Determinants of biological activity of nanomaterials in innate immunity cells: the role of aspect ratio, environmental contaminants and protein corona

As defined by the European Union, “ ’Nanomaterial’ (NM) means a natural, incidental or manufactured material containing particles, in an unbound state or as an aggregate or agglomerate, where, for 50 % or more of the particles in the number size distribution, one or more external dimensions is in the size range 1 nm-100 nm ” (2011/696/UE). Given their peculiar physico-chemical features, nanostructured materials are largely used in many industrial fields (e.g. cosmetics, electronics, agriculture, biomedical) and their applications have astonishingly increased in the last fifteen years. Nanostructured materials are endowed with very large specific surface area that, besides making them very useful in many industrial processes, renders them very reactive towards the biological systems and, hence, potentially endowed with significant hazard for human health. For these reasons, in recent years, many studies have been focused on the identification of toxic properties of nanostructured materials, investigating, in particular, the mechanisms behind their toxic effects as well as their determinants of toxicity. This thesis investigates two types of nanostructured TiO2 materials, TiO2 nanoparticles (NP), which are yearly produced in tonnage quantities, and TiO2 nanofibres (NF), a relatively novel nanomaterial. Moreover, several preparations of MultiWalled Carbon Nanotubes (MWCNT), another nanomaterial widely present in many products, are also investigated.- Although many in vitro and in vivo studies have characterized the toxic properties of these materials, the identification of their determinants of toxicity is still incomplete. The aim of this thesis is to identify the structural determinants of toxicity, using several in vitro models. Specific fields of investigation have been a) the role of shape and the aspect ratio in the determination of biological effects of TiO2 nanofibres of different length; b) the synergistic effect of LPS and TiO2 NP on the expression of inflammatory markers and the role played therein by TLR-4; c) the role of functionalization and agglomeration in the biological effects of MWCNT. As far as biological effects elicited by TiO2 NF are concerned, the first part of the thesis demonstrates that long TiO2 nanofibres caused frustrated phagocytosis, cytotoxicity, hemolysis, oxidative stress and epithelial barrier perturbation. All these effects were mitigated by fibre shortening through ball-milling. However, short TiO2 NF exhibited enhanced ability to activate acute pro-inflammatory effects in macrophages, an effect dependent on phagocytosis. Therefore, aspect ratio reduction mitigated toxic effects, while enhanced macrophage activation, likely rendering the NF more prone to phagocytosis. These results suggest that, under in vivo conditions, short NF will be associated with acute inflammatory reaction, but will undergo a relatively rapid clearance, while long NF, although associated with a relatively smaller acute activation of innate immunity cells, are not expected to be removed efficiently and, therefore, may be associated to chronic inflammatory responses. As far as the relationship between the effects of TiO2 NP and LPS, investigated in the second part of the thesis, are concerned, TiO2 NP markedly enhanced macrophage activation by LPS through a TLR-4-dependent intracellular pathway. The adsorption of LPS onto the surface of TiO2 NP led to the formation of a specific bio-corona, suggesting that, when bound to TiO2 NP, LPS exerts a much more powerful pro-inflammatory effect. These data suggest that the inflammatory changes observed upon exposure to TiO2 NP may be due, at least in part, to their capability to bind LPS and, possibly, other TLR agonists, thus enhancing their biological activities. Finally, the last part of the thesis demonstrates that surface functionalization of MWCNT with amino or carboxylic groups mitigates the toxic effects of MWCNT in terms of macrophage activation and capability to perturb epithelial barriers. Interestingly, surface chemistry (in particular surface charge) influenced the protein adsorption onto the MWCNT surface, allowing to the formation of different protein coronae and the tendency to form agglomerates of different size. In particular functionalization a) changed the amount and the type of proteins adsorbed to MWCNT and b) enhanced the tendency of MWCNT to form large agglomerates. These data suggest that the different biological behavior of functionalized and pristine MWCNT may be due, at least in part, to the different tendency to form large agglomerates, which is significantly influenced by their different capability to interact with proteins contained in biological fluids. All together, these data demonstrate that the interaction between physico-chemical properties of nanostructured materials and the environment (cells + biological fluids) in which these materials are present is of pivotal importance for the understanding of the biological effects of NM. In particular, bio-persistence and the capability to elicit an effective inflammatory response are attributable to the interaction between NM and macrophages. However, the interaction NM-cells is heavily influenced by the formation at the nano-bio interface of specific bio-coronae that confer a novel biological identity to the nanostructured materials, setting the basis for their specific biological activities.

