Dr Hadwen Trust Dr Hadwen House 84A Tilehouse Street Hitchin, Herts. SG5 2DY, UK Registered Charity no. 261096 Dr Hadwen Trust About the Dr Hadwen Trust Telephone 01462 436819 The Dr Hadwen Trust's mission is to advance the development and acceptance of Fax 01462 436844 Science Review non-animal techniques to replace animals in biomedical research. To achieve this we fund a programme of research projects to develop alternative methods, which e-mail [email protected] combine the highest humane principles with the best scientific standards. The Trust 2005 also actively promotes the wider use of non-animal methods through publications, website presentations, workshops and debates. With nearly thirty-five years' experience in the www.drhadwentrust.org.uk THROUGH field of humane research, the Trust is widely consulted for its expertise. General Secretary THE LENS Mr Terry Huxtable Grant Applications The Trust only funds non-animal research that has the potential to replace Scientific Adviser EPLACEMENT Dr Gill Langley, MA R experiments which are currently conducted on living animals. Application enquiries PhD (Cantab) MIBiol IN SIGHT contact: Carol Newman, Scientific Officer. Telephone 01462 436819 or e-mail: [email protected] Patrons Joanna Lumley, OBE David Shepherd, OBE

Dr Hadwen Trust 2005 Research Portfolio NEW EPIDEMIOLOGY SHOWCASING SUCCESSES IN Dr S J Hirst, Dr V A Snetkov & Prof Prof G Pilkington NON-ANIMAL RESEARCH T H Lee Portsmouth University SMARTER King’s College London 2004-2006 Research Assistant DRUG 2005-2008 Research Grant Production and evaluation of three- DEVELOPMENT Pathobiology of airway smooth dimensional live cell imaging muscle from asthmatics. models for the study of novel brain tumour therapies. Dr H Johansen-Berg, Dr T Berens PROBING

Art Today Dr P Stephens & Prof D Higham CANCER CELLS Oxford University Cardiff University Dr G Barnes, Dr A Hillebrand & Dr 2004-2007 PhD Studentship 2005-2008 Postdoctoral Fellowship P Furlong Development of a cell-based Computational approaches to CANCER CELLS Aston University chronic wound bioassay. analysing human brain networks IN CONCERT 2004-2006 Postdoctoral Fellowship and their breakdown in disease. Development of neuropharmacolo- Dr I Tracey & Prof P Matthews gy: towards directed non-invasive FRIMB Centre, Oxford University Prof J Jones human drug testing. 2002-2005 Postdoctoral Fellowship St Barts & the London NHS Trust Understanding pain and its VIRUS CLUE TO Celebrating 2004-2007 Postdoctoral Fellowship Prof T Evans alleviation with pharmacological ASTHMA Glasgow University Development of three-dimensional tools using functional magnetic 35 years 2004-2007 Postdoctoral Fellowship multicellular models of normal resonance imaging in human VACCINE SAFETY of humane Using a novel three-dimensional breast and pre-invasive breast subjects. cell culture model to investigate cancer. IN VITRO research sepsis-induced renal failure. Dr D Xing Prof Y Mahida National Institute for Biological Dr M Griffiths Nottingham University Standards and Control, Royal Brompton Hospital, London 2004-2007 Postdoctoral Fellowship Potters Bar 2006-2008 Research Technician Establishment of model systems to 2003-2006 Research Grant The development of complex in investigate interactions between Identification of gene markers for in vitro models of ventilator- human intestinal mucosa and vitro control test for Pertussis

associated lung injury. Clostridium difficile and its toxins. vaccines. ARS; www.photos.com University; PJ Smith/RJ Errington/Cardiff Front cover photo credits: www.photos.com;

ISSN 1741-0967 Printed on 100% recycled paper. WELCOME PROJECTS SHOWCASE The first word Through the lens

