ROS as signalling molecules in T cells - Evidence for abnormal redox signalling in the autoimmune disease, rheumatoid arthritis

Reactive oxygen species are recognised as important signalling molecules within cells of the immune system. This is, at least in part, due to the reversible activation of kinases, phosphatases and transcription factors by modification of critical thiol residues. However, in the chronic inflammatory disease rheumatoid arthritis, cells of the immune system are exposed to increased levels of oxidative stress and the T cell becomes refractory to growth and death stimuli. This contributes to the perpetuation of the immune response. As many of the effective therapies used in the treatment of rheumatoid arthritis modulate intracellular redox state, this raises the question of whether increased oxidative stress is causative of T-cell hyporesponsiveness. To address this hypothesis, this review considers the putative sources of ROS involved in normal intracellular signalling in T cells and the evidence in support of abnormal ROS fluxes contributing to T-cell hyporesponsiveness. © W. S. Maney & Son Ltd.

A cationic vaccine adjuvant based on a saturated quaternary ammonium lipid have different in vivo distribution kinetics and display a distinct CD4 T cell-inducing capacity compared to its unsaturated analog

Adjuvants are often composed of different constituents that can be divided into two groups based on their primary activity: the delivery system which carries and presents the vaccine antigen to antigen-presenting cells, and the immunostimulator that activates and modulates the ensuing immune response. Herein, we have investigated the importance of the delivery system and in particular its physical characteristics by comparing the delivery properties of two lipids which differ only in the degree of saturation of the acyl chains, rendering the liposomes either rigid (DDA, dimethyldioctadecylammonium) or highly fluid (DODA, dimethyldioleoylammonium) at physiological temperature. We show that these delivery systems are remarkably different in their ability to prime a Th1-directed immune response with the rigid DDA-based liposomes inducing a response more than 100 times higher compared to that obtained with the fluid DODA-based liposomes. Upon injection with a vaccine antigen, DDA-based liposomes form a vaccine depot that results in a continuous attraction of antigen-presenting cells that engulf a high amount of adjuvant and are subsequently efficiently activated as measured by an elevated expression of the co-stimulatory molecules CD40 and CD86. In contrast, the fluid DODA-based liposomes are more rapidly removed from the site of injection resulting in a lower up-regulation of co-stimulatory CD40 and CD86 molecules on adjuvant-positive antigen-presenting cells. Additionally, the vaccine antigen is readily dissociated from the DODA-based liposomes leading to a population of antigen-presenting cells that are antigen-positive but adjuvant-negative and consequently are not activated. These studies demonstrate the importance of studying in vivo characteristics of the vaccine components and furthermore show that physicochemical properties of the delivery system have a major impact on the vaccine-induced immune response. © 2012 Elsevier B.V. All rights reserved.

Regulation of the petrol industry in the UK: issues and evidence

The petrol industry has been investigated twice by the Monopolies and Mergers Commission in the last 20 years. On both occasions the MMC found that the conduct of the companies was not against the public interest. These findings were based on the perceived stable relationship between oil and petrol prices. This paper develops a model of petrol price using a co-integration approach, concluding that one must question the findings of the MMC.

Innovation in the pharmaceutical industry:a study of the effects of regulation on the U.K. pharmaceutical industry

A history of government drug regulation and the relationship between the pharmaceutical companies in the U.K. and the licensing authority is outlined. Phases of regulatory stringency are identified with the formation of the Committees on Safety of Drugs and Medicines viewed as watersheds. A study of the impact of government regulation on industrial R&D activities focuses on the effects on the rate and direction of new product innovation. A literature review examines the decline in new chemical entity innovation. Regulations are cited as a major but not singular cause of the decline. Previous research attempting to determine the causes of such a decline on an empirical basis is given and the methodological problems associated with such research are identified. The U.K. owned sector of the British pharmaceutical industry is selected for a study employing a bottom-up approach allowing disaggregation of data. A historical background to the industry is provided, with each company analysed or a case study basis. Variations between companies regarding the policies adopted for R&D are emphasised. The process of drug innovation is described in order to determine possible indicators of the rate and direction of inventive and innovative activity. All possible indicators are considered and their suitability assessed. R&D expenditure data for the period 1960-1983 is subsequently presented as an input indicator. Intermediate output indicators are treated in a similar way and patent data are identified as a readily-available and useful source. The advantages and disadvantages of using such data are considered. Using interview material, patenting policies for most of the U.K. companies are described providing a background for a patent-based study. Sources of patent data are examined with an emphasis on computerised systems. A number of searches using a variety of sources are presented. Patent family size is examined as a possible indicator of an invention's relative importance. The patenting activity of the companies over the period 1960-1983 is given and the variation between companies is noted. The relationship between patent data and other indicators used is analysed using statistical methods resulting in an apparent lack of correlation. An alternative approach taking into account variations in company policy and phases in research activity indicates a stronger relationship between patenting activity, R&D Expenditure and NCE output over the period. The relationship is not apparent at an aggregated company level. Some evidence is presented for a relationship between phases of regulatory stringency, inventive and innovative activity but the importance of other factors is emphasised.

