Immuno-potentiating activity of surfactant vesicles in DNA and subunit vaccination

Enhanced immune responses for DNA and subunit vaccines potentiated by surfactant vesicle based delivery systems outlined in the present study, provides proof of principle for the beneficial aspects of vesicle mediated vaccination. The dehydration-rehydration technique was used to entrap plasmid DNA or subunit antigens into lipid-based (liposomes) or non-ionic surfactant-based (niosomes) dehydration-rehydration vesicles (DRV). Using this procedure, it was shown that both these types of antigens can be effectively entrapped in DRV liposomes and DRV niosomes. The vesicle size of DRV niosomes was shown to be twice the diameter (~2µm) of that of their liposome counterparts. Incorporation of cryoprotectants such as sucrose in the DRV procedure resulted in reduced vesicle sizes while retaining high DNA incorporation efficiency (~95%). Transfection studies in COS 7 cells demonstrated that the choice of cationic lipid, the helper lipid, and the method of preparation, all influenced transfection efficiency indicating a strong interdependency of these factors. This phenomenon has been further reinforced when 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE): cholesteryl 3b- [N-(N’ ,N’ -dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol)/DNA complexes were supplemented with non-ionic surfactants. Morphological analysis of these complexes using transmission electron microscopy and environmental scanning electron microscopy (ESEM) revealed the presence of heterogeneous structures which may be essential for an efficient transfection in addition to the fusogenic properties of DOPE. In vivo evaluation of these DNA incorporated vesicle systems in BALB/c mice showed weak antibody and cell-mediated immune (CMI) responses. Subsequent mock challenge with hepatitis B antigen demonstrated that, 1-monopalmitoyl glycerol (MP) based DRV, is a more promising DNA vaccine adjuvant. Studying these DRV systems as adjuvants for the Hepatitis B subunit antigen (HBsAg) revealed a balanced antibody/CMI response profile on the basis of the HBsAg specific antibody and cytokine responses which were higher than unadjuvated antigen. The effect of addition of MP, cholesterol and trehalose 6,6’-dibehenate (TDB) on the stability and immuno-efficacy of dimethyldioctadecylammonium bromide (DDA) vesicles was investigated. Differential scanning calorimetry showed a reduction in transition temperature of DDA vesicles by ~12°C when incorporated with surfactants. ESEM of MP based DRV system indicated an increased vesicle stability upon incorporation of antigen. Adjuvant activity of these systems tested in C57BL/6j mice against three subunit antigens i.e., mycobacterial fusion protein- Ag85B-ESAT-6, and two malarial antigens - merozoite surface protein-1, (MSP1), and glutamate rich protein, (GLURP) revealed that while MP and DDA based systems induced comparable antibody responses, DDA based systems induced powerful CMI responses.

Guilty as charged? The effect of interpreting on interviews with suspects

The ways in which an interpreter affects the processes and, possibly, the outcomes of legal proceedings has formed the focus of much recent research, most of it centred upon courtroom discourse. However comparatively little research has been carried out into the effect of interpreting on the interview with a suspect, despite its 'upstream' position in the legal process and vital importance as evidence. As a speech event in the judicial system, the interview differs radically from that which takes place 'downstream', that is, in court. The interview with suspect represents an entirely different construct, in which a range of registers is apparent, and participants use distinctive means to achieve their institutional goals. When a transcript of an interpreter-mediated interview is read out in court, it is assumed that this is a representation of an event, which is essentially identical to a monolingual interview. This thesis challenges that assumption. Using conservation analytic techniques, it examines data from a corpus of monolingual and interpreter-mediated, taped interviews with suspects, in order to identify potentially significant interactional differences and describe ways in which the interpreter affects the processes and may affect the outcomes of the interview. It is argued that although individually, the interactional differences may appear slight, their cumulative effect is significant, particularly since the primary participants in the event are unaware of the full force of the interpreting effect. Finally, the thesis suggests that the insights provided by linguistic analysis of the interpreting on interviews may provide the basis for training, both for interpreters themselves, and for officers in techniques for interpreter-mediated interviews.

