Regulatory T cells (Tregs) play a critical role in the persistence and pathogenesis of Helicobacter pylori infection. However, the mechanisms by which Tregs expand during the infection remain poorly understood. Regulatory B cells (Bregs) have been identified as Treg-inducing, but their role in H. pylori infection is unclear. Flow cytometry was used to characterise Bregs and Tregs in peripheral blood from H. pylori infected and uninfected patients, gastric lymphocytes were analysed by immunohistochemistry. An in vitro B-T cell coculture system was employed to evaluate murine and human Treg differentiation. Higher frequencies of CD19+IL-10+ Bregs (median 85% increase; p = 0.007) and CD4+CD25hi Tregs (129% increase; p = 0.048) were present in the peripheral blood of infected patients. An association was found between numbers of Pax5+ B cells and Foxp3+ Tregs (p < 0.0001) in the infected gastric mucosa, and B and T cells were in close proximity. H. pylori directly activated murine B cells in vitro to produce >6-fold higher IL-10 concentrations (p < 0.04), with enhanced CD25hiFoxp3+ Treg differentiation (p < 0.001). Similar results were found with human B cells. Generation of Tregs was unaffected by anti-IL-10 neutralising antibody, but frequencies of Tregs were halved when B and T cells were separated by a membrane (p < 0.05). VacA and CagA virulence factors did not influence the response. In conclusion, H. pylori induced Breg responses in vitro, promoting Treg expansion, via B-T cell contact and soluble factors other than IL-10. Increased frequencies and proximity of these cell populations in the infected gastric mucosa suggests this mechanism could potentially occur in vivo.

Introduction. Helicobacter pylori is the leading cause of peptic ulcers and gastric cancer. The most common treatment regimens use combinations of two or three antibiotics and a proton pump inhibitor (PPI) to suppress stomach acid. The World Health Organization designated clarithromycin-resistant H. pylori as a high priority pathogen for drug development, due to increasing antibiotic resistance globally.Hypothesis/Gap Statement. There is no routine surveillance of H. pylori primary antimicrobial sensitivities in the UK, and pub-lished data are lacking.Aim. This study aimed to characterize antimicrobial sensitivities of isolates collected in Nottingham, UK, between 2001 and 2018. Methodology. Gastric biopsy samples were collected, with informed written consent and ethics approval, from 162 patients attending the Queen's Medical Centre in Nottingham for an upper GI tract endoscopy. Antibiotic sensitivity was assessed using E -Tests and a more cost-effective disc diffusion test.Results. The clarithromycin, amoxicillin and levofloxacin disc diffusion tests provided identical results to E -Tests on a subset of 30 isolates. Disparities were observed in the metronidazole test results, however. In total, 241 isolates from 162 patients were tested using at least one method. Of all isolates, 28 % were resistant to clarithromycin, 62 % to metronidazole and 3 % to amoxi-cillin, which are used in first -line therapies. For those antibiotics used in second-and third- line therapies, 4 % were resistant to levofloxacin and none of the isolates were resistant to tetracycline. Resistance to more than one antibiotic was found in 27 % of isolates. The frequency of patients with a clarithromycin-resistant strain increased dramatically over time: from 16 % between 2001 and 2005 to 40 % between 2011 and 2018 (P=0.011). For the same time periods, there was also an increase in those with a metronidazole-resistant strain (from 58 to 78 %; P=0.05). The frequencies of clarithromycin and metronidazole resistance were higher in isolates from patients who had previously received eradication therapy, compared to those who had not (40 % versus 77 %, and 80 % versus 92 %, respectively). Of 79 pairs of isolates from the antrum and corpus regions of the same patient's stomach, only six had differences in their antimicrobial susceptibility profiles.Conclusion. Although there was high and increasing resistance to clarithromycin and metronidazole, there was no resistance to tetracycline and the frequencies of amoxicillin and levofloxacin resistance were very low.