DR HADWEN TRUST EQUIPMENT GRANTS 1996 – 2001 ‘Vision’ is a key theme running through our Science Review this year, both overtly and in more subtle ways. Contents Studying cataract in human lenses in vitro In the pragmatic sense, two of our Showcase Projects focus on Grant awarded to Dr A Prescott and Dr R Quinlan, then at the Department of the eye, one featuring cataract studies in vitro and the other our Through the lens 2 historic research that kick-started worldwide efforts to replace Biochemistry, Dundee University the Draize eye test. With Showcase Projects, we revisit past research funded by the Trust to review subsequent progress Probing cancer cells 3 The cell lines were used to study the and impact on replacing animal experiments. intermediate filament proteins involved in cataract, as well as changes in small Works in Progress are vignettes of current projects funded by heat shock proteins (sHSPs). the Dr Hadwen Trust, all of which are selected for their Replacement in sight 5 Intermediate filaments are fibrous contributions to medical progress and replacing laboratory “The lens cell lines… proteins forming a structural network within the cytoplasm. sHSPs protect animals. But the Science Review goes wider than just the are in use in our and Trust’s initiatives, reeling in keynote events and viewpoints from Viral villains in dementia 7 protein structure and activity, and these the wider scientific community. two systems interact in the lens and other labs around the other tissues. Professor Patrick Bateson, who has a long-standing interest in world in situations Drug action — a clearer picture 9 αB-crystallin is a sHSP and a major lens animal issues, is our interviewee this time. He gently chides the where, otherwise, protein. Alan Prescott and Roy Quinlan Trust for expending too much energy on undermining the value suggested that one of the physiological of animal research — a comment we’ve also heard from others. animal cells would have functions of sHSPs like αB-crystallin and The Trust’s primary aim is to develop the scientific tools to Cancer cells in concert 11 HSP27 is to help manage intermediate replace animal research, but we do also criticise the relevance been used — either filament networks in cells [1]. of some animal experiments. The scientific process is robust from abattoir material or enough to encompass fair criticism, and we always cite the Vaccine safety in vitro 13 Disruptions of the α-crystallins can literature to support our concerns. from laboratory animals” reduce lens transparency and lead to cataract, the major cause of blindness Pat Bateson highlights the excitement of solving problems Alan Prescott worldwide. A particular mutation in the Replacement: the Nuffield view 15 α without animal experiments and believes that this research will B-crystallin gene underlies both increasingly attract academic kudos and funding. We agree. cataract and an inherited condition called desmin-related myopathy. And that’s where the second, aspirational meaning of vision is Recently, a lens cell line H36, derived our theme. Practising the best science without causing Biosensors 15 from a 36-week-old donor, provided a suffering to other animals requires an unblinkered vision, an Dr Alan Prescott continues his model to tease apart the processes innovative approach and an embracing ethical perspective. research at the School of Life Sciences involved [2]. Vaccine tests report 16 Research at Dundee University. Dr Roy Quinlan It is an exciting challenge, and the Dr Hadwen Trust hopes that is now at the School of Two important results were that normal Biological and Biomedical Sciences, Pat Bateson is right when he says that researchers can αB-crystallin can assist the assembly of

Durham University. University H Girao/IBILI, Portugal & A Prescott/Dundee become strong allies in this endeavour. Smarter drug development 16 intermediate filaments, supporting the hypothesis originated in the earlier Trust- Exposure of human lens cell line H36 to funded studies. Secondly, mutated αB- 25-hydroxycholesterol causes relocalisa- crystallin forms damaging aggregates in References tion of HSP27 (green) to the collapsed Primate research inquiry 17 vimentin filaments (red). Oxidised association with the filament proteins. 1. Perng MD, Cairns L, van den cholesterol has been implicated in IJssel P et al (1999). Intermediate γ cataract development Genes that code for -crystallin proteins filament interactions can be altered are specifically expressed in the eye HSP27 and αB-crystallin. J. Cell Sci. NC3Rs: replacement centre stage? 17 lens. Several cell types, including a 112:2099-2112. human lens epithelial cell line, were used This research aimed to create 2. Perng MD, Wen FS, van den IJssel to explore the effects of transcription human cell lines from the lenses of Asthma and the common cold 18 P et al (2004). Desmin aggregate factors on the genes [3]. The results formation by R120G αB-crystallin is cataract patients; to make them widely supported the idea that the transcription caused by altered filament interactions available for research; and to develop factor Prox1 is an important activator of and is dependent upon network status an in vitro model of human lens cell dif- the genes that code for γ-crystallin, while ferentiation. Biosimulation network 18 in cells. Mol. Cell Biol. 15:2335-2346. the Six3 factor represses those genes. More than 50 cell lines were established 3. Lengler J, Krausz E, Tomarev S et Other researchers have exploited the al (2001). Antagonistic action of Six3 by immortalising lens cells from cataract Dundee cell lines; one was used to The new epidemiology 19 and Prox1 at the g-crystallin promoter. patients and normal donors. Despite Nucleic Acids Res. 29:515-526. varying the culture conditions, most investigate a key enzyme that protects

