Disinfection of clinical materials using photo-activated tolonium chloride solution

Photo-activated disinfection is beginning to be used in dental surgery to treat deep seated bacterial infection. It works by combining a photosensitiser and light of a specific frequency to generate singlet oxygen which is toxic to many types of bacteria. It is suggested that this technique could be used as a means to help treat infection more generally. To do so, it needs to work with materials and geometries exhibiting different physical and optical characteristics to teeth. In these trials, samples of stainless steel and polymethylmethacrylate were exposed to bacterial solutions of Staphylococcus aureus and Staphylococcus epidermis. These were treated with tolonium chloride-based photo-activated disinfection regimes showing positive results with typically 4 log10 reductions in colony forming units. Tests were also carried out using slotted samples to represent geometric features which might be found on implants. These tests, showed disinfectant effect however to a much lesser degree. © 2011 Inderscience Enterprises Ltd.

Octahedral molybdenum cluster complexes with aromatic sulfonate ligands

This article describes the synthesis, structures and systematic study of the spectroscopic and redox properties of a series of octahedral molybdenum metal cluster complexes with aromatic sulfonate ligands (nBu4N)2[{Mo6X8}(OTs)6] and (nBu4N)2[{Mo6X8}(PhSO3)6] (where X- is Cl-, Br- or I-; OTs- is p-toluenesulfonate and PhSO3 - is benzenesulfonate). All the complexes demonstrated photoluminescence in the red region and an ability to generate singlet oxygen. Notably, the highest quantum yields (>0.6) and narrowest emission bands were found for complexes with a {Mo6I8}4+ cluster core. Moreover, cyclic voltammetric studies revealed that (nBu4N)2[{Mo6X8}(OTs)6] and (nBu4N)2[{Mo6X8}(PhSO3)6] confer enhanced stability towards electrochemical oxidation relative to corresponding starting complexes (nBu4N)2[{Mo6X8}X6].

Aberrant reactive oxygen and nitrogen species generation in rheumatoid arthritis (RA):causes and consequences for immune function, cell survival, and therapeutic intervention

The infiltration and persistence of hematopoietic immune cells within the rheumatoid arthritis (RA) joint results in elevated levels of pro-inflammatory cytokines, increased reactive oxygen (ROS) and -nitrogen (RNS) species generation, that feeds a continuous self-perpetuating cycle of inflammation and destruction. Meanwhile, the controlled production of ROS is required for signaling within the normal physiological reaction to perceived "foreign matter" and for effective apoptosis. This review focuses on the signaling pathways responsible for the induction of the normal immune response and the contribution of ROS to this process. Evidence for defects in the ability of immune cells in RA to regulate the generation of ROS and the consequence for their immune function and for RA progression is considered. As the hypercellularity of the rheumatoid joint and the associated persistence of hematopoietic cells within the rheumatoid joint are symptomatic of unresponsiveness to apoptotic stimuli, the role of apoptotic signaling proteins (specifically Bcl-2 family members and the tumor suppressor p53) as regulators of ROS generation and apoptosis are considered, evaluating evidence for their aberrant expression and function in RA. We postulate that ROS generation is required for effective therapeutic intervention.

Ascorbate and α-tocopherol differentially modulate reactive oxygen species generation by neutrophils in response to FcγR and TLR agonists

Periodontitis, a ubiquitous chronic inflammatory disease, is associated with reduced antioxidant defences and neutrophil hyperactivity in terms of reactive oxygen species (ROS) generation. Its phenotype is thus characterized by oxidative stress. We have determined the effect of antioxidant micronutrients ascorbate and α-tocopherol on neutrophil ROS generation. Peripheral neutrophils from periodontally-healthy individuals (n = 20) were challenged with phorbol myristate acetate, IgG-opsonised Staphylococcus aureus, Fusobacterium nucleatum or PBS in the presence and absence of micronutrients (50 μM). Total and extracellular ROS were measured by luminol and isoluminol chemiluminescence respectively. Total and extracellular unstimulated, baseline ROS generation was unaffected by α-tocopherol, but inhibited by ascorbate and a combination of both micronutrients. Fcγ-receptor (Fcγ-R)-stimulated total or extracellular ROS generation was not affected by the presence of individual micronutrients. However, the combination significantly reduced extracellular FcγR-stimulated ROS release. Neither micronutrient inhibited TLR-stimulated total ROS, but the combination caused inhibition. Ascorbate and the micronutrient combination, but not α-tocopherol, inhibited extracellular ROS release by TLR-stimulated cells. Such micronutrient effects in vivo could be beneficial in reducing collateral tissue damage in chronic inflammatory diseases, such as periodontitis, while retaining immune-mediated neutrophil function. © The Author(s) 2012 Reprints and permissions: sagepub.co.uk/journalsPermissions.nav.

