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

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

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

Effect of bore fluid composition on microstructure and performance of a microporous hollow fibre membrane as a cation-exchange substrate

This is the author accepted manuscript. The final version is available via Elsevier at http://www.sciencedirect.com/science/article/pii/S002196731500432X.Micro-capillary film (MCF) membranes are effective platforms for bioseparations and viable alternatives to established packed bed and membrane substrates at the analytical and preparative chromatography scales. Single hollow fibre (HF) MCF membranes with varied microstructures were produced in order to evaluate the effect of the bore fluid composition used during hollow fibre extrusion on their structure and performance as cation-exchange adsorbers. Hollow fibres were fabricated from ethylene-vinyl alcohol (EVOH) copolymer through solution extrusion followed by nonsolvent induced phase separation (NIPS) using bore fluids of differing composition (100wt.% N-methyl-2-pyrrolidone (NMP), 100wt.% glycerol, 100wt.% water). All HFs displayed highly microporous and mesoporous microstructures, with distinct regions of pore size <1μm, 5-15μm and up to 50μm in diameter, depending upon proximity to the bore fluid. Scanning electron microscopy (SEM) revealed skins of pore size <1μm at the inner surface of HFs produced with water and glycerol, while NMP bore fluid resulted in a skinless inner HF surface. The HFs were modified for chromatography by functionalising the polymer surface hydroxyl groups with sulphonic acid (SP) groups to produce cation-exchange adsorbers. The maximum binding capacities of the HFs were determined by frontal analysis using lysozyme solutions (0.05-100mgml(-1)) for a flow rate of 1.0mlmin(-1). The NMP-HF-SP module displayed the largest maximum lysozyme binding capacity of all the fibres produced (40.3mg lysozyme/ml adsorbent volume), a nearly 2-fold increase over the glycerol and 10-fold increase over the water variants at the same sample flow rate. The importance of NMP as a bore fluid to hollow fibre membrane performance as a result of inner surface porosity was established with a view to applying this parameter for the optimisation of multi-capillary MCF performance in future studies.The authors would like to acknowledge the financial support of the University of Cambridge CHESS scheme. The authors would like to thank Dr. Jeremy Skepper for help with SEM imaging and assistance by Dr. Bart Hallmark and Matthew Townsend

PlantID – DNA-based identification of multiple medicinal plants in complex mixtures

Background An efficient method for the identification of medicinal plant products is now a priority as the global demand increases. This study aims to develop a DNA-based method for the identification and authentication of plant species that can be implemented in the industry to aid compliance with regulations, based upon the economically important Hypericum perforatum L. (St John’s Wort or Guan ye Lian Qiao). Methods The ITS regions of several Hypericum species were analysed to identify the most divergent regions and PCR primers were designed to anneal specifically to these regions in the different Hypericum species. Candidate primers were selected such that the amplicon produced by each species-specific reaction differed in size. The use of fluorescently labelled primers enabled these products to be resolved by capillary electrophoresis. Results Four closely related Hypericum species were detected simultaneously and independently in one reaction. Each species could be identified individually and in any combination. The introduction of three more closely related species to the test had no effect on the results. Highly processed commercial plant material was identified, despite the potential complications of DNA degradation in such samples. Conclusion This technique can detect the presence of an expected plant material and adulterant materials in one reaction. The method could be simply applied to other medicinal plants and their problem adulterants

Salidroside as a Novel Protective Agent to Improve Red Blood Cell Cryopreservation.

Glycerol and trehalose have been widely examined as protective agents in the cryopreservation of red blood cells (RBCs). However, the effectiveness of these reagents alone on cell viability is moderate. Here, the addition of salidroside attenuated oxidative damage of sheep RBCs prior to and post cryostorage. The supplementation of salidroside to the cryopreservation media containing 10% glycerol improved RBC survival by approximately 61.1±4.8% vs 37.9±4.6%. A smaller effect was seen in RBCs cryopreserved in 300 mM trehalose where the addition of salidroside improved survival by 7.6±0.3%. Furthermore, the addition of salidroside to cold storage solution demonstrated a significant reduction of haemolysis after 4 days for RBCs loaded with either glycerol or trehalose, compared to cells incubated without salidroside. RBCs survival was 2-fold greater following freezing in trehalose, compared with glycerol. After 10 days, salidroside enabled a lower haemolysis of 16.7±1.3% compared to 29.0±8.4% for cells incubated without salidroside. However, salidroside had no effect on RBCs which had been frozen in glycerol as the resulting haemolysis rate by day 10 was approximately 60%. Salidroside increased glutathione reductase activity and decreased lactate dehydrogenase activity. Furthermore, it led to reduced carbonylation of proteins in both glycerol and trehalose loaded cells. Finally, no effect on lipid peroxidation was found in the glycerol loaded RBCs although this was reduced in RBCs loaded with trehalose and salidroside. The present findings confirm the potential use of salidroside as a novel protective agent in cryopreservation and refrigerated storage of sheep RBCs.King AbdulAziz City for Science and Technology (KACST), NASAThis is the final version of the article. It first appeared from the Public Library of Science via https://doi.org/10.1371/journal.pone.016274

