Hyperactive gp130/STAT3-driven gastric tumourigenesis promotes submucosal tertiary lymphoid structure development

Tertiary lymphoid structures (TLSs) display phenotypic and functional characteristics of secondary lymphoid organs, and often develop in tissues affected by chronic inflammation, as well as in certain inflammation-associated cancers where they are prognostic of improved patient survival. However, the mechanisms that govern the development of tumour-associated TLSs remain ill-defined. Here, we observed tumour-associated TLSs in a preclinical mouse model (gp130F/F) of gastric cancer, where tumourigenesis is dependent on hyperactive STAT3 signalling through the common IL-6 family signalling receptor, gp130. Gastric tumourigenesis was associated with the development of B and T cell-rich submucosal lymphoid aggregates, containing CD21+ cellular networks and high endothelial venules. Temporally, TLS formation coincided with the development of gastric adenomas and induction of homeostatic chemokines including Cxcl13, Ccl19 and Ccl21. Reflecting the requirement of gp130-driven STAT3 signalling for gastric tumourigenesis, submucosal TLS development was also STAT3-dependent, but independent of the cytokine IL-17 which has been linked with lymphoid neogenesis in chronic inflammation and autoimmunity. Interestingly, upregulated lymphoid chemokine expression and TLS formation were also observed in a chronic gastritis model induced by Helicobacter felis infection. Tumour-associated TLSs were also observed in patients with intestinal-type gastric cancer, and a gene signature linked with TLS development in gp130F/F mice was associated with advanced clinical disease, but was not prognostic of patient survival. Collectively, our in vivo data reveal that hyperactive gp130-STAT3 signalling closely links gastric tumourigenesis with lymphoid neogenesis, and while a TLS gene signature was associated with advanced gastric cancer in patients, it did not indicate a favourable prognosis

A Helicobacter pylori Homolog of Eukaryotic Flotillin Is Involved in Cholesterol Accumulation, Epithelial Cell Responses and Host Colonization.

The human pathogen Helicobacter pylori acquires cholesterol from membrane raft domains in eukaryotic cells, commonly known as "lipid rafts." Incorporation of this cholesterol into the H. pylori cell membrane allows the bacterium to avoid clearance by the host immune system and to resist the effects of antibiotics and antimicrobial peptides. The presence of cholesterol in H. pylori bacteria suggested that this pathogen may have cholesterol-enriched domains within its membrane. Consistent with this suggestion, we identified a hypothetical H. pylori protein (HP0248) with homology to the flotillin proteins normally found in the cholesterol-enriched domains of eukaryotic cells. As shown for eukaryotic flotillin proteins, HP0248 was detected in detergent-resistant membrane fractions of H. pylori. Importantly, H. pylori HP0248 mutants contained lower levels of cholesterol than wild-type bacteria (P < 0.01). HP0248 mutant bacteria also exhibited defects in type IV secretion functions, as indicated by reduced IL-8 responses and CagA translocation in epithelial cells (P < 0.05), and were less able to establish a chronic infection in mice than wild-type bacteria (P < 0.05). Thus, we have identified an H. pylori flotillin protein and shown its importance for bacterial virulence. Taken together, the data demonstrate important roles for H. pylori flotillin in host-pathogen interactions. We propose that H. pylori flotillin may be required for the organization of virulence proteins into membrane raft-like structures in this pathogen

Helicobacter pylori outer membrane vesicle size determines their mechanisms of host cell entry and protein content

Gram-negative pathogens ubiquitously shed outer membrane vesicles (OMVs) that play a central role in initiating and regulating pathogenesis in the host. Due to their highly inflammatory nature, OMVs are extensively being examined for their role in mediating disease in addition to their applications in innovative vaccines. A key mechanism whereby OMVs mediate inflammation and disease progression is dependent on their ability to enter host cells. Currently, the role of OMV size on determining their mechanism of cellular entry and their protein composition remains unknown. In this study, we examined the mechanisms whereby OMV size regulates their mode of entry into epithelial cells, in addition to their protein cargo and composition. We identified that a heterogeneous sized population of Helicobacter pylori OMVs entered epithelial cells via macropinocytosis, clathrin, and caveolin-dependent endocytosis. However, smaller OMVs ranging from 20 to 100 nm in size preferentially entered host cells via caveolin-mediated endocytosis. Whereas larger OMVs ranging between 90 and 450 nm in size entered host epithelial cells via macropinocytosis and endocytosis. Most importantly, we identified the previously unknown contribution that OMV size has on determining their protein content, as fewer and less diverse bacterial proteins were contained within small OMVs compared to larger OMVs. Collectively, these findings identify the importance of OMV size in determining the mechanisms of OMV entry into host cells, in addition to regulating their protein cargo, composition, and subsequent immunogenicity. These findings have significant implications in broadening our understanding of the bacterial regulation of virulence determinants and immunogenic proteins associated with OMVs, their role in mediating pathogenesis and in refining the design and development of OMV-based vaccines

Helicobacter pylori outer membrane vesicle size determines their mechanisms of host cell entry and protein content