The nitric oxide-donating pravastatin derivative, NCX 6550 [(1S-[1α(βS*, δS*), 2α, 6α, 8β-(R*), 8aα]]-1,2,6,7,8,8a-hexahydro-β, δ, 6-trihydroxy-2-methyl-8-(2-methyl-1-oxobutoxy)-1-naphtalene-heptanoic acid 4-(nitrooxy)butyl ester)], reduces splenocyte adhesion and reactive oxygen species generation in normal and atherosclerotic mice

Statins possess anti-inflammatory effects that may contribute to their ability to slow atherogenesis, whereas nitric oxide (NO) also influences inflammatory cell adhesion. This study aimed to determine whether a novel NO-donating pravastatin derivative, NCX 6550 [(1S-[1∝(ßS*,dS*),2∝,6a∝,8ß-(R*),8a∝]]-1,2,6,7,8,8a-hexahydro-ß,δ,6-trihydroxy-2-methyl-8-(2-methyl-1-oxobutoxy)-1-naphthalene-heptanoic acid 4-(nitrooxy)butyl ester)], has greater anti-inflammatory properties compared with pravastatin in normal and atherosclerotic apolipoprotein E receptor knockout (ApoE-/-) mice. C57BL/6 and ApoE-/- mice were administered pravastatin (40 mg/kg), NCX 6550 (48.5 mg/kg), or vehicle orally for 5 days. Ex vivo studies assessed splenocyte adhesion to arterial segments and splenocyte reactive oxygen species (ROS) generation. NCX 6550 significantly reduced splenocyte adhesion to artery segments in both C57BL/6 (8.8 ± 1.9% versus 16.6 ± 6.7% adhesion; P < 0.05) and ApoE-/- mice (9.3 ± 2.9% versus 23.4 ± 4.6% adhesion; P < 0.05) concomitant with an inhibition of endothelial intercellular adhesion molecule-1 expression. NCX 6550 also significantly reduced phorbol 12-myristate 13-acetate-induced ROS production that was enhanced in isolated ApoE-/- splenocytes. Conversely, pravastatin had no significant effects on adhesion in normal or ApoE-/- mice but reduced the enhanced ROS production from ApoE-/- splenocytes. In separate groups of ApoE-/- mice, NCX 6550 significantly enhanced endothelium-dependent relaxation to carbachol in aortic segments precon-tracted with phenylephrine (-logEC50, 6.37 ± 0.37) compared with both vehicle-treated (-logEC50, 5.81 ± 0.15; P < 0.001) and pravastatin-treated (-logEC50, 5.57 ± 0.45; P < 0.05) mice. NCX 6550 also significantly reduced plasma monocyte chemoattractant protein-1 levels (648.8 pg/ml) compared with both vehicle (1191.1 pg/ml; P < 0.001) and pravastatin (847 ± 71.0 pg/ml; P < 0.05) treatment. These data show that NCX 6550 exerts superior anti-inflammatory actions compared with pravastatin, possibly through NO-related mechanisms.

Is the generation of neo-antigenic determinants by free radicals central to the development of autoimmune rheumatoid disease?

Biomolecules are susceptible to many different post-translational modifications that have important effects on their function and stability, including glycosylation, glycation, phosphorylation and oxidation chemistries. Specific conversion of aspartic acid to its isoaspartyl derivative or arginine to citrulline leads to autoantibody production in models of rheumatoid disease, and ensuing autoantibodies cross-react with native antigens. Autoimmune conditions associate with increased activation of immune effector cells and production of free radical species via NADPH oxidases and nitric oxide synthases. Generation of neo-antigenic determinants by reactive oxygen and nitrogen species ROS and RNS) may contribute to epitope spreading in autoimmunity. The oxidation of amino acids by peroxynitrite, hypochlorous acid and other reactive oxygen species (ROS) increases the antigenicity of DNA, LDL and IgG, generating ligands for which autoantibodies show higher avidity. This review focuses on the evidence for ROS and RNS in promoting the autoimmune responses observed in diseases rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). It considers the evidence for ROS/RNS-induced antigenicity arising as a consequence of failure to remove or repair ROS/RNS damaged biomolecules and suggests that an associated defect, probably in T cell signal processing or/or antigen presentation, is required for the development of disease.