Supplementary Table S1 from Toxigenic Helicobacter pylori Infection Precedes Gastric Hypochlorhydria in Cancer Relatives, and H. pylori Virulence Evolves in These Families

High-level expression of decay-accelerating factor, CD55, has previously been found in human gastric cancer (GC) and intestinal metaplasia (IM) tissues. Therapeutic effects of CD55 inhibition in cancer have been reported. However, the role of Helicobacter pylori infection and virulence factors in the induction of CD55 and its association with histological changes of the human gastric mucosa remain incompletely understood. We hypothesised that CD55 would be increased during infection with more virulent strains of H. pylori, and with more marked gastric mucosal pathology. RT-qPCR and immunohistochemical analyses of gastric biopsy samples from 42 H. pylori-infected and 42 uninfected patients revealed that CD55 mRNA and protein were significantly higher in the gastric antrum of H. pylori-infected patients, and this was associated with the presence of IM, but not atrophy, or inflammation. Increased gastric CD55 and IM were both linked with colonisation by vacA i1-type strains independently of cagA status, and in vitro studies using isogenic mutants of vacA confirmed the ability of VacA to induce CD55 and sCD55 in gastric epithelial cell lines. siRNA experiments to investigate the function of H. pylori-induced CD55 showed that CD55 knockdown in gastric epithelial cells partially reduced IL-8 secretion in response to H. pylori, but this was not due to modulation of bacterial adhesion or cytotoxicity. Finally, plasma samples taken from the same patients were analysed for the soluble form of CD55 (sCD55) by ELISA. sCD55 levels were not influenced by IM and did not correlate with gastric CD55 mRNA levels. These results suggest a new link between active vacA i1- type H. pylori, IM, and CD55, and identify CD55 as a molecule of potential interest in the management of IM as well as GC treatment. (c) 2022 The Authors. The Journal of Pathology published by JohnWiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Helicobacter pylori is a bacterial pathogen which commonly colonizes the human gastric mucosa from early childhood and persists throughout life. In the vast majority of cases, the infection is asymptomatic. H. pylori is the leading cause of peptic ulcer disease and gastric cancer, however, and these outcomes occur in 10-15% of those infected. Gastric adenocarcinoma is the third most common cause of cancer-associated death, and peptic ulcer disease is a significant cause of morbidity. Disease risk is related to the interplay of numerous bacterial host and environmental factors, many of which influence chronic inflammation and damage to the gastric mucosa. This chapter summarizes what is known about health and disease in H. pylori infection, and highlights the need for additional research in this area.

Helicobacter pylori infections are usually established in early childhood and continuously stimulate immunity, including T-helper 1 (Th1), Th17, and regulatory T-cell (Treg) responses, throughout life. Although known to be the major cause of peptic ulcer disease and gastric cancer, disease occurs in a minority of those who are infected. Recently, there has been much interest in beneficial effects arising from infection with this pathogen. Published data robustly show that the infection is protective against asthma in mouse models. Epidemiological studies show that H. pylori is inversely associated with human allergy and asthma, but there is a paucity of mechanistic data to explain this. Since Th1 and Treg responses are reported to protect against allergic responses, we investigated if there were links between the human systemic Th1 and Treg response to H. pylori and allergen-specific IgE levels. The human cytokine and T-cell responses were examined using peripheral blood mononuclear cells (PBMCs) from 49 infected and 58 uninfected adult patients. Concentrations of total and allergen-specific plasma IgE were determined by ELISA and ImmunoCAP assays. These responses were analyzed according to major virulence factor genotypes of the patients’ colonizing H. pylori strains. An in vitro assay was employed, using PBMCs from infected and uninfected donors, to determine the role of Treg cytokines in the suppression of IgE. Significantly higher frequencies of IL-10-secreting CD4+CD25hi Tregs, but not H. pylori-specific Th1 cells, were present in the peripheral blood of infected patients. Total and allergen-specific IgE concentrations were lower when there was a strong Treg response, and blocking IL-10 in vitro dramatically restored IgE responses. IgE concentrations were also significantly lower when patients were infected with CagA+ strains or those expressing the more active i1 form of VacA. The systemic IL-10+ Treg response is therefore likely to play a role in H. pylori-mediated protection against allergy in humans.

