Lindsey A. Edwards

Delineation of the Innate and Adaptive T-Cell Immune Outcome in the Human Host in Response to Campylobacter jejuni Infection

Background Campylobacter jejuni is the most prevalent cause of bacterial gastroenteritis worldwide. Despite the significant health burden this infection presents, molecular understanding of C. jejuni-mediated disease pathogenesis remains poorly defined. Here, we report the characterisation of the early, innate immune response to C. jejuni using an ex-vivo human gut model of infection. Secondly, impact of bacterial-driven dendritic cell activation on T-cell mediated immunity was also sought. Methodology Healthy, control paediatric terminal ileum or colonic biopsy tissue was infected with C. jejuni for 8–12 hours. Bacterial colonisation was followed by confocal microscopy and mucosal innate immune responses measured by ELISA. Marked induction of IFNγ with modest increase in IL-22 and IL-17A was noted. Increased mucosal IL-12, IL-23, IL-1β and IL-6 were indicative of a cytokine milieu that may modulate subsequent T-cell mediated immunity. C. jejuni-driven human monocyte-derived dendritic cell activation was followed by analyses of T cell immune responses utilising flow cytometry and ELISA. Significant increase in Th-17, Th-1 and Th-17/Th-1 double-positive cells and corresponding cytokines was observed. The ability of IFNγ, IL-22 and IL-17 cytokines to exert host defence via modulation of C. jejuni adhesion and invasion to intestinal epithelia was measured by standard gentamicin protection assay. Conclusions Both innate and adaptive T cell-immunity to C. jejuni infection led to the release of IFNγ, IL-22 and IL-17A; suggesting a critical role for this cytokine triad in establishing host anti-microbial immunity during the acute and effectors phase of infection. In addition, to their known anti-microbial functions; IL-17A and IL-17F reduced the number of intracellular C. jejuni in intestinal epithelia, highlighting a novel aspect of how IL-17 family members may contribute to protective immunity against C. jejuni.

Gamification for health promotion: systematic review of behaviour change techniques in smartphone apps

Objective Smartphone games that aim to alter health behaviours are common, but there is uncertainty about how to achieve this. We systematically reviewed health apps containing gaming elements analysing their embedded behaviour change techniques. Methods Two trained researchers independently coded apps for behaviour change techniques using a standard taxonomy. We explored associations with user ratings and price. Data sources We screened the National Health Service (NHS) Health Apps Library and all top-rated medical, health and wellness and health and fitness apps (defined by Apple and Google Play stores based on revenue and downloads). We included free and paid English language apps using ‘gamification’ (rewards, prizes, avatars, badges, leaderboards, competitions, levelling-up or health-related challenges). We excluded apps targeting health professionals. Results 64 of 1680 (4%) health apps included gamification and met inclusion criteria; only 3 of these were in the NHS Library. Behaviour change categories used were: feedback and monitoring (n=60, 94% of apps), reward and threat (n=52, 81%), and goals and planning (n=52, 81%). Individual techniques were: self-monitoring of behaviour (n=55, 86%), non-specific reward (n=49, 82%), social support unspecified (n=48, 75%), non-specific incentive (n=49, 82%) and focus on past success (n=47, 73%). Median number of techniques per app was 14 (range: 5–22). Common combinations were: goal setting, self-monitoring, non-specific reward and non-specific incentive (n=35, 55%); goal setting, self-monitoring and focus on past success (n=33, 52%). There was no correlation between number of techniques and user ratings (p=0.07; rs=0.23) or price (p=0.45; rs=0.10). Conclusions Few health apps currently employ gamification and there is a wide variation in the use of behaviour change techniques, which may limit potential to improve health outcomes. We found no correlation between user rating (a possible proxy for health benefits) and game content or price. Further research is required to evaluate effective behaviour change techniques and to assess clinical outcomes. Trial registration number CRD42015029841.