Antioxidants, reactive oxygen and nitrogen species, gene induction and mitochondrial function

Redox-sensitive cell signalling Thiol groups and the regulation of gene expression Redox-sensitive signal transduction pathways Protein kinases Protein phosphatases Lipids and phospholipases Antioxidant (electrophile) response element Intracellular calcium signalling Transcription factors NF-?B AP-1 p53 Cellular responses to oxidative stress Cellular responses to change in redox state Proliferation Cell death Immune cell function Reactive oxygen and nitrogen species – good or bad? Reactive oxygen species and cell death Reactive oxygen species and inflammation Are specific reactive oxygen species and antioxidants involved in modulating cellular responses? Specific effects of dietary antioxidants in cell regulation Carotenoids Vitamin E Flavonoids Inducers of phase II enzymes Disease states affected Oxidants, antioxidants and mitochondria Introduction Mitochondrial generation of reactive oxygen and nitrogen species Mitochondria and apoptosis Mitochondria and antioxidant defences Key role of mitochondrial GSH in the defence against oxidative damage Mitochondrial oxidative damage Direct oxidative damage to the mitochondrial electron transport chain Nitric oxide and damage to mitochondria Effects of nutrients on mitochondria Caloric restriction and antioxidants Lipids Antioxidants Techniques and approaches Mitochondrial techniques cDNA microarray approaches Proteomics approaches Transgenic mice as tools in antioxidant research Gene knockout and over expression Transgenic reporter mice Conclusions Future research needs

Probing molecular changes induced in DNA by reactive oxygen species with monoclonal antibodies

Antibodies reactive with native double stranded DNA are characteristic of the chronic inflammatory disease systemic lupus erythematosus. Native DNA is however, a poor immunogen and the mechanism of anti-DNA antibody production is incompletely understood. Modification of DNA can increase its immunogenicity and in inflammatory disease states reactive oxygen species produced from phagocytic cells have been shown to thus modify DNA. In this study, monoclonal antibodies produced spontaneously by two mice strains with lupus-like disease were used in a competition ELISA to monitor changes to DNA induced by reactive oxygen species. Different procedures for reactive oxygen species generation were found to cause distinct and characteristic changes to DNA involving modifications of base residues, the sugar-phosphate backbone and the gross conformational structure of double-stranded DNA. In view of this, it may be possible to use these antibodies further to probe DNA and infer the source and nature of the reactive oxygen species it has been exposed to, particularly in vivo.

The anti-inflammatory actions of methotrexate are critically dependent upon the production of reactive oxygen species

1. The mechanism of action by which methotrexate (MTX) exerts its anti-inflammatory and immunosuppressive effects remains unclear. The aim of this study is to investigate the hypothesis that MTX exerts these effects via the production of reactive oxygen species (ROS). 2. Addition of MTX (100 nM-10 μM) to U937 monocytes induced a time and dose dependent increase in cytosolic peroxide [peroxide] cyt from 6-16 h. MTX also caused corresponding monocyte growth arrest, which was inhibited (P<0.05) by pre-treatment with N-acetylcysteine (NAC; 10 mM) or glutathione (GSH; 10 mM). In contrast, MTX induction of [peroxide] cyt in Jurkat T cells was more rapid (4 h; P<0.05), but was associated with significant apoptosis at 16 h at all doses tested (P<0.05) and was significantly inhibited by NAC or GSH (P<0.05). 3. MTX treatment of monocytes (10 nM-10 μM) for 16 h significantly reduced total GSH levels (P<0.05) independently of dose (P>0.05). However in T-cells, GSH levels were significantly elevated following 30 nM MTX treatment (P<0.05) but reduced by doses exceeding 1 μM compared to controls (P<0.05). 4. MTX treatment significantly reduced monocyte adhesion to 5 h and 24 h LPS (1 μg ml -1) activated human umbilical vein endothelial cells (HUVEC; P<0.05) but not to resting HUVEC. Pre-treatment with GSH prevented MTX-induced reduction in adhesion. 5. In conclusion, ROS generation by MTX is important for cytostasis in monocytes and cytotoxicity T-cells. Furthermore, MTX caused a reduction in monocyte adhesion to endothelial cells, where the mechanism of MTX action requires the production of ROS. Therefore its clinical efficacy can be attributed to multiple targets.