Molecular regulation of iron uptake in pseudomonas aeruginosa

The microbial demand for iron is often met by the elaboration of siderophores into the surrounding medium and expression of cognate outer membrane receptors for the ferric siderophore complexes. Conditions of iron limitation, such as those encountered in vivo, cause Pseudomonas aeruginosa to express two high-affinity iron-uptake systems based on pyoverdin and pyochelin. These systems will operate both in the organism's natural habitat, soil and water, where the solubility of iron at neutral pH is extremely low, and in the human host where the availability of free iron is too low to sustain bacterial growth due to the iron-binding glycoproteins transferrin and lactoferrin. Cross-feeding and radiolabelled iron uptake experiments demonstrated that pyoverdin biosynthesis and uptake were highly heterogeneous amongst P.aeruginosa strains, that growth either in the presence of pyoverdin or pyochelin resulted in induction of specific IROMPs, and that induction of iron uptake is siderophore-specific. The P.aeruginosa Tn5 mutant PH1 is deficient in ferripyoverdin uptake and resistant to pyocin Sa, suggesting that the site of interaction of pyocin Sa is a ferripyoverdin receptor. Additional Tn5 mutants appeared to exploit different strategies to achieve pyocin Sa-resistance, involving modifications in expression of pyoverdin-mediated iron uptake, indicating that complex regulatory systems exist to enable these organisms to compete effectively for iron. Modulation of expression of IROMPs prompted a study of the mechanism of uptake of a semi-synthetic C(7) α-formamido substituted cephalosporin BRL 41897A. Sensitivity to this agent correlated with expression of the 75 kDa ferri-pyochelin receptor and demonstrated the potential of high-affinity iron uptake systems for targeting of novel antibiotics. Studies with ferri-pyoverdin uptake-deficient mutant PH1 indicated that expression of outer membrane protein G (OprG), which is usually expressed under iron-rich conditions and repressed under iron-deficient conditions, was perturbed. Attempts were made to clone the oprG gene using a degenerate probe based on the N-terminal amino acid sequence. A strongly hybridising HindIll restriction fragment was cloned and sequenced, but failed to reveal an open reading frame correspondmg to OprG. However, there appears to be good evidence that a part of the gene codmg for the hydrophilic membrane-associated ATP-binding component of a hitherto uncharacterised periplasmic- binding-protein-dependent transport system has been isolated. The full organisation and sequence of the operon, and substrate for this putative transport system, are yet: to be elucidated,

Self-regulation of health and safety in a Local Authority with particular reference to safety representatives, supervisors and safety committees

The Report of the Robens Committee (1972), the Health and Safety at Work Act (1974) and the Safety Representatives and Safety Committees Regulations (1977) provide the framework within which this study of certain aspects of health and safety is carried out. The philosophy of self-regulation is considered and its development is set within an historical and an industrial relations perspective. The research uses a case study approach to examine the effectiveness of self-regulation in health and safety in a public sector organisation. Within this approach, methodological triangulation employs the techniques of interviews, questionnaires, observation and documentary analysis. The work is based in four departments of a Scottish Local Authority and particular attention is given to three of the main 'agents' of self-regulation - safety representatives, supervisors and safety committees and their interactions, strategies and effectiveness. A behavioural approach is taken in considering the attitudes, values, motives and interactions of safety representatives and management. Major internal and external factors, which interact and which influence the effectiveness of joint self-regulation of health and safety, are identified. It is emphasised that an organisation cannot be studied without consideration of the context within which it operates both locally and in the wider environment. One of these factors, organisational structure, is described as bureaucratic and the model of a Representative Bureaucracy described by Gouldner (1954) is compared with findings from the present study. An attempt is made to ascertain how closely the Local Authority fits Gouldner's model. This research contributes both to knowledge and to theory in the subject area by providing an in-depth study of self-regulation in a public sector organisation, which when compared with such studies as those of Beaumont (1980, 1981, 1982) highlights some of the differences between the public and private sectors. Both empirical data and hypothetical models are used to provide description and explanation of the operation of the health and safety system in the Local Authority. As data were collected during a dynamic period in economic, political and social terms, the research discusses some of the effects of the current economic recession upon safety organisation.

