Multifunctional receptor-targeted nanocomplexes for the delivery of therapeutic nucleic acids to the brain.

Convection enhanced delivery (CED) is a method of direct injection to the brain that can achieve widespread dispersal of therapeutics, including gene therapies, from a single dose. Non-viral, nanocomplexes are of interest as vectors for gene therapy in the brain, but it is essential that administration should achieve maximal dispersal to minimise the number of injections required. We hypothesised that anionic nanocomplexes administered by CED should disperse more widely in rat brains than cationics of similar size, which bind electrostatically to cell-surface anionic moieties such as proteoglycans, limiting their spread. Anionic, receptor-targeted nanocomplexes (RTN) containing a neurotensin-targeting peptide were prepared with plasmid DNA and compared with cationic RTNs for dispersal and transfection efficiency. Both RTNs were labelled with gadolinium for localisation in the brain by MRI and in brain sections by LA-ICP-MS, as well as with rhodamine fluorophore for detection by fluorescence microscopy. MRI distribution studies confirmed that the anionic RTNs dispersed more widely than cationic RTNs, particularly in the corpus callosum. Gene expression levels from anionic formulations were similar to those of cationic RTNs. Thus, anionic RTN formulations can achieve both widespread dispersal and effective gene expression in brains after administration of a single dose by CED.

Lipid peptide nanocomplexes for gene delivery and magnetic resonance imaging in the brain.

Gadolinium-labelled nanocomplexes offer prospects for the development of real-time, non-invasive imaging strategies to visualise the location of gene delivery by MRI. In this study, targeted nanoparticle formulations were prepared comprising a cationic liposome (L) containing a Gd-chelated lipid at 10, 15 and 20% by weight of total lipid, a receptor-targeted, DNA-binding peptide (P) and plasmid DNA (D), which electrostatically self-assembled into LPD nanocomplexes. The LPD formulation containing the liposome with 15% Gd-chelated lipid displayed optimal peptide-targeted, transfection efficiency. MRI conspicuity peaked at 4h after incubation of the nanocomplexes with cells, suggesting enhancement by cellular uptake and trafficking. This was supported by time course confocal microscopy analysis of transfections with fluorescently-labelled LPD nanocomplexes. Gd-LPD nanocomplexes delivered to rat brains by convection-enhanced delivery were visible by MRI at 6 h, 24 h and 48 h after administration. Histological brain sections analysed by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) confirmed that the MRI signal was associated with the distribution of Gd(3+) moieties and differentiated MRI signals due to haemorrhage. The transfected brain cells near the injection site appeared to be mostly microglial. This study shows the potential of Gd-LPD nanocomplexes for simultaneous delivery of contrast agents and genes for real-time monitoring of gene therapy in the brain.

An evaluation of the safety and feasibility of convection-enhanced delivery of carboplatin into the white matter as a potential treatment for high-grade glioma.

Glioblastoma multiforme (GBM) is the most common and most aggressive form of intrinsic brain tumour. Despite standard treatment involving surgical resection, chemotherapy and radiotherapy this disease remains incurable with the majority of tumours recurring adjacent to the resection cavity. Consequently there is a clear need to improve local tumour control. Convection-enhanced delivery (CED) is a practical technique for administering chemotherapeutics directly into peritumoural brain. In this study, we have tested the hypothesis that carboplatin would be an appropriate chemotherapeutic agent to administer by CED into peritumoural brain to treat GBM. Within this study we have evaluated the relationships between carboplatin concentration, duration of exposure and tumour cell kill in vitro using GBM cell lines and the relationship between carboplatin concentration and clinical and histological evidence of toxicity in vivo. In addition, we have used laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) to evaluate the distribution properties of carboplatin following CED into rat brain and to determine the rate at which carboplatin is cleared from the brain. Finally, we have compared the distribution properties of carboplatin and the MRI contrast agent gadolinium-DTPA in pig brain. The results of these experiments confirm that carboplatin can be widely distributed by CED and that it remains in the brain for at least 24 h after infusion completion. Furthermore, carboplatin provokes a significant GBM cell kill at concentrations that are not toxic to normal brain. Finally, we provide evidence that gadolinium-DTPA coinfusion is a viable technique for visualising carboplatin distribution using T1-weighted MR imaging.