Engineering liposomal systems to develop solubility enhancing technology

This research primarily focused on identifying the formulation parameters which control the efficacy of liposomes as delivery systems to enhance the delivery of poorly soluble drugs. Preliminary studies focused on the drug loading of ibuprofen within vesicle systems. Initially both liposomal and niosomal formulations were screened for their drug-loading capacity: liposomal systems were shown to offer significantly higher ibuprofen loading and thereafter lipid based systems were further investigated. Given the key role cholesterol is known to play within the stability of bilayer vesicles. the optimum cholesterol content in terms of drug loading and release of poorly soluble drugs was then investigated. From these studies a concentration of 11 total molar % of cholesterol was used as a benchmark for all further formulations. Investigating the effect of liposomc composition on several low solubility drugs, drug loading was shown to be enhanced by adopting longer chain length lipids. cationic lipids and. decreasing drug molecular weight. Drug release was increased by using cationic lipids and lower molecular weight of drug; conversely, a reduction was noted when employing longer chain lipids thus supporting the rational of longer chain lipids producing more stable liposomes, a theory also supported by results obtained via Langmuir studies· although it was revealed that stability is also dependent on geometric features associated with the lipid chain moiety. Interestingly, reduction in drug loading appeared to be induced when symmetrical phospholipids were substituted for lipids constituting asymmetrical alkyl chain groups thus further highlighting the importance of lipid geometry. Combining a symmetrical lipid with an asymmetrical derivative enhanced encapsulation of a hydrophobic drug while reducing that of another suggesting the importance of drug characteristics. Phosphatidylcholine liposornes could successfully be prepared (and visualised using transmission electron microscopy) from fatty alcohols therefore offering an alternative liposomal stabiliser to cholesterol. Results obtained revealed that liposomes containing tetradecanol within their formulation shares similar vesicle size, drug encapsulation, surface charge. and toxicity profiles as liposomes formulated with cholesterol, however the tetradecanol preparation appeared to release considerably more drug during stability studies. Langmuir monolayer studies revealed that the condensing influence by tetradecanol is less than compared with cholesterol suggesting that this reduced intercalation by the former could explain why the tetradecanol formulation released more drug compared with cholesterol formulations. Environmental scanning electron microscopy (ESEM) was used to analyse the morphology and stability of liposomes. These investigations indicated that the presence of drugs within the liposomal bilayer were able to enhance the stability of the bilayers against collapse under reduced hydration conditions. In addition the presence of charged lipids within the formulation under reduced hydration conditions compared with its neutral counterpart. However the applicability of using ESEM as a new method to investigate liposome stability appears less valid than first hoped since the results are often open to varied interpretation and do not provide a robust set of data to support conclusions in some cases.

Multiple roles of charged amino acids in cytoplasmic loop 7 for expression and function of the multidrug and organic anion transporter MRP1 (ABCC1)

Multidrug resistance protein MRP1 mediates the ATP-dependent efflux of many chemotherapeutic agents and organic anions. MRP1 has two nucleotide binding sites (NBSs) and three membrane spanning domains (MSDs) containing 17 transmembrane helices linked by extracellular and cytoplasmic loops (CL). Homology models suggest that CL7 (amino acids 1141-1195) is in a position where it could participate in signaling between the MSDs and NBSs during the transport process. We have individually replaced eight charged residues in CL7 with Ala, and in some cases, an amino acid with the same charge, and then investigated the effects on MRP1 expression, transport activity, and nucleotide and substrate interactions. A triple mutant in which Glu(1169), Glu(1170), and Glu(1172) were all replaced with Ala was also examined. The properties of R1173A and E1184A were comparable with those of wild-type MRP1, whereas the remaining mutants were either poorly expressed (R1166A, D1183A) or exhibited reduced transport of one or more organic anions (E1144A, D1179A, K1181A, (1169)AAQA). Same charge mutant D1183E was also not expressed, whereas expression and activity of R1166K were similar to wild-type MRP1. The moderate substrate-selective changes in transport activity displayed by mutants E1144A, D1179A, K1181A, and (1169)AAQA were accompanied by changes in orthovanadate-induced trapping of [alpha-(32)P]azidoADP by NBS2 indicating changes in ATP hydrolysis or release of ADP. In the case of E1144A, estradiol glucuronide no longer inhibited trapping of azidoADP. Together, our results demonstrate the extreme sensitivity of CL7 to mutation, consistent with its critical and complex dual role in both the proper folding and transport activity of MRP1.