NeilTurner/News International NeilTurner/News lines only exhibited early-stage differen- the eye from ultra-violet light. 4. Truscott RJW (2005). Age-related tiation, indicated by the expression of Interestingly, animal models of age- Dr Gill Langley MA PhD MIBiol CBiol Interview: Professor Pat Bateson 21 nuclear cataract — oxidation is the lens fibre-specific crystallins and related cataract are considered highly key. Exp. Eye Res. 80:709-725. imperfect because of species differences Scientific Adviser to the Dr Hadwen Trust for Humane Research cytoskeletal proteins. [4]. October 2005

1 Dr Hadwen Trust Science Review 2005 Dr Hadwen Trust Science Review 2005 2 SHOWCASE PROJECTS 4 et Adv. Cancer Br. J. Br. J. Radiat. Math. Biosci. Pharmacol. 40:280-291. 5:5051-5054. . 57:153-167. (2005). Advanced Org. Lett. Org. Cytometry et al (2004). A mathematical model (2003). Tracking the cell cycle (2003). Tracking 88:1310-1317. Cancer Chemother 61:749-757. (2003). Solid-phase synthesis of 49:1118-1124. et al et al (2000). Characteristics of a novel

microscopy solutions for monitoring solutions microscopy the kinetics and dynamics of drug- DNA in living cells. targeting Rev. Drug Deliv. 9. Councils UK website: Research www.rcuk.ac.uk/basictech/proginfo/ Errington.pdf 189:185-217. 7. Zloh M, Kadom M Malkinson JP, et al the cyclic peptide portion of an inhibitor of p53-MDM2 chlorofusin, interactions. 8. B SM, Ameer-Beg Errington RJ, Vojnovic for the pharmacokinetics of the anti- cancer agent topotecan. origins for escape from topotecan origins for escape from cancer cells. action by breast Cancer 6. Evans ND, Errington RJ, Shelley M 35:403-410. 4. M Smith PJ, Blunt N, Wiltshire al cell- fluorescent deep red/infrared permeant DNA DRAQ5, in probe, intact human cells analyzed by flow and multiphoton confocal cytometry, microscopy. 5. Errington RJ, Wiltshire Feeney GP, M Biol. 3. (1995). Fox ME & Smith PJ Subcellular localisation of the and the mitoxantrone antitumour drug induction of DNA damage in resistant and sensitive human colon carcinoma cells. Res. 2. Falk SJ & Smith PJ (1992). DNA of the effects damaging and cell cycle camptothecin topoisomerase I poison in irradiated human cells. References 1. Smith PJ & Makinson TA of overproduc- Cellular consequences (1989). tion of DNA topoisomerase II in an line. ataxia-telangiectasia cell PJ Smith/RJ Errington/Cardiff University Errington/Cardiff Smith/RJ PJ who co- [email protected] Professor Paul Smith, is now this report, authored of Cancer Biology in the Professor School of Medicine at Cardiff University. Contact in awards for research into for research in awards the size of a single microlasers, human cell, that exploit the drugs of properties fluorescent and dyes in optical biochips [8]. The biochips will be incorporated into hand-held devices, thus miniaturising an optical laboratory onto an instrument the size of a credit goals include The project card. developing these portable devices for use with human cells food safety, in drug discovery, monitoring hazard environmental and the diagnosis of disease in patients from samples directly [9]. grant was The Dr Hadwen Trust an important part of the seed- funding that allowed the early ideas to be developed, in that still hold true to directions for alternativesthe search that of animal the burden reduce experimentation. Fluorescent probes light up dividing cancer cells involves many UK scientists — all the importance of recognising using living human cells as the best place to start when for new medicines. screening Paul has successfully developed (DRAQ5) probe a new fluorescent for tracking the DNA content of living and fixed human cells [4], now used worldwide for for new medicines. screening Recent work has aimed at understanding which cells in a for drug responsible tumour are using cancer, in breast resistance methods that allow the tracking of individual cells [5]. An exciting new development is a collaboration, with engineers and mathematicians at Warwick to develop University, mathematical models to predict cancer cells should how breast to a new anticancer drug respond called topotecan [6]. Armed with these new tools, Paul hopes to leads for novel take forward drugs with collaborators in London [7]. drawing upon directions, Current those early days in Cambridge, than £2.3 million have led to more Dr Hadwen Trust Science Review 2005 Trust Dr Hadwen