AimsHelicobacter pylori infection is the major cause of peptic ulceration and gastric cancer, and an important virulence determinant is its vacuolating cytotoxin vacA. Previously, we have described allelic variation in vacA which determines toxin activity and disease risk. Here we aimed to quantify vacA mRNA expression in the human stomach, define its genetic determinants and assess how well it predicts gastric pathology.Methods Gastric biopsies were donated by 39 patients with H. pylori infection attending for endoscopy at Queen's Medical Centre, Nottingham, UK. Total RNA was extracted, and vacA mRNA quantified by reverse transcriptase quantitative PCR. Separate biopsies were histologically scored for inflammation and atrophy using the updated Sydney system. H. pylori strains were isolated from further biopsies, and the nucleotide sequence upstream of vacA determined.ResultsvacA mRNA levels in human stomachs varied by two orders of magnitude independently of vacA allelic type. Among vacA i1-type (toxic) strains, increased vacA expression was strongly associated with higher grade gastric inflammation (p<0.02), neutrophil infiltration (p<0.005) and the presence of atrophy (p<0.01). A polymorphism at nucleotide +28 near the base of a potential stem-loop structure within the 5 untranslated region was significantly associated with vacA transcript level and inflammation.Conclusions Increased gastric vacA expression during H. pylori infection is associated with inflammation and premalignant pathology. The +28 nucleotide within the vacA 5 stem-loop stratifies disease risk among toxic vacA i1-type strains.

Volume 50, no. 12, p. [3983–3989][1], 2012. Page 3988: The following sentence should be inserted after the first sentence of the Acknowledgments section. “This work was also supported by FEDER funds through Programa Operacional Factores de Competitividade—COMPETE (FCOMP-01-0124-FEDER 021251

The vacuolating cytotoxin, VacA, is an important virulence factor secreted by the gastric pathogen Helicobacter pylori. Certain vacA genotypes are strongly associated with disease risk, but the association is not absolute. The factors determining vacA gene expression are not fully understood, and the mechanisms of its regulation are elusive. We have identified a potential mRNA stem-loop forming structure in the 5' untranslated region (UTR) of the vacA transcript. Using site-directed mutagenesis, we found that disruption of the stem-loop structure reduced steady-state mRNA levels between two-and sixfold (P = 0.0005) and decreased mRNA half-life compared with wild type (P = 0.03). This led to a marked reduction in VacA protein levels and overall toxin activity. Additionally, during stressful environmental conditions of acid pH or high environmental salt concentrations, when general transcription of vacA was decreased or increased respectively, the stabilising effects of the stem-loop were even more pronounced. Our results suggest that the stem-loop structure in the vacA 5' UTR is an important determinant of vacA expression through stabilisation of the vacA mRNA transcript and that the stabilising effect is of particular importance during conditions of environmental stress.

ABSTRACT Persistent Helicobacter pylori infection induces chronic inflammation in the human gastric mucosa, which is associated with development of peptic ulceration, gastric atrophy, and gastric adenocarcinoma. It has been postulated that secretion of immunomodulatory molecules by H. pylori facilitates bacterial persistence, and membrane vesicles (MV), which have the potential to cross the gastric epithelial barrier, may mediate delivery of these molecules to host immune cells. However, bacterial MV effects on human immune cells remain largely uncharacterized to date. In the present study, we investigated the immunomodulatory effects of H. pylori MV with and without the vacuolating cytotoxin, VacA, which inhibits human T cell activity. We show a high degree of variability in the toxin content of vesicles between two H. pylori strains (SS1 and 60190). Vesicles from the more toxigenic 60190 strain contain more VacA (s1i1 type) than vesicles from the SS1 strain (s2i2 VacA), but engineering the SS1 strain to produce s1i1 VacA did not increase the toxin content of its vesicles. Vesicles from all strains tested, including a 60190 isogenic mutant null for VacA, strongly induced interleukin-10 (IL-10) and IL-6 production by human peripheral blood mononuclear cells independently of the infection status of the donor. Finally, we show that H. pylori MV induce T cell apoptosis and that this is enhanced by, but not completely dependent on, the carriage of VacA. Together, these findings suggest a role for H. pylori MV in the stimulation of innate pro- and anti-inflammatory responses and in the suppression of T cell immunity.