Dynamics of a starvation-to-surfeit shift: a transcriptomic and modelling analysis of the bacterial response to zinc reveals transient behaviour of the Fur and SoxS regulators.

We describe a hybrid transcriptomic and modelling analysis of the dynamics of a bacterial response to stress, namely the addition of 200 µM Zn to Escherichia coli growing in severely Zn-depleted medium and of cells growing at different Zn concentrations at steady state. Genes that changed significantly in response to the transition were those reported previously to be associated with zinc deficiency (zinT, znuA, ykgM) or excess (basR, cpxP, cusF). Cellular Zn levels were confirmed by ICP-AES to be 14- to 28-fold greater after Zn addition but there was also 6- to 8-fold more cellular Fe 30 min after Zn addition. Statistical modelling of the transcriptomic data generated from the Zn shift focused on the role of ten key regulators; ArsR, BaeR, CpxR, CusR, Fur, OxyR, SoxS, ZntR, ZraR and Zur. The data and modelling reveal a transient change in the activity of the iron regulator Fur and of the oxidative stress regulator SoxS, neither of which is evident from the steady-state transcriptomic analyses. We hypothesize a competitive binding mechanism that combines these observations and existing data on the physiology of Zn and Fe uptake. Formalizing the mechanism in a differential equation model shows that it can reproduce qualitatively the behaviour seen in the data. This gives new insights into the interplay of these two fundamental metal ions in gene regulation and bacterial physiology, as well as highlighting the importance of dynamic studies to reverse-engineer systems behaviour.

Imaging of phospholipids in formalin fixed rat brain sections by matrix assisted laser desorption/ionization mass spectrometry.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a valuable tool for the analysis of molecules directly from tissue. Imaging of phospholipids is gaining widespread interest, particularly as these lipids have been implicated in a variety of pathologic processes. Formalin fixation (FF) is the standard protocol used in histology laboratories worldwide to preserve tissue for analysis, in order to aid in the diagnosis and prognosis of diseases. This study assesses MALDI imaging of phospholipids directly in formalin fixed tissue, with a view to future analysis of archival tissue. This investigation proves the viability of MALDI-MSI for studying the distribution of lipids directly in formalin fixed tissue, without any pretreatment protocols. High quality molecular images for several phosphatidylcholine (PC) and sphingomyelin (SM) species are presented. Images correspond well with previously published data for the analysis of lipids directly from freshly prepared tissue. Different ionization pathways are observed when analyzing fixed tissue compared with fresh, and this change was found to be associated with formalin buffers employed in fixation protocols. The ability to analyze lipids directly from formalin fixed tissue opens up new doors in the investigation of disease profiles. Pathologic specimens taken for histologic investigation can be analyzed by MALDI-MS to provide greater information on the involvement of lipids in diseased tissue.

Synthesis and characterization of a theranostic vascular disrupting agent for in vivo MR imaging.

