Elasmobranchs (sharks, skates and rays) are the earliest group in phylogeny to have all of the molecules of adaptive immunity also observed in mammals (MHC, TCR, Immunoglobulin). For this reason this group has been extensively studied to try to understand how the adaptive immune system evolved. Work to date has found 3 immunoglobulin isotypes in sharks; the so-called 'conventional' heavy-light chain isotypes IgM (which occurs in a pentameric and monomeric form) and IgW (the shark IgD orthologue). The third, non-conventional, shark specific isotype is called IgNAR (novel antigen receptor) and is of special interest as it is a heavy chain homodimer that naturally lacks light chain. EM studies have shown that each heavy chain has one variable (V) domain tethered to 5 (C) constant domains via a flexible, hinge-like region however dimerization of the V regions is not required for high-affinity antigen binding. Crystal structures of selected IgNAR V regions in complex with their antigen showed that the presence of germline-encoded non-canonical cysteines and a truncation through CDR2/framework region (Fr)2 give the V's a very small size (12 kDa) and extremely compact domain structure. Binding to antigen is mediated mainly by the highly diverse CDR3, which is generated through the rearrangement of 3 D regions. The long (9-24 aa) CDR3 loop protrudes from the top of the V domain to interact with antigen, and in some instances, has been shown to insert itself into clefts and active sites on the antigen surface to provide high affinity binding. In some instances this has the effect of blocking the enzymatic function of the target molecule. Due to their high affinity binding, targeting of active sites, very small size and general robustness in harsh environments there is significant interest within Wyeth towards the development of these molecules as human therapeutics. The shark basic biology group was established within Wyeth Research Aberdeen to support this aim by studying B-cell development and isotype choice in the spiny dogfish (Squalus acanthias), specifically looking at the best way to induce the production of antigen-specific IgNAR V regions, either in vivo or in vitro. Whilst molecular approaches allow us to begin to address this aim there is a real and pressing need to establish methods for the long-term culture of shark B-cells in vitro. For this we need to isolate and study the cytokines which may be involved in lymphocyte growth and development in sharks. Thus, for this work we propose to locate (via gene synteny) and sequence a number of cytokine genes from spiny dogfish which, from their role in other species, should drive B-cell growth and differentiation. We will then focus our attention on a couple of these molecules to produce as recombinant proteins and study their effects on shark B-cells in vitro.

In vertebrates, the primary afferent neurons are specialized to detect chemical, mechanical and thermal stimuli. Much of our knowledge about the function of these primary afferent sensory neurons has been obtained by studying the properties of specific neuronal sub-types innervating soft tissues such as skin and muscle. The general properties of sensory neurons have also been investigated using isolated neurons. Little is known about the properties of sensory nerves innervating bone and joints. We have shown that it is possible to identify the cell bodies of neurons innervating either joint tissues or bone by dye (e.g. True Blue) injection into the target tissue (Fernihough et al. 2004, 2005). Back labelled neurons can then be studied immunohistochemically, electrophysiologically and with functional readouts such as imaging changes in intracellular calcium levels in response to various stimuli. Where possible mice will be used in our study as this offers the possibility to use genetically modified animals to probe the function of various receptors and ion channels expressed by sensory neurons as well as molecules expressed by associated cells in the target organ. The phenotype of identified neurons will be studied using markers (e.g. antibodies) that define sub-specific known sub-types of sensory neurons (e.g. neurofilament protein, sensory neuropeptides and neurotrophin receptors). The expression of specific receptors and ion channels, known or postulated to be involved in sensory transduction, will also be studied using immunolabelling and in situ hydridization of back-labelled neurons in sections of lumbar DRG. The types of receptors and ion channels studied will include those that define nociceptive neurons (e.g. TRPV1, TRPA1, Nav1.8), which signal noxious stimuli. The expression of molecules implicated in mechanotransduction will be explored. The functional properties of enzymatically isolated, identified joint neurons will be studied electrophysiologically (whole cell recording) or using calcium imaging methods. One area of specific interest is the mechanical sensitivity of the neurons. Low and high threshhold mechanically evoked responses have been reported in sensory neurons and we will identify which types of mechanosensors are associated with joint sensory neurons. These combined studies will provide key information about the normal joint sensory neuron phenotype. Sensory neuron properties are influenced by the environment. Increased mechanical sensitivity is seen with joint inflammation. The effects of inflammatory mediators such as TNF-alpha on mechanotransduction are poorly understood. Neurons from animals in which the knee has been experimentally inflamed by intra-articular injection of Complete Freund's Adjuvant will be studied. This results in increased responses to mechanical stimulation of the joint. We will determine if the increased sensitivity is due to a hypersensitivity of the primary transduction process. Identified joint neurons from normal animals will also be exposed to inflammatory mediators and growth factors in vitro (acutely or chronically) to determine if mechanosensitivity is increased. If so we can investigate the molecular processes that underlie changes in sensitivity. The cellular assays will be complemented by behavioural studies that will be carried out in the research organization. Wyeth has expertise in behavioural models that measure responses to mechanical stimulation of the knee. We will exploit information about the types of neurons innervating the joint and bone and use ablation methods to remove sub-types of neurons. Tracers which selectively bind to sub-populations of sensory nerves will be conjugated to saporin (a ribotoxin) and administered into the knee joint. The behavioural consequence of the treatments will be studied and correlated with histological examination to determine the degree and selectivity of nerve ablation.