Synthetic ceramides inhibit CD36 expression in U937 cells through a reduction in cytosolic peroxide

Ceramide (a sphingolipid) and reactive oxygen species (ROS) are each partly responsible for the intracellular signal transduction of a variety of physiological, pharmacological or environmental agents. It has been reported that synthesis of ceramide and ROS are intimately linked, and show reciprocal regulation. The levels of ceramide are reported to be elevated in atherosclerotic plaques providing circumstantial evidence for a pro-atherogenic role for ceramide. Indeed, LDL may be important sources of ceramide from sphingomyelin, where it promotes LDL aggregation. Using synthetic, short chain ceramides to mimic the cellular responses to fluctuations in natural endogenous ceramides, we have investigated ceramide effects on both intracellular redox state (as glutathione and ROS) and redox-sensitive gene expression, specifically the scavenger receptor CD36 (using RT-PCR and flow cytometry), in U937 monocytes and macrophages. We describe that the principal redox altering properties of ceramide are to lower cytosolic peroxide and to increase mitochondrial ROS formation, where growth arrest of U937 monocytes is also observed. In addition, cellular glutathione was depleted, which was independent of an increase in glutathione peroxidase activity. Examination of the effects of ceramide on stress induced CD36 expression in macrophages, revealed a dose dependent reduction in CD36 mRNA and protein levels, which was mimicked by N-acetyl cysteine. Taken together, these data suggest that ceramides differentially affect ROS within different cellular compartments, and that loss of cytosolic peroxide inhibits expression of the redox sensitive gene, CD36. This may attenuate both the uptake of oxidised LDL and the interaction of HDL with macrophages. The resulting sequelae in vivo remain to be determined.

The role of oscillatory brain activity in object processing and figure-ground segmentation in human vision

The perception of an object as a single entity within a visual scene requires that its features are bound together and segregated from the background and/or other objects. Here, we used magnetoencephalography (MEG) to assess the hypothesis that coherent percepts may arise from the synchronized high frequency (gamma) activity between neurons that code features of the same object. We also assessed the role of low frequency (alpha, beta) activity in object processing. The target stimulus (i.e. object) was a small patch of a concentric grating of 3c/°, viewed eccentrically. The background stimulus was either a blank field or a concentric grating of 3c/° periodicity, viewed centrally. With patterned backgrounds, the target stimulus emerged--through rotation about its own centre--as a circular subsection of the background. Data were acquired using a 275-channel whole-head MEG system and analyzed using Synthetic Aperture Magnetometry (SAM), which allows one to generate images of task-related cortical oscillatory power changes within specific frequency bands. Significant oscillatory activity across a broad range of frequencies was evident at the V1/V2 border, and subsequent analyses were based on a virtual electrode at this location. When the target was presented in isolation, we observed that: (i) contralateral stimulation yielded a sustained power increase in gamma activity; and (ii) both contra- and ipsilateral stimulation yielded near identical transient power changes in alpha (and beta) activity. When the target was presented against a patterned background, we observed that: (i) contralateral stimulation yielded an increase in high-gamma (>55 Hz) power together with a decrease in low-gamma (40-55 Hz) power; and (ii) both contra- and ipsilateral stimulation yielded a transient decrease in alpha (and beta) activity, though the reduction tended to be greatest for contralateral stimulation. The opposing power changes across different regions of the gamma spectrum with 'figure/ground' stimulation suggest a possible dual role for gamma rhythms in visual object coding, and provide general support of the binding-by-synchronization hypothesis. As the power changes in alpha and beta activity were largely independent of the spatial location of the target, however, we conclude that their role in object processing may relate principally to changes in visual attention.

