Helicobacter pylori is a common human bacterial infection which usually causes asymptomatic gastritis, but 10-15% of individuals develop peptic ulcer disease (PUD) or gastric cancer. H. pylori stimulates a strong yet ineffective immune response in the gastric mucosa, which allows the infection to persist for many decades. H. pylori modulates the host response to facilitate immune evasion via multiple mechanisms, however regulatory T cells (T regs) are thought to play an important role. Recent epidemiological studies have shown a reduced risk of allergy and autoimmune diseases amongst those with the infection. It was hypothesized that H. pylori-induced local T reg responses protect against PUD and pre-malignant pathology, whilst the systemic T reg response plays a role in suppressing allergy and autoimmunity.

No description available.

No description available.

No description available.

Helicobacter pylori infection is the main cause of peptic ulcer disease, and a significant risk factor for gastric adenocarcinoma, and mucosa-associated lymphoid tissue (MALT) lymphoma. A major virulence determinant is the vacuolating cytotoxin VacA, which causes cytoplasmic vacuolation, and other effects on epithelial cells. Sequence polymorphisms occur throughout vacA, the two most diverse regions being the signal region (s1 or s2) and the mid region (m1 or m2). Type s1/m1 strains are associated with toxigenicity in vitro, and greater disease risk. The published papers presented here aimed to investigate the role of vacA polymorphism on cytotoxin activity. By constructing isogenic hybrid variants of toxigenic and nontoxigenic strains, it was shown that differences within the signal region determined the vacuolating activity of the toxin, while differences in the mid-region conferred the cell-specificity of the vacuolating phenotype. These studies also led to the discovery of a new polymorphic determinant of vacuolating activity, the intermediate region, and it was shown that this region affected toxin activity in vitro, and was a better marker of gastric adenocarcinoma in an Iranian population than signal or mid-region type. Geographical variation in vacA allelic type exists, and it was shown that s1b-type strains, associated with reduced disease risk in the west, predominated in South Africa, which may have implications for the level of gastric disease in this region. The first occurrence of the rare s2/m1 vacA allele was also reported, supporting the view that vacA allelic diversity is due to recombination. Finally, VacA is an autotransporter, and possesses a single pair of cysteine residues near the C-terminus of the mature protein, spaced 11 residues apart. This feature is conserved among several autotransporters, and it is shown that these cysteines are required for high level VacA production, which may have implications for the autotransport process.

The determination of H. pylori in vitro antibiotic susceptibility to the four key antibiotics and the underlying mechanisms of resistance were investigated. Levels of antibiotic resistance in two independent populations in England and Wales were determined. In the urban London-based population, metronidazole resistance was present in 59% and clarithromycin resistance was in 11% of the 101 H. pylori isolates. Of 363 isolates from rural Bangor, North Wales, 23% were metronidazole resistant and 7% were clarithromycin resistant. Combined clarithromycin and metronidazole resistance occurred in 9% of London and in 4% from Bangor isolates. Analysis of patient questionnaires completed at the London centres identified a significant association between metronidazole resistance and birth outside the UK. Analysis of the method used to determine metronidazole susceptibility status indicated that anaerobic pre-incubation gave results falsely indicating susceptibility. This study would recommend the use of Etest strips on 10% Mueller-Hinton blood agar, incubated microaerophilically at 37°C for 48 h. Comparison of detection methods for 23Sr rDNA mutations associated with H. pylori clarithromycin resistance showed a novel 3’-mismatched reverse primer PCR to be the most sensitive approach. Sequence analysis of 16S rDNA in tetracycline resistant isolates identified the A926g mutation for the first time from a UK H. pylori isolate and a novel A926C mutation. The mechanism of unstable amoxicillin resistance was investigated. Novel PCR-RFLP assays, exploiting sequence variability in the outer membrane protein genes hopB and hopC, indicated no association with antibiotic resistance, but have potential for use as a novel genotyping tool for H. pylori. This study highlights the need for H. pylori antibiotic resistance surveillance at the regional level to help guide treatment regimens.