Bacterial-Epithelial Contact Is a Key Determinant of Host Innate Immune Responses to Enteropathogenic and Enteroaggregative Escherichia coli

Background Enteropathogenic (EPEC) and Enteroaggregative (EAEC) E. coli have similar, but distinct clinical symptoms and modes of pathogenesis. Nevertheless when they infect the gastrointestinal tract, it is thought that their flagellin causes IL-8 release leading to neutrophil recruitment and gastroenteritis. However, this may not be the whole story as the effect of bacterial adherence to IEC innate response(s) remains unclear. Therefore, we have characterized which bacterial motifs contribute to the innate epithelial response to EPEC and EAEC, using a range of EPEC and EAEC isogenic mutant strains. Methodology Caco-2 and HEp-2 cell lines were exposed to prototypical EPEC strain E2348/69 or EAEC strain O42, in addition to a range of isogenic mutant strains. E69 [LPS, non-motile, non-adherent, type three secretion system (TTSS) negative, signalling negative] or O42 [non-motile, non-adherent]. IL-8 and CCL20 protein secretion was measured. Bacterial surface structures were assessed by negative staining Transmission Electron Microscopy. The Fluorescent-actin staining test was carried out to determine bacterial adherence. Results Previous studies have reported a balance between the host pro-inflammatory response and microbial suppression of this response. In our system an overall balance towards the host pro-inflammatory response is seen with the E69 WT and to a greater extent O42 WT, which is in fit with clinical symptoms. On removal of the external EPEC structures flagella, LPS, BFP, EspA and EspC; and EAEC flagella and AAF, the host inflammatory response is reduced. However, removal of E69 lymphostatin increases the host inflammatory response suggesting involvement in the bacterial mediated anti-inflammatory response. Conclusion Epithelial responses were due to combinations of bacterial agonists, with host-bacterial contact a key determinant of these innate responses. Host epithelial recognition was offset by the microbes ability to down-regulate the inflammatory response. Understanding the complexity of this host-microbial balance will contribute to improved vaccine design for infectious gastroenteritis.

Characterization of a New Mouse Model of Empyema and the Mechanisms of Pleural Invasion by Streptococcus pneumoniae

Although empyema affects more than 65,000 people each year in the United States and in the United Kingdom, there are limited data on the pathogenesis of pleural infection. We investigated the pathogenesis of empyema using animal and cell culture models of Streptococcus pneumoniae infection. The pathological processes during the development of empyema associated with murine pneumonia due to S. pneumoniae (strain D39) were investigated. Lungs were examined using histology, and pleural fluid and blood bacterial colony-forming units, cytokine levels, and cellular infiltrate were determined over time. Bacterial migration across mesothelial monolayers was investigated using cell culture techniques, flow cytometry, and confocal microscopy. After intranasal inoculation with 10(7) S. pneumoniae D39 strain, mice developed pneumonia associated with rapid bacterial invasion of the pleural space; raised intrapleural IL-8, VEGF, MCP-1, and TNF-α levels; and caused significant intrapleural neutrophilia followed by the development of fibrinous pleural adhesions. Bacterial clearance from the pleural space was poor, and in vitro assays demonstrated that S. pneumoniae crossed mesothelial layers by translocation through cells rather than by a paracellular route. This study describes key events during the development of S. pneumoniae empyema using a novel murine model of pneumonia-associated empyema that closely mimics human disease. The model allows for future assessment of molecular mechanisms involved in the development of empyema and evaluation of potential new therapies. The data suggest that transmigration of bacteria through mesothelial cells could be important in empyema development. Furthermore, upon entry the pleural cavity offers a protected compartment for the bacteria.

Enterotoxin-producing staphylococci cause intestinal inflammation by a combination of direct epithelial cytopathy and superantigen-mediated T-cell activation