Cortical oscillatory activity associated with the perception of illusory and real visual contours

We used magnetoencephalography (MEG) to examine the nature of oscillatory brain rhythms when passively viewing both illusory and real visual contours. Three stimuli were employed: a Kanizsa triangle; a Kanizsa triangle with a real triangular contour superimposed; and a control figure in which the corner elements used to form the Kanizsa triangle were rotated to negate the formation of illusory contours. The MEG data were analysed using synthetic aperture magnetometry (SAM) to enable the spatial localisation of task-related oscillatory power changes within specific frequency bands, and the time-course of activity within given locations-of-interest was determined by calculating time-frequency plots using a Morlet wavelet transform. In contrast to earlier studies, we did not find increases in gamma activity (> 30 Hz) to illusory shapes, but instead a decrease in 10–30 Hz activity approximately 200 ms after stimulus presentation. The reduction in oscillatory activity was primarily evident within extrastriate areas, including the lateral occipital complex (LOC). Importantly, this same pattern of results was evident for each stimulus type. Our results further highlight the importance of the LOC and a network of posterior brain regions in processing visual contours, be they illusory or real in nature. The similarity of the results for both real and illusory contours, however, leads us to conclude that the broadband (< 30 Hz) decrease in power we observed is more likely to reflect general changes in visual attention than neural computations specific to processing visual contours.

Early gamma-band activity as a function of threat processing in the extrastriate visual cortex

Various neuroimaging investigations have revealed that perception of emotional pictures is associated with greater visual cortex activity than their neutral counterparts. It has further been proposed that threat-related information is rapidly processed, suggesting that the modulation of visual cortex activity should occur at an early stage. Additional studies have demonstrated that oscillatory activity in the gamma band range (40-100 Hz) is associated with threat processing. Magnetoencephalography (MEG) was used to investigate such activity during perception of task-irrelevant, threat-related versus neutral facial expressions. Our results demonstrated a bilateral reduction in gamma band activity for expressions of threat, specifically anger, compared with neutral faces in extrastriate visual cortex (BA 18) within 50-250 ms of stimulus onset. These results suggest that gamma activity in visual cortex may play a role in affective modulation of visual processing, in particular with the perception of threat cues.

Sporicidal activity of two disinfectants against Clostridium difficile spores

The sporicidal activity of an odour-free peracetic acid-based disinfectant (Wofasteril®) and a widely-used dichloroisocyanurate preparation (Chlor-clean®) was assessed against spores of the hyper-virulent strain of Clostridium difficile (ribotype 027), in the presence and absence of organic matter. In environmentally clean conditions, dichloroisocyanurate achieved a >3 log10 reduction in 3 minutes, but a minimum contact time of 9 minutes was required to reduce the viable spore load to below detection levels. Peracetic acid achieved a >3 log10 reduction in 30 minutes and was overall significantly less effective (P<0.05). However, in the presence of organic matter - which reflects the true clinical environment - there was no significant difference between the sporicidal activity of dichloroisocyanurate and peracetic acid over a 60-minute period (P=0.188). Given the greater occupational health hazards generally associated with chlorine-releasing agents, odour-free peracetic acid-based disinfectants may offer a suitable alternative for environmental disinfection.

Bio-inspired carbon electro-catalysis for the oxygen reduction reaction

We report the synthesis, characterisation and catalytic performance of two nature-inspired biomass-derived electro-catalysts for the oxygen reduction reaction in fuel cells. The catalysts were prepared via pyrolysis of a real food waste (lobster shells) or by mimicking the composition of lobster shells using chitin and CaCO3 particles followed by acid washing. The simplified model of artificial lobster was prepared for better reproducibility. The calcium carbonate in both samples acts as a pore agent, creating increased surface area and pore volume, though considerably higher in artificial lobster samples due to the better homogeneity of the components. Various characterisation techniques revealed the presence of a considerable amount of hydroxyapatite left in the real lobster samples after acid washing and a low content of carbon (23%), nitrogen and sulphur (<1%), limiting the surface area to 23 m2/g, and consequently resulting in rather poor catalytic activity. However, artificial lobster samples, with a surface area of ≈200 m2/g and a nitrogen doping of 2%, showed a promising onset potential, very similar to a commercially available platinum catalyst, with better methanol tolerance, though with lower stability in long time testing over 10,000 s.

Do changes in transglutaminase activity alter latent transforming growth factor beta activation in experimental diabetic nephropathy?