A study of the properties and applications of electrostatic charged particle oscillators

This investigation originated from work by Dr. A.H. McIlraith of the National Physical Laboratory who, in 1966, described a new type of charged particle oscillator. This makes use of two equal cylindrical electrodes to constrain the particles in such a way that they follow extremely long oscillatory paths between the electrodes under the influence of an electrostatic field alone. The object of this work has been to study the principle of the oscillator in detail and to investigate its properties and applications. Any device which is capable of creating long electron trajectories has potential application in the field of ultra high vacuum technology. It was therefore considered that a critical review of the problems associated with the production and measurement of ultra high vacuum was relevant in the initial stages of the work. The oscillator has been applied with a considerable degree of success as a high energy electrostatic ion source. This offers several advantages over existing ion sources. It can be operated at much lower pressures without the need of a magnetic field. The oscillator principle has also been applied as a thermionic ionization gauge and has been compared with other ionization gauges to pressures as low as 5 x 10- 11 torr.. This new gauge exhibited a number of advantages over most of the existing gauges. Finally the oscillator has been used in an evaporation ion pump and has exhibited fairly high pumping speeds for argon gas relative to those for nitrogen. This investigation supports the original work of Dr. A.H. McIlraith and shows that his proposed oscillator has considerable potential in the fields of vacuum technology and electron physics.

Investigating the role of cholesterol in the formation of non-ionic surfactant based bilayer vesicles:thermal analysis and molecular dynamics

The aim of this research was to investigate the molecular interactions occurring in the formulation of non-ionic surfactant based vesicles composed monopalmitoyl glycerol (MPG), cholesterol (Chol) and dicetyl phosphate (DCP). In the formulation of these vesicles, the thermodynamic attributes and surfactant interactions based on molecular dynamics, Langmuir monolayer studies, differential scanning calorimetry (DSC), hot stage microscopy and thermogravimetric analysis (TGA) were investigated. Initially the melting points of the components individually, and combined at a 5:4:1 MPG:Chol:DCP weight ratio, were investigated; the results show that lower (90 C) than previously reported (120-140 C) temperatures could be adopted to produce molten surfactants for the production of niosomes. This was advantageous for surfactant stability; whilst TGA studies show that the individual components were stable to above 200 C, the 5:4:1 MPG:Chol:DCP mixture show ∼2% surfactant degradation at 140 C, compared to 0.01% was measured at 90 C. Niosomes formed at this lower temperature offered comparable characteristics to vesicles prepared using higher temperatures commonly reported in literature. In the formation of niosome vesicles, cholesterol also played a key role. Langmuir monolayer studies demonstrated that intercalation of cholesterol in the monolayer did not occur in the MPG:Chol:DCP (5:4:1 weight ratio) mixture. This suggests cholesterol may support bilayer assembly, with molecular simulation studies also demonstrating that vesicles cannot be built without the addition of cholesterol, with higher concentrations of cholesterol (5:4:1 vs 5:2:1, MPG:Chol:DCP) decreasing the time required for niosome assembly. © 2013 Elsevier B.V.