Carriage of Helicobacter pylori strains producing more active (s1/i1) forms of VacA is strongly associated with gastric adenocarcinoma. To our knowledge, we are the first to determine effects of different polymorphic forms of VacA on inflammation and metaplasia in the mouse stomach. Bacteria producing the less active s2/i2 form of VacA colonized mice more efficiently than mutants null for VacA or producing more active forms of it, providing the first evidence of a positive role for the minimally active s2/i2 toxin. Strains producing more active toxin forms induced more severe and extensive metaplasia and inflammation in the mouse stomach than strains producing weakly active (s2/i2) toxin. We also examined the association in humans, controlling for cagPAI status. In human gastric biopsy specimens, the vacA i1 allele was strongly associated with precancerous intestinal metaplasia, with almost complete absence of intestinal metaplasia in subjects infected with i2-type strains, even in a vacA s1, cagA+ background.

Background Helicobacter pylori-induced peptic ulceration is less likely to occur in patients with a strong gastric anti-inflammatory regulatory T cell (Treg) response. Migration of Tregs into the gastric mucosa is therefore important.Objective To identify the homing receptors involved in directing Tregs to the gastric mucosa, and investigate how H pylori stimulates the relevant chemokine responses.Design Gastric biopsy samples and peripheral blood were donated by 84 H pylori-infected and 46 uninfected patients. Luminex assays quantified gastric biopsy chemokine concentrations. Flow cytometry was used to characterise homing receptors on CD4(+)CD25(hi) Tregs. H pylori wild-type and isogenic mutants were used to investigate the signalling mechanisms behind CCL20 and IL-8 induction in gastric epithelial cell lines. Transwell assays were used to quantify Treg migration towards chemokines in vitro.Results CCL20, CXCL1-3 and IL-8 concentrations were significantly increased in gastric biopsy samples from H pylori-infected patients. CCR6 (CCL20 receptor), CXCR1 and CXCR2 (IL-8 and CXCL1-3 receptors) were expressed by a higher proportion of peripheral blood Tregs in infected patients. Most gastric Tregs expressed these receptors. H pylori induced CCL20 production by gastric epithelial cells via cag pathogenicity island (cagPAI)-dependent NF-kappa B signalling. Foxp3(+), but not Foxp3(-), CD4 cells from infected mice migrated towards recombinant CCL20 in vitro.Conclusions As well as increasing Treg numbers, H pylori infection induces a change in their characteristics. Expression of CCR6, CXCR1 and CXCR2 probably enables their migration towards CCL20 and IL-8 in the infected gastric mucosa. Such qualitative changes may also explain how H pylori protects against some extragastric inflammatory disorders.