miniaturised systems to assess miniaturised systems the use of candidate drugs without worked by animals. The reader to shining light into each microwell chemicals excite the fluorescent of interest features and reveal inside living human cells. One of the most interesting is, of course, DNA. Some features anticancer drugs act by causing for example DNA strand breaks, by influencing the formation of complexes between DNA and the nuclear topoisomerase enzymes was reader [1]. The fluorescence used to rapidly analyse enhanced DNA-unwinding in alkaline strand the from solutions, resulting or caused by added drugs breaks chemicals [2]. acorn grant The Dr Hadwen Trust’s — the first such grant that Paul — as a researcher had received enabled him to exploit the fact that naturally some drugs are be and can therefore fluorescent tracked on their journey into the Using cell until they hit their target. Paul reader, the fluorescence tested ideas for using human cells of drugs, and to track the effects to understand why some drugs fail. The grant promising also helped his PhD student Mary Fox to develop a method of detecting the DNA-damaging action of drugs in small samples of human colon cancer cells [3], setting her on a cancer research career. transfer to group 1995 saw Paul’s new facilities in Cardiff, of excellence establishing a centre for live human cell-imaging using time-lapse, confocal and multi- The original photon microscopy. concepts have developed into a that also effort personal research PJ Smith/RJ Errington/Cardiff University Errington/Cardiff Smith/RJ PJ Dr Hadwen Trust Science Review 2005 Trust Dr Hadwen The Dr Hadwen Trust in support to Paul Smith provided 1987 when he worked for the Council in Medical Research Cambridge, in a unit dedicated to of cancer the improvement treatments. Paul has been active in the fields drug development of DNA repair, and advanced cell detection than 20 technologies for more years — with his focus remaining His early work on on cancer. understanding how cells respond to anticancer drugs was a technology supported through ‘acorn’ the Dr Hadwen grant from Trust. a The grant was used to purchase reader, microplate fluorescence for measuring light emitted by chemicals added to fluorescent in 96 human cells, growing on a plate the size of a microwells The aim was to postcard. establish human cell-based, Paul Smith experimentation” The optical biochip: a red light-emitting microlaser the size of a single human cell the burden of animal true to the search for “The Dr Hadwen Trust directions that still hold alternatives that reduce ideas to be developed in grant… allowed the early DR HADWEN TRUST EQUIPMENT 1987 – 1990 GRANT DNA and anticancer drugs in human cells DNA and anticancer then at the MRC Clinical N Bleehen, Dr P Smith and Professor to Grant awarded University Radiotherapeutics Unit, Cambridge Oncology and Probing cells cancer