Colchicine, a known tubulin binding agent and vascular disrupting agent, causes rapid vascular shut down and central necrosis in tumors. The binding of tubulin results in tubulin destabilization, with characteristic cell shape changes and inhibition of cell division, and results in cell death. A gadolinium(III) labeled derivative of colchicine (Gd·DOTA·Colchicinic acid) was synthesized and characterized as a theranostic agent (enabling simultaneous diagnostic/real time MRI contrast imaging). In vitro, Gd·DOTA·Colchicinic acid was shown to initiate cell changes characteristic of tubulin-destabilization in both OVCAR-3 and IGROV-1 ovarian carcinoma cell lines in vitro over a period of 24 h, while maintaining the qualities of the MR imaging tracer. In vivo, Gd·DOTA·Colchicinic acid (200 mg/kg) was shown to induce the formation of central necrosis, which was confirmed ex vivo by histology, in OVCAR-3 subcutaneous tumor xenografts, while simultaneously acting as an imaging agent to promote a significant reduction in the MR relaxation time T(1) (p < 0.05) of tumors 24 h post-administration. Morphological changes within the tumor which corresponded with areas derived from the formation of central necrosis were also present on MR images that were not observed for the same colchicine derivate that was not complexed with gadolinium that also presented with central necrosis ex vivo. However, Gd·DOTA·Colchicinic acid accumulation in the liver, as shown by changes in liver T(1) (p < 0.05), takes place within 2 h. The implication is that Gd·DOTA·Colchicinic acid distributes to tissues, including tumors, within 2 h, but enters tumor cells to lower T(1) times and promotes cell death over a period of up to 24 h. As the biodistribution/pharmacokinetic and pharmacodynamics data provided here is similar to that of conventional colchicines derivatives, such combined data are a potentially powerful way to rapidly characterize the complete behavior of drug candidates in vivo.

A low molecular weight folate receptor targeted contrast agent for magnetic resonance tumor imaging.

PURPOSE: This study aims to develop a low molecular weight folate receptor (FR) contrast agent for MR tumor imaging. PROCEDURES: Gadolinium-tetraazacyclododecane tetraacetic acid (Gd.DOTA) was conjugated to folic acid to create Gd.DOTA.Folate. The efficacy of Gd.DOTA.Folate to bind FR was evaluated in vitro by inductively coupled mass spectrometry (ICP-MS) and in vivo by magnetic resonance imaging (MRI) tumor enhancement over 14 h, utilizing an overexpressing α-FR cell line (IGROV-1), compared to an α-FR-negative cell line (OVCAR-3). Gd.DOTA.Folate localization ex vivo was verified by laser ablation ICP-MS. RESULTS: ICP-MS confirmed Gd.DOTA.Folate uptake by IGROV-1 cells and competitive binding with free folic acid inhibited binding. IGROV-1 tumors showed an increase in R (1) at 2 h, which increased significantly over 14 h post-Gd.DOTA.Folate with clear enhancement on MR images. This was not observed in controls. CONCLUSION: These data support the use of FR-targeted small molecular weight MRI contrast agents for tumor imaging in vivo.

Manganese enhancement in non-CNS organs.

Manganese-enhanced magnetic resonance imaging (MEMRI) is a novel imaging technique capable of monitoring calcium influx, in vivo. Manganese (Mn2+) ions, similar to calcium ions (Ca2+), are taken up by activated cells where their paramagnetic properties afford signal enhancement in T(1)-weighted MRI methodologies. In this study we have assessed Mn2+ distribution in mice using magnetization-prepared rapid gradient echo (MP-RAGE) based MRI, by measuring changes in T(1)-effective relaxation times (T(1)-eff), effective R(1)-relaxation rates (R(1)-eff) and signal intensity (SI) profiles over time. The manganese concentration in the tissue was also determined using inductively coupled plasma atomic emission spectrometry (ICP-AES). Our results show a strong positive correlation between infused dose of MnCl2 and the tissue manganese concentration. Furthermore, we demonstrate a linear relationship between R(1)-eff and tissue manganese concentration and tissue-specific Mn2+ distribution in murine tissues following dose-dependent Mn2+ administration. This data provides an optimized MnCl2 dose regimen for an MP-RAGE based sequence protocol for specific target organs and presents a potential 3D MRI technique for in vivo imaging of Ca2+ entry during Ca2+-dependent processes in a wide range of tissues.

Imaging of gadolinium spatial distribution in tumor tissue by laser ablation inductively coupled plasma mass spectrometry.