Background. Diabetic nephropathy is the leading cause of end-stage kidney failure worldwide. It is characterized by excessive extracellular matrix accumulation. Transforming growth factor beta 1 (TGF-ß1) is a fibrogenic cytokine playing a major role in the healing process and scarring by regulating extracellular matrix turnover, cell proliferation and epithelial mesanchymal transdifferentiation. Newly synthesized TGF-ß is released as a latent, biologically inactive complex. The cross-linking of the large latent TGF-ß to the extracellular matrix by transglutaminase 2 (TG2) is one of the key mechanisms of recruitment and activation of this cytokine. TG2 is an enzyme catalyzing an acyl transfer reaction leading to the formation of a stable e(?-glutamyl)-lysine cross-link between peptides.Methods. To investigate if changes in TG activity can modulate TGF-ß1 activation, we used the mink lung cell bioassay to assess TGF-ß activity in the streptozotocin model of diabetic nephropathy treated with TG inhibitor NTU281 and in TG2 overexpressing opossum kidney (OK) proximal tubular epithelial cells.Results. Application of the site-directed TG inhibitor NTU281 caused a 25% reduction in kidney levels of active TGF-ß1. Specific upregulation of TG2 in OK proximal tubular epithelial cells increased latent TGF-ß recruitment and activation by 20.7% and 19.7%, respectively, in co-cultures with latent TGF-ß binding protein producing fibroblasts.Conclusions. Regulation of TG2 directly influences the level of active TGF-ß1, and thus, TG inhibition may exert a renoprotective effect by targeting not only a direct extracellular matrix deposition but also TGF-ß1 activation and recruitment.

Production of phenol-formaldehyde resin composites

The main objective of this work was to examlne the various stages of the production of industrial laminates based on phenol-formaldehyde resins, with a view of suggesting ways of improving the process economics and/or the physical properties of the final product. Aspects of impregnation, drying, and lamination were investigated. The resins used in all experiments were ammonia-catalysed. Work was concentrated on the lamination stage since this is a labour intensive activity. Paper-phenolic lay-ups were characterised in terms of the temperatures experienced during cure, and a shorter cure-cycle is proposed, utilising the exothermic heat produced during pressing of 25.5 mm thick lay-ups. Significant savings in production costs and improvements in some of the physical properties have been achieved. In particular, water absorption has been reduced by 43-61%. Work on the drying stage has shown that rapid heating of the wet impregnated substrate results in resin solids losses. Drying at lower temperatures by reducing the driving force leads to more resin (up to 6.5%) being retained by the prepregs and therefore more effective use of an expensive raw material. The impregnation work has indicated that residence times above 6 seconds in the varnish bath enhance the insulation resistance of the final product, possibly due to improved resin distribution and reduction in water absorption. In addition, a novel process which involves production of laminates by in situ polymerisation of the phenolic resin on the substrate has been examined. Such a process would eliminate the solvent recovery plant - a necessary stage in current industrial processes. In situ polymerisation has been shown to be chemically feasible.