Gram-negative pathogens ubiquitously shed outer membrane vesicles (OMVs) that play a central role in initiating and regulating pathogenesis in the host. Due to their highly inflammatory nature, OMVs are extensively being examined for their role in mediating disease in addition to their applications in innovative vaccines. A key mechanism whereby OMVs mediate inflammation and disease progression is dependent on their ability to enter host cells. Currently, the role of OMV size on determining their mechanism of cellular entry and their protein composition remains unknown. In this study, we examined the mechanisms whereby OMV size regulates their mode of entry into epithelial cells, in addition to their protein cargo and composition. We identified that a heterogeneous sized population of Helicobacter pylori OMVs entered epithelial cells via macropinocytosis, clathrin, and caveolin-dependent endocytosis. However, smaller OMVs ranging from 20 to 100 nm in size preferentially entered host cells via caveolin-mediated endocytosis. Whereas larger OMVs ranging between 90 and 450 nm in size entered host epithelial cells via macropinocytosis and endocytosis. Most importantly, we identified the previously unknown contribution that OMV size has on determining their protein content, as fewer and less diverse bacterial proteins were contained within small OMVs compared to larger OMVs. Collectively, these findings identify the importance of OMV size in determining the mechanisms of OMV entry into host cells, in addition to regulating their protein cargo, composition, and subsequent immunogenicity. These findings have significant implications in broadening our understanding of the bacterial regulation of virulence determinants and immunogenic proteins associated with OMVs, their role in mediating pathogenesis and in refining the design and development of OMV-based vaccines

A Helicobacter pylori homolog of eukaryotic flotillin is involved in cholesterol accumulation, epithelial cell responses and host colonization

The human pathogen Helicobacter pylori acquires cholesterol from membrane raft domains in eukaryotic cells, commonly known as “lipid rafts.” Incorporation of this cholesterol into the H. pylori cell membrane allows the bacterium to avoid clearance by the host immune system and to resist the effects of antibiotics and antimicrobial peptides. The presence of cholesterol in H. pylori bacteria suggested that this pathogen may have cholesterol-enriched domains within its membrane. Consistent with this suggestion, we identified a hypothetical H. pylori protein (HP0248) with homology to the flotillin proteins normally found in the cholesterol-enriched domains of eukaryotic cells. As shown for eukaryotic flotillin proteins, HP0248 was detected in detergent-resistant membrane fractions of H. pylori. Importantly, H. pylori HP0248 mutants contained lower levels of cholesterol than wild-type bacteria (P &lt; 0.01). HP0248 mutant bacteria also exhibited defects in type IV secretion functions, as indicated by reduced IL-8 responses and CagA translocation in epithelial cells (P &lt; 0.05), and were less able to establish a chronic infection in mice than wild-type bacteria (P &lt; 0.05). Thus, we have identified an H. pylori flotillin protein and shown its importance for bacterial virulence. Taken together, the data demonstrate important roles for H. pylori flotillin in host-pathogen interactions. We propose that H. pylori flotillin may be required for the organization of virulence proteins into membrane raft-like structures in this pathogen

Prophylactic biological mesh reinforcement versus standard closure of stoma site (ROCSS): a multicentre, randomised controlled trial

Background: Closure of an abdominal stoma, a common elective operation, is associated with frequent complications; one of the commonest and impactful is incisional hernia formation. We aimed to investigate whether biological mesh (collagen tissue matrix) can safely reduce the incidence of incisional hernias at the stoma closure site. Methods: In this randomised controlled trial (ROCSS) done in 37 hospitals across three European countries (35 UK, one Denmark, one Netherlands), patients aged 18 years or older undergoing elective ileostomy or colostomy closure were randomly assigned using a computer-based algorithm in a 1:1 ratio to either biological mesh reinforcement or closure with sutures alone (control). Training in the novel technique was standardised across hospitals. Patients and outcome assessors were masked to treatment allocation. The primary outcome measure was occurrence of clinically detectable hernia 2 years after randomisation (intention to treat). A sample size of 790 patients was required to identify a 40% reduction (25% to 15%), with 90% power (15% drop-out rate). This study is registered with ClinicalTrials.gov, NCT02238964. Findings: Between Nov 28, 2012, and Nov 11, 2015, of 1286 screened patients, 790 were randomly assigned. 394 (50%) patients were randomly assigned to mesh closure and 396 (50%) to standard closure. In the mesh group, 373 (95%) of 394 patients successfully received mesh and in the control group, three patients received mesh. The clinically detectable hernia rate, the primary outcome, at 2 years was 12% (39 of 323) in the mesh group and 20% (64 of 327) in the control group (adjusted relative risk [RR] 0·62, 95% CI 0·43–0·90; p=0·012). In 455 patients for whom 1 year postoperative CT scans were available, there was a lower radiologically defined hernia rate in mesh versus control groups (20 [9%] of 229 vs 47 [21%] of 226, adjusted RR 0·42, 95% CI 0·26–0·69; p<0·001). There was also a reduction in symptomatic hernia (16%, 52 of 329 vs 19%, 64 of 331; adjusted relative risk 0·83, 0·60–1·16; p=0·29) and surgical reintervention (12%, 42 of 344 vs 16%, 54 of 346: adjusted relative risk 0·78, 0·54–1·13; p=0·19) at 2 years, but this result did not reach statistical significance. No significant differences were seen in wound infection rate, seroma rate, quality of life, pain scores, or serious adverse events. Interpretation: Reinforcement of the abdominal wall with a biological mesh at the time of stoma closure reduced clinically detectable incisional hernia within 24 months of surgery and with an acceptable safety profile. The results of this study support the use of biological mesh in stoma closure site reinforcement to reduce the early formation of incisional hernias. Funding: National Institute for Health Research Research for Patient Benefit and Allergan