Helicobacter pylori (Hp) is a major cause of peptic ulcer disease (PUD) and gastric cancer, yet the infection remains asymptomatic in most people. One factor that influences the outcome of Hp infection is the host immune response. Expression of the immune-stimulating cytokine interleukin-17 (IL-17) is increased in the human Hp-infected gastric mucosa, but its cellular source and role in pathology are unclear. In this study dendritic cell cytokine responses to Hp stimulation were studied, and relative IL-12p70 and IL-23 concentrations compared, to assess the potential of Hp to promote differentiation of IL-17-secreting T-helper cells (Th17). The effect of Hp virulence factors on cytokine secretion was assessed and monocyte-derived DC (MoDCs) and CD1c+ myeloid DC (MyDC) responses compared. MoDCs produced high concentrations of IL-12p70 upon Hp stimulation. There was also an~ IL-23 MoDCs response, but this was >10-fold lower than the IL-12p70 response. Both IL-12p70 and IL-23 responses were significantly reduced when Hp isogenic mutants for the virulence factor dupA were used, although the effect on MoDC IL-12p70 and IL-23 secretion was less marked than previously reported for monocytes. MyDCs produced lower concentrations of IL-23 than MoDCs, and no detectable IL-12p70. It is known that Hp infection can have systemic effects, so next peripheral blood mononuclear cells (PBMCs) from 21 Hp+ and 13 uninfected patients were stimulated with Hp or control antigen and Th17 and Th1 cell frequencies analyzed by flow cytometry. A systemic Hp-specific Th17 response was identified with higher Th17 cell frequencies in the Hp+ patients compared to the uninfected controls (2.0-fold, p=0.027). A variable proportion of these cells also secreted IFNgamma (median 33%, n=21), but there was no significant correlation between Th17 and Th1 cell frequencies. Peripheral blood Th1 cells were also increased in Hp+ patients (2.1-fold, p=0.018). No significant difference was found between peripheral blood Th17 and Th1 frequencies in the Hp+ patients. The concentrations of Th17, Th1, Th2 and Treg cytokines in the gastric mucosa of Hp+ patients and uninfected controls were investigated using luminex and real-time PCR. High levels of Il-17 expression in the infected compared to uninfected gastric mucosa were confirmed at both the mRNA and protein level (mRNA: 42.6-fold, p<0.0001; protein 3.5-fold, p<0.0001). Il-17 concentrations correlated with the levels of Il-17F (p=0.80, p<0.0001) and the chemokines CCl20 (p=0.59, p<0.0001) and Il-8 (p=0.49, p=0.0004). Concentrations of the Th17-differentiating cytokines IL-1beta, IL-6, IL-21 and IL-23 were not increased in the Hp+ gastric biopsies, although IL-23 was present at high concentrations in all samples regardless of Hp infection status. IL-17 was present at higher concentrations than IFNgamma (3.9-fold, p<0.0001), IL-4 (3.0-fold, p<0.0001) and IL-10 (6.8-fold, p<0.0001) in Hp+ gastric biopsies. IFNG mRNA was also more highly expressed than IL17 mRNA (3.3-fold, p=0.016). To identify the cellular source of the IL-17 mononuclear cells were extracted from gastric biopsies, stimulated with PMA/ionomycin and analyzed by flow cytometry. Amongst biopsy CD3+ T cells from 10 Hp+ patients, IL-17 was produced mainly by CD4+ Th17 cells (68.5%), although CD8+/l-17+ (24.7%) and CD4-CD8- (18.2%) cells also made a significant contribution. High IL-17 concentrations were associated with increased inflammation (2.4-fold, p=0.024) and neutrophil infiltration (2.4-fold, p=0.031). RORC2 mRNA expression was weakly associated with PUD (p=0.046, 1.4-fold) but, surprisingly, no association was found between the IL-17 response and incidence of PUD or precancerous changes. In conclusion Hp can stimulate DCs to produce IL-23 in vitro, and high levels of this Th17-differentiating cytokine were found in gastric mucosal biopsies. Stimulation with dupA null Hp strains led to reduced IL-12p70 and IL-23 secretion, suggesting a possible mechanism of action for this recently discovered virulence factor. Culturing Hp with MoDCs and MyDCs yielded quite different results, and it remains unknown whether either model closely reflects gastric mucosal DC responses. Hp-specific Th17 responses were identified in the peripheral blood of patients with active Hp infection for the first time. Th17 cells were identified as the main cellular source of IL-17 in the Hp-infected gastric mucosa but there were also significant numbers of CDS+IL-17+ and CD4-CDS-IL-17+ T cells, which have not previously been described in this context. High mucosal concentrations of IL-17 and association of this cytokine with infiltration of immune cells indicate that it is an important component of the human immune response to Hp. However, no association between IL-17 and risk of disease was detected in this study, although RORC2 expression was weakly associated with PUD. The role of I L-17/Th 17 in Hp related disease warrants further investigation.