Formulation of a live bacterial vaccine for stable room temperature storage results in loss of acid, bile and bile salt resistance

Live bacterial vaccines have great promise both as vaccines against enteric pathogens and as heterologous antigen vectors against diverse diseases. Ideally, room temperature stable dry formulations of live bacterial vaccines will allow oral vaccination without cold-chain storage or injections. Attenuated Salmonella can cross the intestinal wall and deliver replicating antigen plus innate immune activation signals directly to the intestinal immune tissues, however the ingested bacteria must survive firstly gastric acid and secondly the antimicrobial defences of the small intestine. We found that the way in which cells are grown prior to formulation markedly affects sensitivity to acid and bile. Using a previously published stable storage formulation that maintained over 10% viability after 56 days storage at room temperature, we found dried samples of an attenuated S. typhimurium vaccine lost acid and bile resistance compared to the same bacteria taken from fresh culture. The stable formulation utilised osmotic preconditioning in defined medium plus elevated salt concentration to induce intracellular trehalose accumulation before drying. Dried bacteria grown in rich media without osmotic preconditioning showed more resistance to bile, but less stability during storage, suggesting a trade-off between bile resistance and stability. Further optimization is needed to produce the ultimate room-temperature stable oral live bacterial vaccine formulation

An Exploration into Christian Engagement in Freedom of Religion or Belief

This study explores the challenges and opportunities facing Christian organisations engaged in the pursuit of Freedom of Religion or Belief (FoRB). The thesis suggests that FoRB is consistent with the mission of God and demanded by it. Chapter one sets out the method and narrative for the research. Chapters two to four provide a case study of the research subject, Christian Solidarity Worldwide (CSW), an evangelical human rights charity engaged in FoRB. The challenges, opportunities, and ambiguities facing Christian organisations in this field are here explored. Chapter five considers Christian ideas behind Article 1 and Article 18 of the 1948 Universal Declaration of Human Rights (UDHR). Chapter six identifies historic examples of theological thought that flowed into Christian activism in the Declaration. The thesis aims to provide a reflection which supports Christian praxis in this field: crucially, chapters seven and eight attempt to lay this foundation. The material considers selected texts which explore human dignity, and the universal character of God’s mission that responds to all human suffering. My final chapter offers some practical thoughts for Christians engaged in FoRB. This qualitative ethnographic study explored the organisation’s understanding of the biblical drivers behind their praxis through a series of twenty-nine semi-structured interviews. Interviews were supplemented by primary material from the World Council of Churches (WCC), the United Nations and the Evangelical Alliance UK. My own study journal provided opportunities for reflexivity. Ultimately, this study aims to make a contribution to an area of ministry with scope for more specific theological reflection

Transient marine electromagnetic responses of 3-D resistive structures: Implications for navigation

The marine controlled-source electromagnetic method has become a popular technique for mapping the electrical resistivity structure of the seafloor. Electromagnetic fields, produced by an electric dipole transmitter, diffuse through the earth and are recorded on the seabed by nodal or cable based electric dipole receivers. Accurate information on transmitter and receiver geometry is extremely important for proper interpretation; errors in the position and orientation of the transmitter and/or the receivers propagate into errors in the predicted seafloor resistivity. We consider the special situation where a receiver cable is towed in the vicinity of a transmitter which is located on the seafloor with a known position and orientation. We theoretically and numerically examine the response of this system for a selection of 1D, 2D, and 3D models, and show that although the electromagnetic field recorded at early times contains information about the subsurface resistivity, the late time decay of the transient response is only a function of the seawater conductivity and receiver position for all the models we consider. In fact, the shape of the late time decay is almost exactly the same as that of a dipole in a wholespace having the same resistivity as seawater. This shows that late time transient information can be used to determine the position of the receiver cable, whereas early time information can be used to determine the resistivity structure of the seafloor

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 microbe's ability to down-regulate the inflammatory response. Understanding the complexity of this host-microbial balance will contribute to improved vaccine design for infectious gastroenteritis

Public-private partnerships for hospitals.

While some forms of public-private partnerships are a feature of hospital construction and operation in all countries with mixed economies, there is increasing interest in a model in which a public authority contracts with a private company to design, build and operate an entire hospital. Drawing on the experience of countries such as Australia, Spain, and the United Kingdom, this paper reviews the experience with variants of this model. Although experience is still very limited and rigorous evaluations lacking, four issues have emerged: cost, quality, flexibility and complexity. New facilities have, in general, been more expensive than they would have been if procured using traditional methods. Compared with the traditional system, new facilities are more likely to be built on time and within budget, but this seems often to be at the expense of compromises on quality. The need to minimize the risk to the parties means that it is very difficult to "future-proof" facilities in a rapidly changing world. Finally, such projects are extremely, and in some cases prohibitively, complex. While it is premature to say whether the problems experienced relate to the underlying model or to their implementation, it does seem that a public-private partnership further complicates the already difficult task of building and operating a hospital