Consideration of the efficacy of non-ionic vesicles in the targeted delivery of oral vaccines

The fundamentals of this research were to exploit non-ionic surfactant technology for delivery and administration of vaccine antigens across the oral route and to gain a better understanding of vaccine trafficking. Using a newly developed method for manufacture of non-ionic surfactant vesicles (niosomes and bilosomes) lower process temperatures were adopted thus reducing antigen exposure to potentially damaging conditions. Vesicles prepared by this method offered high protection to enzymatic degradation, with only ~10 % antigen loss measured when vesicles incorporating antigen were exposed to enzyme digestion. Interestingly, when formulated using this new production method, the addition of bile salt to the vesicles offered no advantage in terms of stability within simulated gastro-intestinal conditions. Considering their ability to deliver antigen to their target site, results demonstrated that incorporation of antigen within vesicles enhanced delivery and targeting of the antigen to the Peyer's Patch, again with niosomes and bilosomes offering similar efficiency. Delivery to both the Peyer's patches and mesentery lymphatics was shown to be dose dependent at lower concentrations, with saturation kinetics applying at higher concentrations. This demonstrates that in the formulation of vaccine delivery systems, the lipid/antigen dose ratio is not only a key factor in production cost, but is equally a key factor in the kinetics of delivery and targeting of a vaccine system. © 2013 Controlled Release Society.

Consideration of the efficacy of non-ionic vesicles in the targeted delivery of oral vaccines

The fundamentals of this research were to exploit non-ionic surfactant technology for delivery and administration of vaccine antigens across the oral route and to gain a better understanding of vaccine trafficking. Using a newly developed method for manufacture of non-ionic surfactant vesicles (niosomes and bilosomes) lower process temperatures were adopted thus reducing antigen exposure to potentially damaging conditions. Vesicles prepared by this method offered high protection to enzymatic degradation, with only ~10 % antigen loss measured when vesicles incorporating antigen were exposed to enzyme digestion. Interestingly, when formulated using this new production method, the addition of bile salt to the vesicles offered no advantage in terms of stability within simulated gastro-intestinal conditions. Considering their ability to deliver antigen to their target site, results demonstrated that incorporation of antigen within vesicles enhanced delivery and targeting of the antigen to the Peyer's Patch, again with niosomes and bilosomes offering similar efficiency. Delivery to both the Peyer's patches and mesentery lymphatics was shown to be dose dependent at lower concentrations, with saturation kinetics applying at higher concentrations. This demonstrates that in the formulation of vaccine delivery systems, the lipid/antigen dose ratio is not only a key factor in production cost, but is equally a key factor in the kinetics of delivery and targeting of a vaccine system. © 2013 Controlled Release Society.

Physical and functional characterisation of apoptotic cell-derived extracellular vesicles

Damaged, aged or unwanted cells are removed from the body by an active process known as apoptosis. This highly orchestrated programme results in the exposure of 'flags' at the dying cell surface and the release of attractive signals to recruit phagocytes. Together these changes ensure efficient phagocytic removal of dying cells and prevention of inflammatory and autoimmune disorders. Extracellular vesicles (EV) are released from a variety of cells (both viable and apoptotic) and they serve as a novel means of intercellular communication. They range in size: 70-100nm ('exosomes') through 100-1000nm ('microparticles') to large vesicles released from dying cells ('apoptotic bodies'). Release of apoptotic cell-derived extracellular vesicles (acdEV) of less than 1000nm is an important mechanism by which phagocytes are attracted to sites of cell death. Using a variety of approaches we characterize the release, physical characteristics and function of acdEV. Using fluorescence microscopy we demonstrate release of ICAM-3 on acdEV from dying leukocytes and, through the use of resistive pulse technology (qNano, IZON Science), we accurately size and quantitate acdEV release. The function of acdEV is revealed through the use of both horizontal chemotaxis assays (Dunn chambers) and vertical transwell migration assays (Cell-IQ, CM Technologies). These assays reveal potent chemoattractive capacity of acdEV and associated ICAM-3. Additionally we demonstrate an additional novel function of acdEV as anti-inflammatory immune-modulators. These data support an integrated approach to the physical and functional analyses of EV.