Biochemical aspects of Tourette's Syndrome

Kynurenine (KYN) is the first stable metabolite of the kynurenine pathway, the major route of tryptophan. (TRP) metabolIsm. In the liver, cortisol-inducible tIyptophan-2,3-dioxygenase (TDO) is the first enzyme and rate limiting step. In extrahepatic tissues, it is superceded by indoleamine-2,3-dioxygenase (IDO), an enzyme with a wider substrate specificity. Earlier work in this research group has found substantial elevations in plasma KYN in fasting Tourette's Syndrome (TS) patients with normal TRP and neopterin. The aim of our initial pilot study was to confirm this increase in KYN in fasting human TS patients compared with normal controls, and to see how changes in diet :ay influence certain kynurenine pathway variables. However, we failed to detect a change in plasma KYN, TRP, kynurenic acid (KYNA), neopterin or cortisol between the fasting TS and control groups. Moreover, none of the variables was affected by dietary status, and thus candidates selected for the larger cross-sectional study were permitted to eat and drink freely on the day that blood samples were submitted, but were requested to avoid products containing caffeine, aspirin or nicotine. In the cross-sectional study, TS patients exhibited significantly higher plasma KYN concentrations than controls, although the magnitude of the change was much smaller than originally found. This may be due to differences in detection procedure and the seasonal fluctuation of some biochemical variables, notably cortisol. The generalised increase in neopterin in the TS subject group, suggests a difference in the activity of cytokine-inducible IDO as a likely source for this elevated KYN. Other kynurenine pathway metabolites, specifIcally TRP, 3-hydroxykynurenine (HKY), 3-hydroxyanthranilic acid (HAA) and KYNA were unchanged. In view of recent speculation of the potential therapeutic effects of nicotine in TS, the lower KYN concentrations observed in TS smokers, compared with non-smoking TS patients, was another interesting finding. Tic-like movements, such as head-shakes (HS), which occur in rodents both spontaneously and following diverse drug treatments, closely resemble tic behaviours in humans. The animal tic model was used to examine what effects nicotine may have on shaking behaviours and on selected TRP metabolites. Acute systemic administration of nicotine to mice, produced a dose-dependent reduction in HS frequency (induced by the 5-HT2A/2C agonist DOl), which appeared to be mediated via central nicotinic cholinergic receptors, since mecamylamine pretreatment abolished this effect. Conversely, twice daily subcutaneous injections of nicotine for 7 days, led to an increase in spontaneous and DOI-induced HS. Chronic nicotine also caused a significant elevation m plasma and whole brain KYN concentrations, but plasma TRP, HKY, HAA and KYNA were unaltered. In addition, no change in brain 5-HT or 5-HIAA concentrations or 5-HT turnover, was found. Despite contrasting results from human and animal studIes, a role for nicotine in the mediation of tic-like movements is indicated. The relevance of the kynurenine pathway to TS and the potential role played by nicotine in modifying tic-like behaviours is discussed.

The effects of optical activity and absorption on two wave mixing in Bi12SiO20

We consider non-degenerate two-wave mixing in photorefractive Bi12SiO20. It is shown theoretically that the presence of absorption and optical activity in the photorefractive media may result in a number of maxima for the gain as the frequency detuning between the two beams is varied. Further, when the beam interaction is used for optical amplification, there may also exist an optimum crystal length beyond which there is a reduction in the useful gain obtainable. Experimental results are presented in confirmation of the theory.

The effects of substrate water availability on the isolation and growth of fungi

The work reported in this thesis was carried out to contribute to the knowledge of the effects of substrate water availability or water activity (a ) on fungal growth parameters and its implications in the preparationw of materials susceptible to biodeterioration. Fungi were isolated from soils of different ecological sites at a range of substrate aw levels controlled by sodium chloride (NaCl). Three groups of fungi were isolated : firstly, those isolated only at high a (aw about 0.997).secondly, those isolated at high and decreasing aw (aw 0.997 to 0.85) and finally, those isolated at only decreased aw (aw O.95 to 0.80). From these isolations, test fungi were selected to study the effects of pH, temperature, exo-enzyme production and biocide efficacy at decreased aw levels, with glycerol and NaCl as a controlling solutes. The linear extension rates of the fungi increased at all test pH values near optimum a of growth. Test fungi of the Aspergillus glaucus group were found to be most resistant to low aw. Growth and survival of vegetative and fruiting bodies at elevated temperatures were enhanced with the addition of a controlling solutes. A. flavus, A. fumigatus displayed high heat resistance and A. amstelodami, A. versicolor and Penicillium citrinum displayed low heat resistance at high aw levels and vice versa at low aw levels. Amylase, lipase and protease activities were studied at lowered aw , using modifications of the test tube method of Raute11a and Cowling. Amylase and protease production in most xerophilic fungi ceased around 0.80 aw , but lipase production in some xerophilic fungi, including A. glatlcus fungi, was up to and including 0.70 aw with g1ycero1.

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.