The formation and impact of hazard control policy:a study of the regulation of white lead paint in Britain

Government regulation of industrial hazards is examined in the context of the economic and technical processes of industrial development. Technical problems and costs of control are considered as factors in both the formation and impact of regulation. This thesis focuses on an historical case-study of the regulation of the hazard to painting workers from the use of lead pigments in paint. A regulatory strategy based on the prohibition of lead paints gained initial acceptance within the British state in 1911, but was subsequently rejected in favour of a strategy that allowed continued use of lead paint subject to hygiene precautions. The development of paint technology and its determinants, including concern about health hazards, are analysed, focusing on the innovation and diffusion into the paint industry of the major white pigments: white lead (PbC03 .PB(OH)2)and its substitutes. The process of regulatory development is examined, and the protracted and polarised regulatory d~bate contrasted to the prevailing 'consensual' methods of workplace regulation. The rejection of prohibition is analysed in terms of the different political and technical resources of those groups in conflict over this policy. This highlights the problems of consensus formation around such a strategy, and demonstrates certain constraints on state regulatory activity, particularly regarding industrial development. Member-states of the International Labour Organisation agreed to introduce partial prohibition of lead paint in 1921. Whether this was implemented is related to the economic importance of lead and non-lead metal and pigment industries to a nation. An analysis is made of the control of lead poisoning. The rate of control is related to the economic and technological trajectory of the regulated industry. Technical and organisational characteristics are considered as well as regulatory factors which range from voluntary compliance and informal pressures to direct legal requirements. The implications of this case-study for the analysis of the development and impacts of regulation are assessed.

Copper N2S2 Schiff base macrocycles:the effect of structure on redox potential

A series of bis-salicylidene based N2S2 copper macrocycles were prepared, structurally characterised and subjected to electrochemical analysis. The aim was to investigate the effects of length of polymethylene chains between either the imine donors or the sulfur donors on redox state and potential of the metal. The complexes structurally characterised had either distorted square planar or tetrahedral geometries depending on their oxidation state (Cu2+ or Cu+, respectively), and the N-(CH2)n-N bridge was found to be most critical moiety in determining the redox potential and oxidation state of the copper macrocycles, with relatively little change in these properties caused by lengthening the S-(CH2)n-S bridge from two to three carbons. In fact, a weakness was observed in the complexes at the sulfur donor, as further lengthening of the S-(CH2)n-S methylene bridge to four carbons caused fission of the carbon-sulfur bond to give dimeric rings and supramolecular assemblies. Cu+ complexes could be oxidised to Cu2+ by tert-butylhydroperoxide, with a corresponding change in the spectrophotometric properties, and likewise Cu2+ complexes could be reduced to Cu+ by treatment with ß-mercaptoethylamine. However, repeated redox cycles appeared to compromise the stability of the macrocycles, most probably by a competing oxidation of the ligand. Thus the copper N2S2 macrocycles show potential as redox sensors, but further development is required to improve their performance in a biochemical environment.

Redox signalling and detection of protein oxidation by mass spectrometry

It is now recognised that redox control of proteins plays an important role in many signalling pathways both in health and disease. Proteins can undergo a wide variety of oxidative post-translational modifications (oxPTM); while the reversible modifications are thought to be most important in physiological processes, non-reversible oxPTM may contribute to pathological situations and disease. The oxidant is also important in determining the type of oxPTM (chlorination, nitration, etc.), and the susceptibilities of residues vary depending on their structural location. The best characterized oxPTMs involved in signalling modulation are partial oxidations of cysteine to the disulfide, glutathionylated or sulfenic acid forms, but there is increasing evidence that specific oxidations of methionine and tyrosine may have some biological roles. Well understood examples of oxidative regulation include protein tyrosine phosphatases, e.g. PTP1B/C, and members of the MAPK pathways such as MEKK1 and ASK1. Transcription factors such as NFkB and Nrf-2 are also regulated by redox-active cysteines. Improved methods for analysing specific oxPTMs in biological samples are critical for understanding the physiological and pathological roles of these changes, and tandem or MS3 mass spectrometry techniques interfaced with nano-LC separation are being now used. MS3 fragmentation markers for a variety of oxidized residues including tyrosine, tryptophan and proline have been identified, and a precursor ion scanning method that allows the selective identification of these oxPTMs in complex samples has been developed. Such advances in technology offer potential for biomarker development, disease diagnosis and understanding pathology.