Background: Enterotoxin‐producing Staphylococcus aureus may cause severe inflammatory intestinal disease, particularly in infants or immunodeficient or elderly patients. They are also recognized to be associated with sudden infant death syndrome. Little is known, however, about mucosal responses to staphylococci. Methods: The mucosal lesion in three infants with staphylococcal enterocolitis was assessed by immunohistochemistry and electron microscopy. The organisms underwent extensive molecular analysis. Their toxins were assessed for capacity to induce T‐cell activation and host mucosal responses examined by in vitro organ culture. Epithelial responses were studied by coculture with HEp‐2 and Caco‐2 cells. Results: Intestinal biopsies from the patients showed marked epithelial damage with mucosal inflammation. The three staphylococci, representing two distinct clones, were methicillin‐sensitive, producing SEG/I enterotoxins and Rho‐inactivating EDIN toxins. Their enterotoxins potently activated T cells, but only whole organisms could induce in vitro enteropathy, characterized by remarkable epithelial desquamation uninhibited by tacrolimus. EDIN‐producing staphylococci, but not their supernatants, induced striking cytopathy in HEp‐2 epithelial cells but not in Caco‐2 cells. Although HEp‐2 and Caco‐2 cells produced similar IL‐8, CCL20, and cathelicidin LL37 responses upon bacterial exposure, only Caco‐2 cells expressed mRNA for the &bgr;‐defensins HBD2 and HBD3, while HEp‐2 cells were unable to do so. Conclusions: Staphylococci induce enterocolitis by a combination of direct enterocyte cytopathy mediated by EDIN toxins, disrupting the epithelial barrier, and enterotoxin superantigen‐induced mucosal T‐cell activation. Gut epithelial production of &bgr;‐defensins may contribute to host defense against invasive staphylococcal disease. (Inflamm Bowel Dis 2011;)

Aberrant response to commensal Bacteroides thetaiotaomicron in Crohn's disease: an ex vivo human organ culture study.

Background: Human ex vivo evidence indicating that an inappropriate immune response(s) to nonpathogenic bacteria contributes to disease pathogenesis in pediatric Crohns disease (CD) is limited. The aim of the present study was to compare and contrast the early innate immune response of pediatric “healthy” versus CD mucosa to pathogenic, probiotic, and commensal bacteria. Methods: “Healthy control” and CD pediatric mucosal biopsies (terminal ileum and transverse colon) were cocultured for 8 hours with E. coli O42, Lactobacillus GG (LGG), Bacteroides thetaiotaomicron (B. theta), or stimulated with interleukin (IL)‐1&bgr; (positive control). Matched nonstimulated biopsies served as experimental controls. IL‐8 was the immune marker of choice. IL‐8 mRNA and protein levels were quantified by quantitative polymerase chain reaction and sandwich enzyme‐linked immunosorbent assay, respectively. Results: IL‐8 secretion was observed when control, ileal biopsies were exposed to pathogenic O42 and probiotic LGG, with no response noted to commensal B. theta. In comparison, Crohns ileal biopsies showed impaired ability to induce IL‐8 in response to O42 and LGG. Control colonic tissue showed a limited response to O42 or B. theta and LGG significantly reduced IL‐8 secretion. Unlike control tissue, however, Crohns ileal and colonic tissue did respond to B. theta, with more enhanced expression in the colon. Conclusions: We provide the first ex vivo data to support the notion that aberrant mucosal recognition of commensal bacteria may contribute to pediatric CD. While IL‐8 responses to O42 and LGG varied with disease status and anatomical location, B. theta consistently induced significant IL‐8 both in ileal and colonic CD tissue, which was not seen in control, healthy tissue. (Inflamm Bowel Dis 2010;)

Importance of Bacterial Replication and Alveolar Macrophage-Independent Clearance Mechanisms during Early Lung Infection with Streptococcus pneumoniae

ABSTRACT Although the importance of alveolar macrophages for host immunity during early Streptococcus pneumoniae lung infection is well established, the contribution and relative importance of other innate immunity mechanisms and of bacterial factors are less clear. We have used a murine model of S. pneumoniae early lung infection with wild-type, unencapsulated, and para-amino benzoic acid auxotroph mutant TIGR4 strains to assess the effects of inoculum size, bacterial replication, capsule, and alveolar macrophage-dependent and -independent clearance mechanisms on bacterial persistence within the lungs. Alveolar macrophage-dependent and -independent (calculated indirectly) clearance half-lives and bacterial replication doubling times were estimated using a mathematical model. In this model, after infection with a high-dose inoculum of encapsulated S. pneumoniae, alveolar macrophage-independent clearance mechanisms were dominant, with a clearance half-life of 24 min compared to 135 min for alveolar macrophage-dependent clearance. In addition, after a high-dose inoculum, successful lung infection required rapid bacterial replication, with an estimated S. pneumoniae doubling time of 16 min. The capsule had wide effects on early lung clearance mechanisms, with reduced half-lives of 14 min for alveolar macrophage-independent and 31 min for alveolar macrophage-dependent clearance of unencapsulated bacteria. In contrast, with a lower-dose inoculum, the bacterial doubling time increased to 56 min and the S. pneumoniae alveolar macrophage-dependent clearance half-life improved to 42 min and was largely unaffected by the capsule. These data demonstrate the large effects of bacterial factors (inoculum size, the capsule, and rapid replication) and alveolar macrophage-independent clearance mechanisms during early lung infection with S. pneumoniae.