A study in the reduction of efficiency of nitrogen oxide removal catalysts for automobiles

Automotive catalysts are the most effective short-term answer to air pollution from automobiles. Since strict control of exhaust emissions is, or will be,covered by legislation in most developed countries in the world, catalytic devices will be increasingly fitted to cars. There is consequently an urgent need for the development of catalysts that will not compete for scarce precious metal resources. A number of problems have already been identified in connection with base metal catalysts but quantitative investigations are lacking. The base metal reduction catalysts developed by Imperial Chemical Industries Limited, catalysts and Chemical Group, in collaboration with the Air Pollution Control Laboratory, B L Cars Limited for automotive emission control, are susceptible to de-activation by three major mechanisms. These are: physical loss of the wash-coat (a high surface area coating which supports the active species), aggregation of the active species and poisoning by fuel and engine oil additives. This thesis is especially concerned with the first two of these and attempts to indicate the relative magnitude .of their effect on the activity of. the catalysts. Aggregation of the active species or sintering, as it is loosely called, was studied by using impregnated granules to overcome effects due to the loss of the wash-coat. Samples were aged in a synthetic exhaust gas, free from poisons, and metal crystallite sizes were measured by scanning-electron microscopy. The increase in particle size was correlated with the loss in catalytic activity. In order to maintain a link with the real conditions of service a number of monolithic catalysts were tested in an engine-dynamometer and several previously tested endurance catalysts were examined. A mechanism is proposed for the break-up and subsequent 10s.5 of the wash-coat and suggestions for improved resistance to loss of the' coating and active species are proposed.

Antibacterial activity of low amperage and low voltage electric current (DC)

Infection is a major clinical problem associated with the use of intravenous catheters.The efficacy of a direct electric current (10µA, 9V) via electrode-conducting carbon impregnated catheters to prevent colonisation of catheters by micro-organisms was investigated. The range of organisms susceptible to 10µA was determined by a zone of inhibition test. The catheters acting as the anode and the cathode were inserted into a nutrient agar plate inoculated with a lawn of bacteria. There was no zone of inhibition observed around the anode. Organisms susceptible to 10µA at the cathode were Staphylococcus aureus (2 strains), Staphylococcus epidermidis (5 strains), Escherichia coli and Klebsiella pneumoniae (2 strains each), and one strain of the following micro-organisms: Staphylococcus hominis, Proteus mirabilis, Pseudomonas aeruginosa and Candida albicans. The zones ranged from 6 to 16 mm in diameter according to the organisms under test. The zone size was proportional to the amperage (10 - 100 µA) and the number of organisms on the plate. Ten µA did not prevent adhesion of staphylococci to the cathode nor did it affect their growth in nutrient broth. However, it was bactericidal to adherent bacteria on the cathodal catheter and significantly reduced the number of bacteria on the catheter after 4 to 24 h application of electricity. The antimicrobial activity of low amperage electric current under anaerobic conditions and in the absence of chloride ions against bacteria attached to the surface of a current carrying electrode was also investigated.The mechanisms of the bactericidal activity associated with the cathode were investigated with S. epidermidis and S. aureus. The inhibition zone was greatly reduced in the presence of catalase. There was no zone around the cathode when the test was carried out under anaerobic conditions. Hydrogen peroxide was produced at the cathode surface under aerobic conditions, but not in the absence of oxygen. A salt-bridge apparatus was used to demonstrate further that hydrogen peroxide was produced at the cathode, and chlorine at the anode. The antimicrobial activity of low amperage electric current under anaerobic conditions and in the absence of chloride ions against bacteria attached to the surface of a current carrying electrode was also investigated. Antibacterial activity was reduced under anaerobic conditions, which is compatible with the role of hydrogen peroxide as a primary bactericidal agent of electricity associated with the cathode. A reduction in chloride ions did not significantly reduce the antibacterial activity suggesting chlorine plays only a minor role in the bactericidal activity against organisms attached to anodal electrode surfaces. The bactericidal activity of electric current associated with the cathode and H202 was greatly reduced in the presence of 50 μM to 0.5 mM magnesium ions in the test menstrum. Ten μA applied via the catheters did not prevent the initial biofilm growth by the adherent bacteria but reduced the number of bacteria in the biofilm by 2 log order aiter 24 h. The results suggested that 10 μA may prevent the colonisation of catheters by both the extra~ and intra-luminal routes. The localised production of hydrogen peroxide and chlorine and the intrinsic activity due to electric current may offer a useful method for the eradication of bacteria from catheter surfaces.