Serotonin is a chemical that can act on the blood vessels of the lungs to cause them to thicken. This can lead to high blood pressure in the lungs. The enzyme responsible for the synthesis of serotonin that affects the lungs is tryptophan hydroxlase 1. Blood vessels have three layers, an outer layer of adventitial cells, a muscular layer and an inner layer of cells called endothelial cells. We will determine if tryptophan hydroxlase 1 causes synthesis of serotonin by the endothelial cells and if this serotonin can then act on the adjacent muscle and adventitial cells to make them grow and cause thickening of the artery. We will also determine if hypoxia (a reduction in oxygen) can stimulate an increase in endothelial cell serotonin synthesis and release and that this contributes to hypoxia-induced thickening of the blood vessels. We will use genetically modified mice which lack tryptophan hydroxlase 1 in our studies as well as isolated smooth muscle, adventitial and endothelial cells. We will also examine the feasilbility of knocking out tryptophan hydroxlase 1 in the endothelial cells in the whole animal using novel gene transfer techniques.

Inhaled microbes are likely drivers of lung inflammation and function and understanding airway responses to microbial challenge will provide insights to identify novel drugs. In vivo modelling of the airway responses is therefore critical. Lipopolysaccharide (LPS) is a major component of gram negative bacterial membranes, whose inhalation mimics bacterial inhalation by interacting with epithelial toll-like receptors (TLR-4) resulting in induction of NF-kappaB and transcription of proinflammatory genes and cytokines. We showed that single LPS inhalations by guinea-pigs cause inflammatory cell influx to the lungs and immediate airways hyperreactivity (AHR). After repeated LPS exposure, there was persistent AHR and goblet cell hyperplasia. Parainfluenza3 virus (PIV3) inoculation alone caused airways inflammation and AHR but no goblet cell hyperplasia indicating differential responses to microbial challenges. We hypothesise that combining LPS and PIV3 will induce heightened or differential responses. Lung function, AHR, inflammatory cell influx and goblet cell histology in guinea-pigs will identify any modified microbial responses in vivo. Influenza virus (H1N1 strains WSN33 and PR8/34) alone and with acute or chronic LPS challenge will be examined as well as measles (wildtype and vaccine virus) (mWTFB, mEdmonston) and poly[I:C]. Lung histology after these treatments will determine gross histology (haematoxylin and eosin), lung remodelling from collagen staining (Gomori's stain) and goblet cell mucin (Alcian blue/periodic acid Schiff stain). TLR-4 are implicated in innate immune responses to infection and inflammation after LPS, but not inflammation from influenza infection. Invasion of airways epithelial cells by viruses like influenza, release proinflammatory and antiviral products including IL-8, IL-6 and IFN-beta and we will measure NF-kappaB levels as an index of TLR-4 receptor activity and BAL fluid IL-8 as a marker of viral infection. These assays will depend upon sufficient cross-reactivity between reagents for guinea-pig and human. Studies will determine whether exacerbations are associated with changes in viral load using culture of live virus from lung tissue and lavage fluid, supported by real time PCR to detect viral nucleic acids. Corticosteroid use in viral inflammatory exacerbations is counter-intuitive since they should reduce immune responses and worsen viral infection. However, steroids have the advantage of being anti-inflammatory. The effects of injected dexamethasone and inhaled budesonide will be examined against functional and histological responses to LPS/virus and against viral load to assess any pro- or antiviral effects. Do steroids affect viral entry to epithelial cells and subsequent replication, and if so, what is the mechanism? A non-steroidal anti-inflammatory (NSAI) COX inhibitor, indomethacin, on viral infection and inflammation will also be examined. Mucociliary clearance will be examined from airway function and gamma scintigraphy. Inhaled mucus secretagogues (UTP, histamine) cause prolonged reduction in airways conductance, recovery of which will be an index of mucociliary clearance. Does UTP affect mucociliary clearance as well as mucus secretion? Drug interventions will be examined after inducing mucus secretion. Gamma scintigraphy will monitor clearance of radiolabelled 99mTc-Sn colloid particles instilled into anaesthetized guinea-pig trachea. Mucociliary clearance will be measured following UTP inhalation in guinea-pigs receiving LPS alone and superimposed virus. At Novartis, primary cultures of human and guinea-pig epithelial cells will enable the student to examine in vitro viral effects on cytokine production to relate to in vivo studies. Do prior exposure to IL-13, bacterial products (LPS) or oxidant stressors alter viral responses? Repeated LPS exposure desensitizes toll-like receptors. Steroids and NSAIs will be examined on viral entry into cells and subsequent replication.

Ca2+ and cAMP are delivered to cells as spatially organised signals and reciprocal interactions between them are widespread. In bronchial airways, for example, Ca2+ is the primary control of contraction, via activation of myosin light chain kinase, while receptors that evoke cAMP formation cause relaxation. Both pathways are targets of drugs used to treat asthma and chronic obstructive pulmonary disease (COPD), with agonists of beta2-receptors and antagonists of M3 muscarinic receptors in widespread clinical use. In combination therapy, these drugs act at least additively and perhaps synergistically,but the nature of the interactions is undefined. In human bronchial airway smooth muscle cells (BASMC), cAMP attenuates Ca2+ signals and the Ca2+-sensitivity of the contractile apparatus; and Ca2+ attenuates cAMP signalling. The mechanisms underlying these physiologically and clinically important interactions are unresolved. Hitherto interactions between M3 and beta2 receptors have relied on heterologous expression in cell lines, but they need to be explored in native human tissues that retain an appropriate cohort of signalling proteins. Novartis is developing combination therapies for COPD, but there is presently no human pre-clinical model for assessment of their efficacy, and limited knowledge from which to identify possible drug targets downstream of receptors. Human bronchial airway smooth muscle cells (BASMC) in culture largely retain their native phenotype for up to 10 passages, but transfection is required to maintain normal levels of M3 receptors. These cells currently provide the best model for signalling analyses in BASMC. Our aims are to identify sites of interaction between cAMP and Ca2+ in human BASMC stimulated with agonists of beta2 and M3 receptors.

While bulk chemicals are generally manufactured in efficient continuous processes, generating relatively little waste (E-factor <1-5), high value chemical products (fine chemicals, agrochemicals, pharmaceuticals) are usually manufactured in batch units, generating enormous waste (E-factor ca. 25-100). There is therefore a clear business advantage, if continuous manufacturing techniques could be adapted for use in these industries. Continuous processing can facilitate safe manipulation of potentially hazardous reagents that can nevertheless effect low-waste transformations, e.g. by efficient heat-transfer in very exothermic processes & minimising local inventory of hazardous material. Whilst most oxidations of bulk chemicals can be performed continuously using molecular oxygen in the gas phase, safety considerations (exotherm, formation of explosive mixtures) preclude its use in batch processes, particularly for thermally sensitive, involatile substrates found in fine chemical manufacture. This leads to two undesirable behaviours: a) adoption of costly, atom inefficient, waste-generating stoichiometric oxidants, with consequent negative environmental impact, and b) a tendency to avoid oxidative transformations by using inevitably less efficient 'workarounds'. It is therefore clear that oxidation is a potentially key enabling technology. The demonstration of cost-effective, safe, and efficient use of aerobic oxidation in an organic solvent would be transformative since a wide and increasing range of catalytic organic transformations would be considered seriously by industry, and not avoided due to safety concerns. The potential to drive further efficiency by 'telescoping' subsequent transformations would also be facilitated by continuous, work-up free oxidation. Thus, this project aims to instigate a step change in efficiency in the manufacture of many products by developing safe continuous oxidative transformations.