The incidence of psychiatric disorders, neurodegenerative diseases, and pain is on the increase. In terms of therapeutic intervention, this is self-evidently an area that requires the identification of novel pathways and neuronal targets. The CB1 and CB2 receptors are being investigated as potential therapeutic targets, however there are also likely to be considerable opportunities for modulating the synthesis of the most abundant endocannabinoid in the adult brain, 2-AG. The molecule is generated from diacylglycerol by the sn1-specific DAG lipases (DAGL-alpha and DAGL-beta) that we recently cloned (Bisogno et al. 2003). To fully exploit DAGL as a target, we need to understand how its function is regulated by protein-protein interactions. These are often mediated by linear peptides sequences and we have used a comparative genomic approach to identify the most highly conserved sequences in the DAGLs. We have synthesised four peptides (as well as reverse control peptides) in tandem with the antennapedia peptide sequence that mediates uptake into the cytosol of live cells. We now have evidence that three of these sequences represent functional motifs that have the same biological effects on cells as two conventional DAGL inhibitors (RHC80267 and THL); in this context they specifically inhibit DAGL dependent neurite outgrowth and also DAGL dependent proliferation of neural stem cells. The student will test the hypothesis that the peptides are inhibiting the function of the DAGLs by measuring their effects on DAGL activity in collaboration with scientists at Wyeth. The student will next use alanine scanning to identify key residues required for inhibitory activity and then introduce mutations to these residues in full length constructs of DAGL and determine their effects on enzyme activity following transfection into a variety of cell types. The student will also use two approaches to identify proteins that the peptides, and by implication, the DAGLs interact with. In the first approach the DAGL peptides (both the functionally blocking sequence and the reverse control) will be coupled directly to a sepahrose column. Brain extracts will be run over the columns, and specifically interacting proteins eluted and separated by conventional gel electrophoresis. Proteins that interact with the blocking but not control peptide will be identified following silver staining of the gels. Mass spectrometry methods will be used to identify proteins that interact with the blocking peptide, but not the 'reverse' control peptides. In the second approach, mini-constructs encoding the active inhibitory peptides will be used as bait in conventional yeast two hybrid screens. We anticipate that antibodies will be available for some of the interacting proteins, but if they are not we will outsource the generation of antibodies. Finally, we will test the hits for co-immunoprecipitation with DAGLs in a number of tissues and cell types.