A sulphonate based hydrogel for nucleus pulposus repair

Introduction: Lower back pain treatment and compensation costs >$80 billion overall in the US. 75% of back pain is due to disc degeneration in the lumbar region of the spine. Current treatment comprises of painkillers and bed rest or as a more radical solution – interbody cage fusion. In the early stages of disc degeneration the patient would benefit from addition of an injectable gel which polymerises in situ to support the degenerated nucleus pulposus. This involves a material which is an analogue of the natural tissue capable of restoring the biomechanical properties of the natural disc. The nucleus pulposus of the intervertebral disc is an example of a natural proteoglycan consisting of a protein core with negatively charged keratin and chondroitin sulphate attached. As a result of the high fixed charge density of the proteoglycan, the matrix exerts an osmotic swelling pressure drawing sufficient water into support the spinal system. Materials and Methods: NaAMPs (sodium 2- acrylamido 2-methyl propane sulphonic acid) and KSPA (potassium 3- sulphopropyl acrylate) were selected as monomers, the sulphonate group being used to mimic the natural sulphate group. These are used in dermal applications involving chronic wounds and have acceptably low cytotoxicity. Other hydrophilic carboxyl, amide and hydroxyl monomers such as 2-hydroxyethyl acrylamide, ß-carboxyethyl acrylate, acryloyl morpholine, and polyethylene glycol (meth)acrylate were used as diluents together with polyethyleneglycol di(meth)acrylate and hydrophilic multifunctional macromers as cross-linker. Redox was the chosen method of polymerisation and a range of initiators were investigated. Components were packaged in two solutions each containing a redox pair. A dual syringe method of injection into the cavity was used, the required time for polymerisation is circa 3-7 minutes. The final materials were tested using a Bohlin CVO Rheometer cycling from 0.5-25Hz at 37oC to measure the modulus. An in-house compression testing method was developed, using dialysis tubing to mimic the cavity, the gels were swelled in solutions of various osmolarity and compressed to ~ 20%. The pre-gel has also been injected into sheep spinal segments for mechanical compression testing to demonstrate the restoration of properties upon use of the gel. Results and Discussion: Two systems resulted using similar monomer compositions but different initiation and crosslinking agents. NaAMPs and KSPA were used together at a ratio of ~1:1 in both systems with 0.25-2% crosslinking agent, diacrylate or methacrylate. The two initiation systems were ascorbic acid/oxone, and N,N,N,N - tetramethylethylenediamine (TEMED)/ potassium persulphate. These systems produced gelation within 3-7 and 3-5 minutes respectively. Storage of the two component systems was shown to be stable for approximately one month after mixing, in the dark, refrigerated at 1-4oC. The gelation was carried out at 37oC. Literature values for the natural disc give elastic constants ranging from 3-8kPa. The properties of the polymer can be tailored by altering crosslink density and monomer composition and are able to match those of the natural disc. It is possible to incorporate a radio-opaque (histodenz) to enable x-ray luminescence during and after injection. At an inclusion level of 5% the gel is clearly visible and polymerisation and mechanical properties are not altered. Conclusion: A two-pac injection system which will polymerise in situ, that can incorporate a radio-opaque, has been developed. This will reinforce the damaged nucleus pulposus in degenerative disc disease restoring adequate hydration and thus biomechanical properties. Tests on sheep spine segments are currently being carried out to demonstrate that a disc containing the gel has similar properties to an intact disc in comparison to one with a damaged nucleus.

Is a little sunshine all we need? On the impact of sunshine regulation on profits, productivity and prices in the Dutch drinking water sector

This paper analyzes the performance of Dutch drinking water utilities before and after the introduction of sunshine regulation, which involves publication of the performance of utilities but no formal price regulation. By decomposing profit change into its economic drivers, our results suggest that, in the Dutch political and institutional context, sunshine regulation was effective in improving the productivity of publicly organised services. Nevertheless, while sunshine regulation did bring about a moderate reduction in water prices, sustained and substantial economic profits suggest that it may not have the potential to fully align output prices with economic costs in the long run. In methodological terms, the DEA based profit decomposition is extended to robust and conditional non-parametric efficiency measures, so as to account better for both uncertainty and differences in operating environment between utilities.