Reactive oxygen and nitrogen species production in cardiomyoblasts during hypoxia and reoxygenation

Hypoxia is a stress condition in which tissues are deprived of an adequate O2 supply; this may trigger cell death with pathological consequences in cardiovascular or neurodegenerative disease. Reperfusion is the restoration of an oxygenated blood supply to hypoxic tissue and can cause more cell injury. The kinetics and consequences of reactive oxygen and nitrogen species (ROS/RNS) production in cardiomyoblasts are poorly understood. The present study describes the systematic characterization of the kinetics of ROS/RNS production and their roles in cell survival and associated protection during hypoxia and hypoxia/reperfusion. H9C2 cells showed a significant loss of viability under 2% O2 for 30min hypoxia and cell death; associated with an increase in protein oxidation. After 4h, apoptosis induction under 2% O2 and 10% O2 was dependent on the production of mitochondrial superoxide (O2-•) and nitric oxide (•NO), partly from nitric oxide synthase (NOS). Both apoptotic and necrotic cell death during 2% O2 for 4h could be rescued by the mitochondrial complex I inhibitor; rotenone and NOS inhibitor; L-NAME. Both L-NAME and the NOX (NADPH oxidase) inhibitor; apocynin reduced apoptosis under 10% O2 for 4h hypoxia. The mitochondrial uncoupler; FCCP significantly reduced cell death via a O2-• dependent mechanism during 2% O2, 30min hypoxia. During hypoxia (2% O2, 4h)/ reperfusion (21% O2, 2h), metabolic activity was significantly reduced with increased production of O2-• and •NO, during hypoxia but, partially restored during reperfusion. O2-• generation during hypoxia/reperfusion was mitochondrial and NOX- dependent, whereas •NO generation depended on both NOS and non-enzymatic sources. Inhibition of NOS worsened metabolic activity during reperfusion, but did not effect this during sustained hypoxia. Nrf2 activation during 2% O2, a sustained hypoxia and reperfusion was O2-•/•NO dependent. Inhibition of NF-?B activation aggravated metabolic activity during 2% O2, 4h hypoxia. In conclusion, mitochondrial O2-•, but, not ATP depletion is the major cause of apoptotic and necrotic cell death in cardiomyoblasts under 2% O2, 4h hypoxia, whereas apoptotic cell death under 10% O2, 4h, is due to NOS-dependent •NO. The management of ROS/RNS rather than ATP is required for improved survival during hypoxia. O2-• production from mitochondria and NOS is cardiotoxic during hypoxia/reperfusion. NF-?B activation during hypoxia and NOS activation during reperfusion is cardiomyoblast protective.

Ceramide and reactive oxygen species (ROS) as signal transduction molecules in inflammation