Recent research is highlighting the important role of mesenchymal cells in tissue differentiation [1]. These cells generate the extracellular matrix environment and secrete chemokines and cytokines that direct and maintain the differentiation of other cells. Thus, mesenchymal cells are critical to the development and maintenance of tissue phenotype at all stages, from controlling stem cells (both embryonic and adult) to controlling normal tissue homeostasis. Accordingly, inappropriate mesenchymal activity contributes to the major disease states such as fibrogenesis and cancer [1]. This project will examine the role of a mesenchymal cell within the liver - termed the myofibroblast - on the growth and differentiation of a liver progenitor cell. The C1-3 antibody reagent - developed by the Academic Supervisor [2] (and licensed to the Industrial Collaborator following a previously successful BBSRC CASE award) - will be used to deplete myofibroblasts from the liver in vivo [3,4] in order to examine their role on progenitor cell proliferation and differentiation. The major functional cell of the liver is the hepatocyte. Recent work in the academic lab includes the use of an in vitro model of hepatocyte differentiation from a progenitor cell [5,6]. This precursor has now been stably transfected with selectable vectors that encode a gene for visualising these cells in vitro or in vivo (green fluorescent protein and luciferase). The role of liver myofibroblast interactions on the activity of the labelled hepatocyte pre-cursor will initially be investigated in co-cultures in vitro. Culture vessels will be seeded with myofibroblasts with or without pre-treatment with a range of factors suspected to affect myofibroblasts and/or progenitor cell activity (e.g. fibrogenic factors TGFb, TNFa; growth factors such as EGF, HGF; cytokines; anti-fibrogenic factors such as NF-kB inhibitors, PXR activators, anti-TGFb, anti-TIMP1). GFP-labelled progenitor cells will then be cultured alone or with myofibroblasts (with or without direct physical contact) and the effect of myofibroblasts on progenitor cell proliferation, response to growth factors and promoters of apoptosis examined. Confocal microscopy will be used to distinguish between myofibroblasts and progenitor cells and to co-localise expression of selected genes. FACS analysis will also be used to separate myofibroblasts and progenitor cells for quantitative analysis of any effects. To examine the effect of myofibroblasts on progenitor cells in vivo, luciferase-labelled progenitor cells will be injected (via the hepatic portal vein but other potential routes, such as the spleen will be examined) into SCID mice. Location and growth of progenitor cells and derived mature cells, will be monitored using a CCD (1.4M pixels) camera [Lightools Inc, USA] with close focus zoom lens. Myofibroblast activity will be modulated using a variety of established protocols (carbon tetrachloride treatment, bile duct ligation, concanavalin A, methionine and choline-deficient diet) and the effects on progenitor cell growth monitored. Myofibroblast depletion and its effects on progenitor cell function will be achieved using the C1-3 antibody technology [3,4]. At appropriate timepoints, dependent on imaging data, tissue will be harvested and the interactions of myofibroblasts and progenitor cells examined by histological and immunocytochemical analyses. This project will establish how myofibroblast activity impacts on liver progenitor cell activity and will determine whether therapeutics designed to modulate myofibroblast activity may impact on normal tissue regenerative potential. 1 Wallace K et al Biochem J 2008;411:1-18 2 Elrick LJ et al J Hepatol 2005;42:888-96 3 Douglass A et al J Hepatol 2008;49:88-98 4 Douglass A. et al Hepatol Int. 2008 in press 5 Marek CJ et al Biochem J 2003; 6 Wallace K et al 2008 submitted.

It is now widely accepted that up to ten years are needed to take a drug from discovery to availability for general healthcare treatment. This means that only a limited time is available where a company is able to recover its very high investment costs in making a drug available via exclusivity in the market and via patents. The next generation drugs will be even more complex and difficult to manufacture. If these are going to be available at affordable costs via commercially viable processes then the speed of drug development has to be increased while ensuring robustness and safety in manufacture. The research in this proposal addresses the challenging transition from bench to large scale where the considerable changes in the way materials are handled can severely affect the properties and ways of manufacture of the drug. The research will combine novel approaches to scale down with automated robotic methods to acquire data at a very early stage of new drug development. Such data will be relatable to production at scale, a major deliverable of this programme. Computer-based bioprocess modelling methods will bring together this data with process design methods to explore rapidly the best options for the manufacture of a new biopharmaceutical. By this means those involved in new drug development will, even at the early discovery stage, be able to define the scale up challenges. The relatively small amounts of precious discovery material needed for such studies means they must be of low cost and that automation of the studies means they will be applicable rapidly to a wide range of drug candidates. Hence even though a substantial number of these candidates may ultimately fail clinical trials it will still be feasible to explore process scale up challenges as safety and efficency studies are proceeding. For those drugs which prove to be effective healthcare treatments it will be possible then to go much faster to full scale operation and hence recoup the high investment costs.As society moves towards posing even greater demands for effective long-term healthcare, such as personalised medicines, these radical solutions are needed to make it possible to provide the new treatments which are going to be increasingly demanding to manufature.