PURPOSE: Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was utilized in postmortem imaging of gadolinium (Gd) spatial distribution in a mouse tumor model postadministration of PEGylated Gd liposomal nanoparticles. PROCEDURES: PEGylated liposomal nanoparticles were formulated using a paramagnetic lipid incorporating Gd, in addition to a fluorescent lipid, and injected intravenously into Balb/C nude mice bearing IGROV-1 tumors. At postinjection (2 h), the tumors and selective organs were imaged by magnetic resonance imaging (MRI) and, after excision, by histology and LA-ICP-MS. RESULTS: The presence of Gd within tumor tissue was confirmed by LA-ICP-MS and when correlated to histology was found to be prevalent in regions of higher vascularity. The presence of Gd in the kidneys was also confirmed. CONCLUSIONS: We have demonstrated, in a novel manner, the use of LA-ICP-MS for the spatial detection of Gd in tumor tissue. LA-ICP-MS is valuable in providing spatio-specific information of MRI contrast agents and more importantly Gd in tumor tissue.

A novel method for exploring elemental composition of microbial communities: laser ablation-inductively coupled plasma-mass spectrometry of intact bacterial colonies.

Bacterial colonies are spatially complex structures whose physiology is profoundly dependent on interactions between cells and with the underlying semi-solid substratum. Here, we use bacterial colonies as a model of a microbial community to evaluate the potential of laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) to delineate elemental distributions within colonies with minimal pre-treatment. To reduce water content of the colony and limit undesirable absorption of laser energy, we compared methods of preparing 24h-old colonies of Escherichia coli TG1 on agar for laser ablation. Colonies on excised agar segments dried on chromatography paper were superior to colonies dried in a dessicator or by prolonged incubation, with respect to signal magnitude, signal:noise ratio and background signal. Having optimised laser scan speed (10 microm s(-1)) and laser beam diameter (100 microm), further improvements were achieved by growing colonies on nylon membranes over agar, which were then transferred to the ablation chamber without further treatment. Repeated line rasters across individual membrane-supported colonies yielded three-dimensional elemental maps of colonies, revealing a convex morphology consistent with visual inspection. By normalising isotope counts for P, Mn, Zn, Fe and Ca against Mg, the most abundant cellular divalent cation, we sought elemental heterogeneity within the colony. The normalised concentration of Mn in the perimeter was higher than in the colony interior, whereas the converse was true for Ca. LA-ICP-MS is a novel and powerful method for probing elemental composition and organisation within microbial communities and should find numerous applications in, for example, biofilm studies.

Severe zinc depletion of Escherichia coli: roles for high affinity zinc binding by ZinT, zinc transport and zinc-independent proteins.

Zinc ions play indispensable roles in biological chemistry. However, bacteria have an impressive ability to acquire Zn(2+) from the environment, making it exceptionally difficult to achieve Zn(2+) deficiency, and so a comprehensive understanding of the importance of Zn(2+) has not been attained. Reduction of the Zn(2+) content of Escherichia coli growth medium to 60 nm or less is reported here for the first time, without recourse to chelators of poor specificity. Cells grown in Zn(2+)-deficient medium had a reduced growth rate and contained up to five times less cellular Zn(2+). To understand global responses to Zn(2+) deficiency, microarray analysis was conducted of cells grown under Zn(2+)-replete and Zn(2+)-depleted conditions in chemostat cultures. Nine genes were up-regulated more than 2-fold (p < 0.05) in cells from Zn(2+)-deficient chemostats, including zinT (yodA). zinT is shown to be regulated by Zur (zinc uptake regulator). A mutant lacking zinT displayed a growth defect and a 3-fold lowered cellular Zn(2+) level under Zn(2+) limitation. The purified ZinT protein possessed a single, high affinity metal-binding site that can accommodate Zn(2+) or Cd(2+). A further up-regulated gene, ykgM, is believed to encode a non-Zn(2+) finger-containing paralogue of the Zn(2+) finger ribosomal protein L31. The gene encoding the periplasmic Zn(2+)-binding protein znuA showed increased expression. During both batch and chemostat growth, cells "found" more Zn(2+) than was originally added to the culture, presumably because of leaching from the culture vessel. Zn(2+) elimination is shown to be a more precise method of depleting Zn(2+) than by using the chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine.

