their virulence potential possibly by regulating their gene expression. This has important implications and could explain why only some CagA strains cause disease. Although, vacA sl- and il-type strains are good markers of disease, determination of an independent virulence marker of H pylori- associated disease is difficult.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:580532 |
| Date | January 2012 |
| Creators | Memon, Ameer Afzal |
| Publisher | University of Nottingham |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
Page generated in 0.0142 seconds