Introduction Patients with gastric carcinoma often suffer from malnutrition after curative gastrectomy. It is difficult to identify patients who require further nutritional support as part of their long-term post-operative care because factors influencing weight loss are unknown, and it is also inefficient to directly evaluate the nutritional status of every patient using available screening tools. This study aims to identify factors influencing weight loss after curative gastrectomy, which can be used to efficiently identify at-risk patients to improve outcomes. Methods 163 patients (71.8% of those eligible) who underwent curative gastrectomy from 2000 to 2010 at the National University Hospital were identified from a prospectively maintained database. Factors predicting severe malnutrition at 6 months post-gastrectomy (defined as ≥10% loss of pre-operative weight), and factors influencing gain/plateau vs further weight loss over the following 6 months in the patients who were malnourished at 6 months post-gastrectomy were identified with multiple logistic regression. Results Severe malnutrition was present in 52.8% (n=86) at 6 months post-gastrectomy. For these patients, those who had anaemia (OR 3.662, p=0.006), gastrointestinal bleeding (OR 2.532; p=0.038) or weight loss (OR 1.052; p=0.010) at presentation, histopathologies of poorly and moderately differentiated tumours (OR 3.632 and 1.757 respectively; p=0.029), adjuvant therapy (OR 8.464; p=0.000), and those requiring step-down care upon discharge (OR 5.739; p=0.021) were at increased risk of weight loss. However, male gender (OR 0.288; p=0.012), vomiting at presentation (OR 0.219; p=0.014), and neo-adjuvant therapy (OR 0.225; p=0.027) were associated with decreased risk. Age, race and operation length were not significant factors at 6 months. Of the patients who were malnourished at 6 months, 54.9% (n=39) developed further weight loss over the subsequent 6 months. Pre-operative total lymphocyte count (OR 2.141; p=0.033) and tumour size (OR 1.235; p=0.054) were associated with increased risk. Age, gender and race were not significant factors at 12 months. Conclusion Malnutrition is a significant problem post-gastrectomy. More nutritional care should be provided to patients presenting with acute presentation, poor tumour biology and those requiring adjuvant therapy and step-down care as these are risk factors at 6 months. At 1 year, patients who initially had larger tumour sizes and higher pre-operative total lymphocyte counts are at greater risk of continued weight loss, and should receive further nutritional support as part of their long-term post-operative care. Competing interests None declared.

Introduction The Gram negative bacterium Helicobacter pylori persistently colonises the stomachs of around half the world9s population. It causes chronic asymptomatic gastritis, and leads to peptic ulceration and gastric cancer in a small proportion of cases. One of the key factors influencing the development of more severe pathology and disease is the expression of virulence factors by the colonising strain. The vacuolating cytotoxin, VacA, is a major determinant of H pylori pathogenicity and is highly polymorphic. Strains producing more active forms of VacA (i1 types) are more strongly associated with gastric cancer than strains producing less toxigenic VacA (i2 types).1 The i-region polymorphism is characterised by 10 amino acid substitutions and a serine-asparagine-glutamine (SNQ) tri-peptide insertion in i2. We aimed to further characterise the structure and function of this important region. Methods The SNQ tri-peptide from i2 was engineered into H pylori strain 60190 (VacA s1/i1/m1) to make strain 60190/SNQ. The vacuolating activities of 60190/SNQ and wild type strains were compared by incubating AGS cells with broth culture supernatants, and counting vacuolated cells in random fields by microscopy. To allow further structure-function characterisation of i-region variants, we modified a recombinant VacA system2 by expressing VacA p33 and p55 subunits in Escherichia coli and purifying both under denaturing conditions by metal affinity chromatography, then refolding by stepwise dialysis. Recombinant VacA was tested for vacuolating activity and previously described effects on Jurkat T-cells.3 Results Introduction of the i2-specific SNQ insertion into i1 VacA in the H pylori 60190 strain resulted in a 1.8-fold decrease in native toxin vacuolating activity on AGS cells lines (pH pylori—they induced extensive vacuolation of gastric epithelial cell lines at concentrations down to 0.2 μg/ml. The recombinant toxin also inhibited proliferation of, and IL-2 production by Jurkat cells. Engineering the SNQ insertion into recombinant i1 VacA resulted in modest reductions in the stability of the toxin. Conclusion A naturally-occurring three amino acid insertion in the VacA i-region renders the toxin less active in vitro. We speculate that this small insertion is a major contributor to the reduced association with cancer of strains with type i2 VacA. Recombinant VacA toxin is highly active on both epithelial and T cells and can be genetically modified to explore VacA structure-function relationships. We aim to use this system to further characterise the structure and function of i-region variants. Competing interests None declared. References 1. Rhead JL, Letley DP, Mohammadi M, et al. A new Helicobacter pylori vacuolating cytotoxin determinant, the intermediate region, is associated with gastric cancer. Gastroenterology 2007;133:926–36. 2. González-Rivera C, Gangwer KA, McClain MS, et al. Reconstitution of Helicobacter pylori VacA toxin from purified components. Biochemistry 2010;49:5743–52. 3. Gebert B, Fischer W, Weiss E, et al. Helicobacter pylori vacuolating cytotoxin inhibits T lymphocyte activation. Science 2003;301:1099–102.