Characteristics of water-soluble inorganic chemical components in size-resolved airborne particulate matters--Sheffield, UK.

To characterise the water-soluble inorganic components of PM(10) in the urban area of Sheffield, size-resolved aerosol samples were collected using an electric low pressure impactor (ELPI) during a 13-day sampling campaign in October and November 2006. Cl(-), NO(3)(-), SO(4)(2-), and NH(4)(+) were determined by ion chromatography, and Na(+), K(+), Mg(2+), and Ca(2+) by inductively coupled plasma-mass spectrometry. Back trajectories analyses revealed that the air masses could be classified into two main groups. In the "maritime regime", the air masses moved mostly over the sea and just short distances (ca. 100 to 150 km) over land; in the "terrestrial regime", the air masses had moved long distances (ca. 300 to 600 km) over land before reaching the sampling site. The chemical composition of the particles was strongly influenced by the origins of the air masses. Air mass belonging to the maritime regime brought more sea-salt species such as Na(+), Cl(-), and Mg(2+). Air mass belonging to the terrestrial regime carried more K(+) and secondary components like SO(4)(2-), NO(3)(-), and NH(4)(+). Sulfate showed bimodal distribution, peaking both in the fine mode (particle size <0.96 microm) and the coarse mode (particle size >0.96 microm). Nitrate exhibited a bimodal distribution in the terrestrial regime but only a coarse mode in maritime regime. Ammonium displayed a unimodal size distribution, peaking in the fine size range, mainly bound to sulfate and nitrate. In the maritime regime, chloride showed a unimodal distribution, peaking in the coarse size range. In the terrestrial regime, chloride appeared to be bimodal with one peak in the fine mode, reflecting the presence of chloride sources from industries, and another one in the coarse mode, mainly from sea spray. Although in general the air mass trajectories can be grouped into "maritime" and "terrestrial" regimes, the results suggest that air masses reaching Sheffield have been impacted by both maritime and terrestrial sources.

Carbon monoxide-releasing antibacterial molecules target respiration and global transcriptional regulators.

Carbon monoxide, a classical respiratory inhibitor, also exerts vasodilatory, anti-inflammatory, and antiapoptotic effects. CO-releasing molecules have therapeutic value, increasing phagocytosis and reducing sepsis-induced lethality. Here we identify for the first time the bacterial targets of Ru(CO)(3)Cl(glycinate) (CORM-3), a ruthenium-based carbonyl that liberates CO rapidly under physiological conditions. Contrary to the expectation that CO would be preferentially inhibitory at low oxygen tensions or anaerobically, Escherichia coli cultures were also sensitive to CORM-3 at concentrations equimolar with oxygen. CORM-3, assayed as ruthenium, was taken up by bacteria and rapidly delivered CO intracellularly to terminal oxidases. Microarray analysis of CORM-3-treated cells revealed extensively modified gene expression, notably down-regulation of genes encoding key aerobic respiratory complexes. Genes involved in metal metabolism, homeostasis, or transport were also differentially expressed, and free intracellular zinc levels were elevated. Probabilistic modeling of transcriptomic data identified the global transcription regulators ArcA, CRP, Fis, FNR, Fur, BaeR, CpxR, and IHF as targets and potential CO sensors. Our discovery that CORM-3 is an effective inhibitor and global regulator of gene expression, especially under aerobic conditions, has important implications for administration of CO-releasing agents in sepsis and inflammation.

Expression of the PitA phosphate/metal transporter of Escherichia coli is responsive to zinc and inorganic phosphate levels.