Optimal protection of stabilised dry live bacteria from bile toxicity in oral dosage forms by bile acid adsorbent resins

We previously found that dried live bacteria of a vaccine strain can be temporarily sensitive to bile acids and suggested that Bile Adsorbing Resins (BAR) can be used in oral vaccine tablets to protect dried bacteria from intestinal bile. Here, we report a quantitative analysis of the ability of BAR to exclude the dye bromophenol blue from penetrating into matrix tablets and also sections of hard capsule shells. Based on this quantitative analysis, we made a fully optimised formulation, comprising 25% w/w of cholestyramine in Vcaps™ HPMC capsules. This gave effectively 100% protection of viability from 4% bile, with 4200-fold more live bacteria recovered from this formulation compared to unprotected dry bacteria. From the image analysis, we found that the filler material or compaction force used had no measurable effect on dye exclusion but did affect the rate of tablet hydration. Increasing the mass fraction of BAR gave more exclusion of dye up to 25% w/w, after which a plateau was reached and no further dye exclusion was seen. More effective dye exclusion was seen with smaller particle sizes (i.e. cholestyramine) and when the BAR was thoroughly dried and disaggregated. Similar results were found when imaging dye penetration into capsule sections or tablets. The predictions of the dye penetration study were tested using capsules filled with dried attenuated Salmonella vaccine plus different BAR types, and the expected protection from bile was found, validating the imaging study. Surprisingly, depending on the capsule shell material, some protection was given by the capsule alone without adding BAR, with Vcaps™ HPMC capsules providing up to 174-fold protection against 1% bile; faster releasing Vcaps Plus™ HPMC capsules and Coni Snap™ gelatin capsules gave less protection

Protection of live bacteria from bile acid toxicity using bile acid adsorbing resins

We previously demonstrated that a dry, room temperature stable formulation of a live bacterial vaccine was highly susceptible to bile, and suggested that this will lead to significant loss of viability of any live bacterial formulation released into the intestine using an enteric coating or capsule. We found that bile and acid tolerance is very rapidly recovered after rehydration with buffer or water, raising the possibility that rehydration in the absence of bile prior to release into the intestine might solve the problem of bile toxicity to dried cells. We describe here a novel formulation that combines extensively studied bile acid adsorbent resins with the dried bacteria, to temporarily adsorb bile acids and allow rehydration and recovery of bile resistance of bacteria in the intestine before release. Tablets containing the bile acid adsorbent cholestyramine release 250-fold more live bacteria when dissolved in a bile solution, compared to control tablets without cholestyramine or with a control resin that does not bind bile acids. We propose that a simple enteric coated oral dosage form containing bile acid adsorbent resins will allow improved live bacterial delivery to the intestine via the oral route, a major step towards room temperature stable, easily administered and distributed vaccine pills and other bacterial therapeutic

Sphingosine 1-phosphate receptors and sphingosine kinase 1 : novel biomarkers for clinical prognosis in breast, prostate, and hematological cancers

There is substantial evidence for a role in cancer of the bioactive lipid sphingosine 1-phosphate (S1P), the enzyme sphingosine kinase 1 (that catalyses S1P formation) and S1P-specific G protein-coupled receptors. This perspective highlights recent findings demonstrating that sphingosine kinase 1 and S1P receptors are new important biomarkers for detection of early cancer and progression to aggressive cancer. The impact of the sub-cellular distribution of S1P metabolizing enzymes and S1P receptors and their spatial functional interaction with oncogenes is considered with respect to prognostic outcome. These findings suggest that S1P, in addition to being a biomarker of clinical prognosis, might also be a new therapeutic target for intervention in cancer

Measuring the health-related quality of life of children with impaired mobility:examining correlation and agreement between children and parent proxies

Abstract Objective The objective of this research project was to evaluate the validity of proxy health-related quality of life measures in the context of paediatric mobility impairment. Accurate health-related quality of life data is essential for quality-adjusted life year calculation; a key outcome in economic evaluation. Thirteen child-parent dyads (13 children with mobility impairments, 13 parent proxies) were asked to complete a range of outcome measures (EQ-5D-Y, VAS and HUI2/3) relating to the child’s health. The relationship between respondent outcomes was examined using tests of respondent type effect (Wilcoxon signed-rank), correlation (Spearman’s rank-order) and agreement (Bland–Altman plots). Results Parent proxies significantly undervalued the health-related quality of life of their mobility-impaired children: children rated their health-related quality of life higher than their parents by proxy on all measures. The VAS had the highest overall mean score for children and proxies (79.50 [SD = 15.01] and 75.77 [SD = 14.70] respectively). Child and proxy results were significantly different (p < 0.05) for all measures besides the VAS (p = 0.138). Strong correlation and acceptable agreement were observed for equivalent child/proxy VAS and HUI measures. The EQ-5D-Y exhibited the least agreement between children and proxies. Sufficient association between child/proxy VAS and HUI measures indicated a degree of interchangeability