Chronic Rejection of Cardiac Allografts Is Associated With Increased Lymphatic Flow and Cellular Trafficking

Background: Cardiac transplantation is an excellent treatment for end-stage heart disease. However, rejection of the donor graft, in particular, by chronic rejection leading to cardiac allograft vasculopathy, remains a major cause of graft loss. The lymphatic system plays a crucial role in the alloimmune response, facilitating trafficking of antigen-presenting cells to draining lymph nodes. The encounter of antigen-presenting cells with T lymphocytes in secondary lymphoid organs is essential for the initiation of alloimmunity. Donor lymphatic vessels are not anastomosed to that of the recipient during transplantation. The pathophysiology of lymphatic disruption is unknown, and whether this disruption enhances or hinders the alloimmune responses is unclear. Although histological analysis of lymphatic vessels in donor grafts can yield information on the structure of the lymphatics, the function following cardiac transplantation is poorly understood. Methods: Using single-photon emission computed tomography/computed tomography lymphoscintigraphy, we quantified the lymphatic flow index following heterotrophic cardiac transplantation in a murine model of chronic rejection. Results: Ten weeks following transplantation of a minor antigen (HY) sex-mismatched heart graft, the lymphatic flow index was significantly increased in comparison with sex-matched controls. Furthermore, the enhanced lymphatic flow index correlated with an increase in donor cells in the mediastinal draining lymph nodes; increased lymphatic vessel area; and graft infiltration of CD4+, CD8+ T cells, and CD68+ macrophages. Conclusions: Chronic rejection results in increased lymphatic flow from the donor graft to draining lymph nodes, which may be a factor in promoting cellular trafficking, alloimmunity, and cardiac allograft vasculopathy.

Immunotoxin Against a Donor MHC Class II Molecule Induces Indefinite Survival of Murine Kidney Allografts

Rejection of donor organs depends on the trafficking of donor passenger leukocytes to the secondary lymphoid organs of the recipient to elicit an immune response via the direct antigen presentation pathway. Therefore, the depletion of passenger leukocytes may be clinically applicable as a strategy to improve graft survival. Because major histocompatibility complex (MHC) class II+ cells are most efficient at inducing immune responses, selective depletion of this population from donor grafts may dampen the alloimmune response and prolong graft survival. In a fully MHC mismatched mouse kidney allograft model, we describe the synthesis of an immunotoxin, consisting of the F(ab′)2 fragment of a monoclonal antibody against the donor MHC class II molecule I‐Ak conjugated with the plant‐derived ribosomal inactivating protein gelonin. This anti–I‐Ak gelonin immunotoxin depletes I‐Ak expressing cells specifically in vitro and in vivo. When given to recipients of kidney allografts, it resulted in indefinite graft survival with normal graft function, presence of Foxp3+ cells within donor grafts, diminished donor‐specific antibody formation, and delayed rejection of subsequent donor‐type skin grafts. Strategies aimed at the donor arm of the immune system using agents such as immunotoxins may be a useful adjuvant to existing recipient‐orientated immunosuppression.

Therapeutic plasma exchange as a novel treatment for severe intrahepatic cholestasis of pregnancy: Case series and mechanism of action

Intrahepatic cholestasis of pregnancy is characterised by pruritus and elevated serum bile acids. The pruritus can be severe, and pharmacological options achieve inconsistent symptomatic improvement. Raised bile acids are linearly associated with adverse fetal outcomes, with existing management of limited benefit. We hypothesised that therapeutic plasma exchange removes pruritogens and lowers total bile acid concentrations, and improves symptoms and biochemical abnormalities in severe cases that have not responded to other treatments.