Escherichia coli possesses two major systems for inorganic phosphate (P(i)) uptake. The Pst system (pstSCAB) is inducible by low phosphate concentrations whereas the low-affinity transporter (pitA) has been described as constitutively expressed. PitA catalyses transport of metal [Mg(II), Ca(II)]-phosphate complexes, and mutations in pitA confer Zn(II) resistance. Here we report that pitA transcription is not constitutive; activity of a single-copy pitA-lacZ transcriptional fusion (monolysogen) was maximal at high extracellular Zn(II) (150 microM), in the absence of added P(i), and in a well-defined pitA mutant strain. Intracellular zinc levels were unaffected by adding Zn(II) to the medium for both the wild-type and mutant strains. However, in the wild-type strain, Mg levels (per gram of dry biomass) fell by eightfold in cells grown with added Zn(II) and by 20-fold when Zn(II) and P(i) were added to cultures. Mutation of pitA reduced the effects of external Zn(II) and phosphate levels on Mg pools, consistent with competition or inhibition by Zn(II) of PitA. The mechanism of pitA regulation by extracellular Zn(II) and P(i) is unknown but appears not to involve Fur or other well-characterized regulators.

Combination of immunohistochemistry and laser ablation ICP mass spectrometry for imaging of cancer biomarkers.

Laser ablation (LA) ICP-MS has been developed as a new tool for imaging of cancer biomarkers in tissue sections. The distribution of two breast cancer-associated proteins, MUC-1 and HER2 was studied based on multiple line rastering of tissue sections and measurement of relevant Au/Ag tagged antibodies bound to the tissue. Comparisons with optical microscopy indicated extremely high sensitivity for the LA technique and sufficiently good resolution to permit fine scale feature mapping at the cellular level. Application to the quantitative assessment of HER2 expression in tissue microarrays was demonstrated.

Development and certification of the new NIES CRM 28: urban aerosols for the determination of multielements.

A new environmental certified reference material (CRM) for the determination of multielements in aerosol particulate matter has been developed and certified by the National Institute for Environmental Studies (NIES), Japan, based on analyses by a network of laboratories using a wide range of methods. The origin of the material was atmospheric particulate matter collected on filters in a central ventilating system in a building in Beijing city centre. The homogeneity and stability of this material were sufficient for its use as a reference material. Values for elemental mass fractions in the material were statistically determined based on the analytical results of the participating laboratories. Eighteen certified values and 14 reference values were obtained. The diameters, obtained from a micrographic image using image analysis software, of 99% of the particles were less than 10 microm, demonstrating that almost all the particles in the material could be classified as particles of 10 microm or less in aerodynamic diameter. The chemical composition and particle size distribution of this material were close to those of an authentic aerosol collected in Beijing. NIES CRM 28 is appropriate for use in analytical quality control and in the evaluation of methods used in the analysis of aerosols, particularly those collected in urban environments in northeast Asia.

Heavy coal combustion as the dominant source of particulate pollution in Taiyuan, China, corroborated by high concentrations of arsenic and selenium in PM10.

Coal burning generates toxic elements, some of which are characteristic of coal combustion such as arsenic and selenium, besides conventional coal combustion products. Airborne particulate samples with aerodynamic diameter less than 10 microm (PM(10)) were collected in Taiyuan, China, and multi-element analyses were performed by inductively coupled plasma atomic emission spectrometry (ICP-AES) and inductively coupled plasma mass spectrometry (ICP-MS). Concentrations of arsenic and selenium from ambient air in Taiyuan (average 43 and 58 ng m(-3), respectively) were relatively high compared to what is reported elsewhere. Arsenic and selenium were found to be highly correlated (r=0.997), indicating an overwhelmingly dominant source. Correlation between these two chalcophile elements and the lithophile element Al is high (r is 0.75 and 0.72 for As and Se, respectively). This prompted the hypothesis that the particles were from coal combustion. The enrichment of the trace elements could be explained by the volatilization-condensation mechanism during coal combustion process. Even higher correlations of arsenic and selenium with PM(10) (r=0.90 and 0.88) give further support that airborne particulate pollution in Taiyuan is mainly a direct result of heavy coal consumption. This conclusion agrees with the results from our previous study of individual airborne particles in Taiyuan.

Determination of (234)U/ (238)U isotope ratios in environmental waters by quadrupole ICP-MS after U stripping from alpha-spectrometry counting sources.