Impact of diet composition on blood glucose regulation

Abstract Nutritional management of blood glucose levels is a strategic target in the prevention and management of type 2 diabetes mellitus (T2DM). To implement such an approach it is essential to understand the effect of food on glycaemic regulation and on the underlying metabolic derangements. This comprehensive review summarises the results from human dietary interventions exploring the impact of dietary components on blood glucose levels. Included are the major macronutrients; carbohydrate, protein and fat, micronutrient vitamins and minerals, non-nutrient phytochemicals and additional foods including low-calorie sweeteners, vinegar and alcohol. Based on the evidence presented in this review, it is clear that dietary components have significant and clinically relevant effects on blood glucose modulation. An integrated approach that includes reducing excess body weight, increased physical activity along with a dietary regime to regulate blood glucose levels will not only be advantages in T2DM management, but will benefit the health of the population and limit the increasing worldwide incidence of T2DM.

Oxidised phospholipid regulation of toll-like receptor signalling

Toll-like receptors (TLRs) serve to initiate inflammatory signalling in response to the detection of conserved microbial molecules or products of host tissue damage. Recent evidence suggests that TLR-signalling plays a considerable role in a number of inflammatory diseases, including atherosclerosis and arthritis. Agents which modulate TLR-signalling are, therefore, receiving interest in terms of their potential to modify inflammatory disease processes. One such family of molecules, the oxidised phospholipids (OxPLs), which are formed as a result of inflammatory events and accumulate at sites of chronic inflammation, have been shown to modulate TLR-signalling in both in vitro and in vivo systems. As the interaction between OxPLs and TLRs may play a significant role in chronic inflammatory disease processes, consideration is given in this review to the potential role of OxPLs in the regulation of TLR-signalling.

NADPH oxidase-dependent redox signalling in cardiac hypertrophy, remodelling and failure

Markers of increased oxidative stress are known to be elevated following acute myocardial infarction and in the context of chronic left ventricular hypertrophy or heart failure, and their levels may correlate with the degree of contractile dysfunction or cardiac deficit. An obvious pathological mechanism that may account for this correlation is the potential deleterious effects of increased oxidative stress through the induction of cellular dysfunction, energetic deficit or cell death. However, reactive oxygen species have several much more subtle effects in the remodelling or failing heart that involve specific redox-regulated modulation of signalling pathways and gene expression. Such redox-sensitive regulation appears to play important roles in the development of several components of the phenotype of the failing heart, for example cardiomyocyte hypertrophy, interstitial fibrosis and chamber remodelling. In this article, we review the evidence supporting the involvement of reactive oxygen species and redox signalling pathways in the development of cardiac hypertrophy and heart failure, with a particular focus on the NADPH oxidase family of superoxide-generating enzymes which appear to be especially important in redox signalling.

Glutaredoxin-1 up-regulation induces soluble vascular endothelial growth factor receptor 1, attenuating post-ischemia limb revascularization

Glutaredoxin-1 (Glrx) is a cytosolic enzyme that regulates diverse cellular function by removal of GSH adducts from S-glutathionylated proteins including signaling molecules and transcription factors. Glrx is up-regulated during inflammation and diabetes. Glrx overexpression inhibits VEGF-induced endothelial cell (EC) migration. The aim was to investigate the role of up-regulated Glrx in EC angiogenic capacities and in vivo revascularization in the setting of hind limb ischemia. Glrx overexpressing EC from Glrx transgenic mice (TG) showed impaired migration and network formation and secreted higher level of soluble VEGF receptor 1 (sFlt), an antagonizing factor to VEGF. After hind limb ischemia surgery Glrx TG mice demonstrated impaired blood flow recovery, associated with lower capillary density and poorer limb motor function compared to wild type littermates. There were also higher levels of anti-angiogenic sFlt expression in the muscle and plasma of Glrx TG mice after surgery. Non-canonical Wnt5a is known to induce sFlt. Wnt5a was highly expressed in ischemic muscles and EC from Glrx TG mice, and exogenous Wnt5a induced sFlt expression and inhibited network formation in human microvascular EC. Adenoviral Glrx-induced sFlt in EC was inhibited by a competitive Wnt5a inhibitor. Furthermore, Glrx overexpression removed GSH adducts on p65 in ischemic muscle and EC, and enhanced nuclear factor kappa B (NF-kB) activity which was responsible for Wnt5a-sFlt induction. Taken together, up-regulated Glrx induces sFlt in EC via NF-kB -dependent Wnt5a, resulting in attenuated revascularization in hind limb ischemia. The Glrx-induced sFlt may be a part of mechanism of redox regulated VEGF signaling.