The language of acute pain assessment: a corpus-based critical discourse analysis

Title: The language of acute pain assessment: a corpus-based Critical Discourse Analysis approach Aim: Through use of real time interactions between healthcare workers and patients in an acute hospital setting this study sets out to investigate how health care workers help or hinder patients to express their pain during the pain assessment process. Background: Pain has long been an issue for investigation and there are a multitude of assessment options available. However, despite using an assessment framework, the ability of patients to use language to express pain has been shown to be more problematic than might be first considered. This study sets out to investigate how both patients and healthcare workers use language in this assessment process. Method: Real time data was recorded in an acute hospital in-patient setting. The use of corpus based critical discourse analysis enabled specific instances of word use and phrases related to pain experience to be identified and analysed. Findings: Two key areas were identified in the analysis of these interactions. The first area related to the traditional aspects of pain assessment relating to terminology used, location and function of pain. The second more important area related to how healthcare professionals presented a certain ‘mentality’ about the assessment process in how they appeared to be patient centred but through the use of brevity of interaction and trivialisation of the issues actually presented an opposite view. Conclusion: The primary conclusion is that although healthcare workers apply pain assessment processes, their use of language can show that they are both patient-centred and have their own motivations and agendas

Measurement of prompt D+ and D+s production in pPb collisions at √sNN= 5.02 TeV

The production of prompt D+ and D+s mesons is studied in proton-lead collisions at a centre-of-mass energy of √sNN = 5.02 TeV. The data sample corresponding to an integrated luminosity of (1.58 ± 0.02)nb−1 is collected by the LHCb experiment at the LHC. The differential production cross-sections are measured using D+ and D+s candidates with transverse momentum in the range of 0 &lt; pT &lt; 14 GeV/c and rapidities in the ranges of 1.5 &lt; y∗ &lt; 4.0 and –5.0 &lt; y∗ &lt; –2.5 in the nucleon-nucleon centre-of-mass system. For both particles, the nuclear modification factor and the forward-backward production ratio are determined. These results are compared with theoretical models that include initial-state nuclear effects. In addition, measurements of the cross-section ratios between D+, D+s and D0 mesons are presented, providing a baseline for studying the charm hadronization in lead-lead collisions at LHC energies

The language of acute pain assessment: a corpus-based critical discourse analysis

Title: The language of acute pain assessment: a corpus-based Critical Discourse Analysis approach Aim: Through use of real time interactions between healthcare workers and patients in an acute hospital setting this study sets out to investigate how health care workers help or hinder patients to express their pain during the pain assessment process. Background: Pain has long been an issue for investigation and there are a multitude of assessment options available. However, despite using an assessment framework, the ability of patients to use language to express pain has been shown to be more problematic than might be first considered. This study sets out to investigate how both patients and healthcare workers use language in this assessment process. Method: Real time data was recorded in an acute hospital in-patient setting. The use of corpus based critical discourse analysis enabled specific instances of word use and phrases related to pain experience to be identified and analysed. Findings: Two key areas were identified in the analysis of these interactions. The first area related to the traditional aspects of pain assessment relating to terminology used, location and function of pain. The second more important area related to how healthcare professionals presented a certain ‘mentality’ about the assessment process in how they appeared to be patient centred but through the use of brevity of interaction and trivialisation of the issues actually presented an opposite view. Conclusion: The primary conclusion is that although healthcare workers apply pain assessment processes, their use of language can show that they are both patient-centred and have their own motivations and agendas