The 234U/238U isotope ratio has been widely used as a tracer for geochemical processes in underground aquifers. Quadrupole-based inductively coupled plasma mass spectrometry (ICP-MS) equipped with a high-efficiency nebulizer and a membrane desolvator was employed for the determination of 234U/238U isotope ratios in natural water samples. The instrumental limit of detection for 234U was at the low pg L(-1) level with very low sample consumption. Measurement precision (234U/238U) was 3-5% for bottled mineral water with elevated uranium concentration (>1 microg L(-1)). For the analysis of groundwater samples from the Almonte-Marisma underground aquifer (Huelva, Spain), uranium was stripped from stainless steel planchets that had previously been used as radiometric counting sources for alpha-particle spectrometry. Potential spectral interferences from other metals introduced during the dissolution were investigated. Matrix-matched blank solutions were needed to subtract the background on 234U due to the formation of platinum argides, and to allow for mass bias correction and background correction. The Pt appears to be an impurity present in the stainless steel, either as a minor component by itself or after extraction from the anode and a subsequent uranium electrodeposition. The 234U/238U isotope ratio data were in very good agreement with those of alpha spectrometry, while precision was improved by a factor of up to 10 and counting time was reduced down to approximately 20 min (10 replicate measurements).

A protocol for investigation of the effects of outdoor air pollution on stroke incidence, phenotypes and survival using the South London Stroke Register.

Stroke is a major cause of death and disability. About 5.3 million people die every year from stroke worldwide with over 9 million people surviving at any one time after suffering a stroke. About 1 in 4 men and 1 in 5 women aged 45 years will suffer a stroke if they live to their 85th year. It is estimated that by 2023 there will be an absolute increase in the number of people experiencing a first ever stroke of about 30% compared with 1983. In the UK, stroke is the third commonest cause of death and the most common cause of adult physical disability and consumes 5% of the health and social services budget. Stroke is assuming strategic public health importance because of increased awareness in society, an ageing population and emerging new treatments. It is an NHS health service and research priority, being identified as a target in Our Healthier Nation and the NSF for Older People for prevention and risk factor control and in the NHS Plan as a disease requiring intermediate care planning and reduction in inequalities of care. Whilst a number of risk factors for stroke are well known (e.g. increasing age, ethnicity, socioeconomic deprivation, hypertension), the potential importance of outdoor air pollution as a modifiable risk factor is much less well recognised. This is because studies to date are inconclusive or have methodological limitations. In Sheffield, we estimated that 11% of stroke deaths may be linked to current levels of outdoor air pollution and this high figure is explained by the fact that so many people are exposed to air pollution.We plan to study the effects of outdoor air pollution on stroke using a series of epidemiological (i.e. population based) studies. The purpose of this project is: to examine if short term increases in pollution can trigger a stroke in susceptible individuals, to investigate if the occurrence of stroke is higher amongst people living in more polluted areas (which would be explained by a combination of exposure to short term increases and longer term exposure to higher pollution levels), and to see if people living in more polluted areas have reduced survival following their stroke. We will use geographical information systems, robust statistical methods and powerful grid computing facilities to link and analyse the data. The datasets we will use are the South London Stroke Register database, daily monitored pollution data from national monitoring networks and modelled pollution data for London from the Greater London Authority. The South London Stroke Register records information on all patients who suffer a stroke ("incident" cases) living within a defined area. This stroke incidence dataset offers major advantages over previous studies examining the effects of pollution on hospital admissions and mortality, as not all patients with stroke are admitted or die and there may be a delay between the onset of stroke and admission or death. In addition, it contains other useful information, particularly the type of stroke people have suffered. Air pollution is a potentially modifiable risk factor for stroke. This study will provide robust population level evidence regarding the effects of outdoor air pollution on stroke. If it confirms the link, it will suggest to policy-makers at national and international levels that targeting policy interventions at high pollution areas may be a feasible option for stroke prevention.