Reactive oxygen species (ROS) and the sphingolipid ceramide are each partly responsible for the intracellular signal transduction of a variety of physiological, pharmacological or environmental agents. Furthermore, the enhanced production of many of these agents, that utilise ROS and ceramide as signalling intermediates, is associated with the aetiologies of several vascular diseases (e.g. atherosclerosis) or disorders of inflammatory origin (e.g. rheumatoid arthritis; RA). Excessive monocyte recruitment and uncontrolled T cell activation are both strongly implicated in the chronic inflammatory responses that are associated with these pathologies. Therefore the aims of this thesis are (1) to further elucidate the cellular responses to modulations in intracellular ceramide/ROS levels in monocytes and T cells, in order to help resolve the mechanisms of progression of these diseases and (2) to examine both existing agents (methotrexate) and novel targets for possible therapeutic manipulation. Utilising synthetic, short chain ceramide to mimic the cellular responses to fluctuations in natural endogenous ceramide or, stimulation of CD95 to induce ceramide formation, it is described here that ceramide targets and manipulates two discrete sites responsible for ROS generation, preceding the cellular responses of growth arrest in U937 monocytes and apoptosis in Jurkat T-cells. In both cell types, transient elevations in mitochondrial ROS generation were observed. However, the prominent redox altering effects appear to be the ceramide-mediated reduction in cytosolic peroxide, the magnitude of which dictates in part the cellular response in U937 monocytes, Jurkat T-cells and primary human peripheral blood resting or PHA-activated T-cells in vitro. The application of synthetic ceramides to U937 monocytes for short (2 hours) or long (16 hours) treatment periods reduced the membrane expression of proteins associated with cell-cell interaction. Furthermore, ceramide treated U937 monocytes demonstrated reduced adhesion to 5 or 24 hour LPS activated human umbilical vein endothelial cells (HUVEC) but not resting HUVEC. Consequently it is hypothesised that the targeted treatment of monocytes from patients with cardiovascular diseases with short chain synthetic ceramide may reduce disease progression. Herein, the anti-inflammatory and immunosuppressant drug, methotrexate, is described to require ROS production for the induction of cytostasis or cytotoxicity in U937 monocytes and Jurkat T-cells respectively. Further, ROS are critical for methotrexate to abrogate monocyte interaction with activated HUVEC in vitro. The histological feature of RA of enhanced infiltration, survivability and hyporesponsiveness of T-cells within the diseased synovium has been suggested to arise from aberrant signalling. No difference in the concentrations of endogenous T-cell ceramide, the related lipid diacylglycerol (DAG) and cytosolic peroxide ex vivo was observed. TCR activation following PHA exposure in vitro for 72 hours did not induced maintained perturbations in DAG or ceramide in T-cells from RA patients or healthy individuals. However, T-cells from RA patients failed to upregulate cytosolic peroxide in response to PHA, unlike those from normals, despite expressing identical levels of the activation marker CD25. This inability to upregulate cytosolic peroxide may contribute to the T-cell pathology associated with RA by affecting the signalling capacity of redox sensitive biomolecules. These data highlight the importance of two distinctive cellular pools of ROS in mediating complex biological events associated with inflammatory disease and suggest that modulation of cellular ceramides represents a novel therapeutic strategy to minimise monocyte recruitment.

Techno-economic assessment and uncertainty analysis of thermochemical processes for second generation biofuels

Biomass-To-Liquid (BTL) is one of the most promising low carbon processes available to support the expanding transportation sector. This multi-step process produces hydrocarbon fuels from biomass, the so-called “second generation biofuels” that, unlike first generation biofuels, have the ability to make use of a wider range of biomass feedstock than just plant oils and sugar/starch components. A BTL process based on gasification has yet to be commercialized. This work focuses on the techno-economic feasibility of nine BTL plants. The scope was limited to hydrocarbon products as these can be readily incorporated and integrated into conventional markets and supply chains. The evaluated BTL systems were based on pressurised oxygen gasification of wood biomass or bio-oil and they were characterised by different fuel synthesis processes including: Fischer-Tropsch synthesis, the Methanol to Gasoline (MTG) process and the Topsoe Integrated Gasoline (TIGAS) synthesis. This was the first time that these three fuel synthesis technologies were compared in a single, consistent evaluation. The selected process concepts were modelled using the process simulation software IPSEpro to determine mass balances, energy balances and product distributions. For each BTL concept, a cost model was developed in MS Excel to estimate capital, operating and production costs. An uncertainty analysis based on the Monte Carlo statistical method, was also carried out to examine how the uncertainty in the input parameters of the cost model could affect the output (i.e. production cost) of the model. This was the first time that an uncertainty analysis was included in a published techno-economic assessment study of BTL systems. It was found that bio-oil gasification cannot currently compete with solid biomass gasification due to the lower efficiencies and higher costs associated with the additional thermal conversion step of fast pyrolysis. Fischer-Tropsch synthesis was the most promising fuel synthesis technology for commercial production of liquid hydrocarbon fuels since it achieved higher efficiencies and lower costs than TIGAS and MTG. None of the BTL systems were competitive with conventional fossil fuel plants. However, if government tax take was reduced by approximately 33% or a subsidy of £55/t dry biomass was available, transport biofuels could be competitive with conventional fuels. Large scale biofuel production may be possible in the long term through subsidies, fuels price rises and legislation.