The present report describes a novel method for genotyping the virulence-associated vacA intermediate (i) region of Helicobacter pylori in archive material. vacA i-region genotypes as determined by the novel method were completely concordant with those of sequence analysis and with those of functional vacuolation activity. The method was further validated directly in gastric biopsy specimens of 386 H. pylori-positive cases, and effective characterization of the vacA i region was obtained in 191 of 192 (99.5%) frozen and in 186 of 194 (95.9%) formalin-fixed paraffin-embedded gastric biopsy specimens, respectively. The genotyping method was next used to address the relationship between the vacA genotypes and the cagA status. The vacA i1 genotype was associated with vacA s1 (where s indicates signal region), vacA m1 (where m indicates middle region), and cagA-positive genotypes (P < 0.0001), while the vacA i2 genotype was closely related with vacA s2, vacA m2, and cagA-negative genotypes (P < 0.0001). The relationship between H. pylori vacA i-region genotypes and gastric disease development was subsequently evaluated in the Portuguese population. Patients infected with vacA i1 strains showed an increased risk for gastric atrophy and for gastric carcinoma, with odds ratios of 8.0 (95% confidence interval [CI], 2.3 to 27) and of 22 (95% CI, 7.9 to 63), respectively. Taken together, the results show that this novel H. pylori vacA i-region genotyping method can be applied directly to archive material, providing a fast evaluation of strain virulence determinants without the need of culture. The results further emphasize that the characterization of the vacA i region may be useful to identify patients at higher risk of gastric carcinoma development.

(3.7%) and stenosis occurred in seven patients (8.5%). Post-operative stasis occurred in one patient (1.2%). No anastomotic bleeding was encountered. Recurrence at the site of the anastomosis was not detected during routine follow-up endoscopy in any of the patients. Conclusion Intracorporeal oesophagojejunostomy using our anvil suture pull-through technique is feasible, safe and associated with good outcomes in the context of early gastric cancer. It is a reliable fi rst-line technique and a useful alternative strategy when overlap is not possible or oral anvil insertion is contraindicated.

BACKGROUND:We examine the effect of eradicating Helicobacter in idiopathic parkinsonism (IP). Marked deterioration, where eradication-therapy failed, prompted an interim report in the first 20 probands to reach de-blinding. The null-hypothesis, "eradication has no effect on principal outcome, mean stride length at free-walking speed," was rejected. We report on study completion in all 30 who had commenced post-treatment assessments.METHODS:This is a randomized, placebo-controlled, parallel-group efficacy study of eradicating biopsy-proven (culture and/or organism on histopathology) Helicobacter pylori infection on the time course of facets of IP, in probands taking no, or stable long-t(1/2), anti-parkinsonian medication. Persistent infection at de-blinding (scheduled 1-year post-treatment) led to open active eradication-treatment.RESULTS:Stride length improved (73 (95% CI 14-131) mm/year, p = .01) in favor of "successful" blinded active over placebo, irrespective of anti-parkinsonian medication, and despite worsening upper limb flexor rigidity (237 (57-416) Nm x 10(-3)/year, p = .01). This differential effect was echoed following open active, post-placebo. Gait did not deteriorate in year 2 and 3 post-eradication. Anti-nuclear antibody was present in all four proven (two by molecular microbiology only) eradication failures. In the remainder, it marked poorer response during the year after eradication therapy, possibly indicating residual "low-density" infection. We illustrate the importance of eradicating low-density infection, detected only by molecular microbiology, in a proband not receiving anti-parkinsonian medication. Stride length improved (424 (379-468) mm for 15 months post-eradication, p = .001), correction of deficit continuing to 3.4 years. Flexor rigidity increased before hydrogen-breath-test positivity for small intestinal bacterial overgrowth (208 (28-388) Nm x 10(-3), p = .02), increased further during (171 (67-274), p = .001) (15-31 months), and decreased (136 (6-267), p = .04) after restoration of negativity (32-41 months).CONCLUSION:Helicobacter is an arbiter of progression, independent of infection-load.