SHOWCASE PROJECTS 3 HWAEPROJECTS SHOWCASE HWAEPROJECTS SHOWCASE Replacement in sight

DR HADWEN TRUST RESEARCH FELLOWSHIP 1982 – 1986

tested 52 compounds representing a In vitro methods for assessing eye irritancy broad range of chemical structure, Grant awarded to Dr B Northover and Dr C Muir, then at the Department of physical form and aqueous solubility. They concluded that the assay is Pharmacology, Leicester Polytechnic reproducible and easy to use without special expertise or training. In addition, the endpoints (opacity, permeability, and changes in guishing between shampoos of thickness of the cornea) are differing eye irritancy using cell quantifiable and are complementary cultures, with cell viability as the for a better prediction of ocular endpoint. It laid the foundations for irritancy. the development of cytotoxicity assays, which remain important Since then the BCOP test has among current approaches to replace undergone several other evaluation Draize eye test. and validation processes, and is still being improved. In the last year, for Subsequent research, funded by the example, a new corneal holder has Trust through the 1980s, pursued a been designed that maintains the second and complementary angle normal curvature of the tissue, with even greater success. Dr Colin further enhancing the test’s repro- Muir, a research fellow at Leicester ducibility. The use of laser light Polytechnic, was an innovative instead of white light may improve its scientist. His was the first eye performance in ranking mild and

irritation test based on corneal moderate eye irritants. Aid Iain Green/Animal opacification using the isolated Replacing rabbit eye tests: a goal finally in sight

www.photos.com bovine cornea — the forerunner of The assay is considered highly suitable for screening moderate and Eyes need protection from chemical damage the now widely used bovine corneal opacity and permeability (BCOP) severe eye irritants. The Belgian assay. Because bovine corneas are Pharmaceutical Commission accepts other single non-animal method is a 2. Reviewed by Simons PJ (1980). An obtained from the slaughterhouse, the test for identifying drug stand-alone replacement for the alternative to the Draize eye test. In: the method does not cause the death formulations that are eye irritants. rabbit eye test in regulatory The Use of Alternatives in Drug Research. or suffering of animals, unlike other Positive BCOP data are accepted in toxicology. However, through its Publ. Macmillan Press. “The Draize test… has 19,100 experiments involving organotypic models such as the the EU for pesticides and other application in-house, and the the “application of substances to the isolated rabbit eye. products. In France, Germany, the acceptance by regulators of positive 3. Muir CK (1987). Surfactant- two other major eyes of rabbits” were conducted in Netherlands, the UK and Ireland, BCOP data for severe eye irritants, it induced opacity of bovine isolated Britain in 1982. Twenty-one years shortcomings: …low Colin developed the opacitometer, a BCOP results are considered valid has already contributed significantly cornea: an epithelial phenomenon? later, the number of Draize eye tests for classifying new and existing set-up which shines a beam of light to a massive reduction in rabbit eye Toxicol Lett. 38:51-54. interlaboratory had fallen to 1,107, with a similar through the isolated cornea enabling substances that are severe eye tests. pattern in many other countries. irritants [7]. The assay is also used an objective measurement of changes 4. Muir CK (1984). A simple method routinely in-house by companies to It is widely agreed that a major reproducibility, and in its opacity. He tested several to assess surfactant-induced bovine How did this dramatic change come assess the safety of products such barrier to fully replacing the rabbit surfactants and industrial chemicals corneal opacity in vitro: preliminary differences in about? A political campaign, launched as air fresheners, insect repellants, eye test has been “…a lack of in vivo in his new assay, and also identified findings. Toxicol. Lett. 22:199-203. in 1980 by a global coalition of animal the corneal epithelium, rather than cleaning and laundry products, data of sufficient quality for use in sensitivity… between protection groups, highlighted the the stroma or endothelium, as a key cosmetics, hair dye formulations and validation studies” [7]. Despite this, 5. Muir CK (1985). Opacity of bovine ethical and scientific limitations of the site of opacification [3]. His pharmaceutical intermediates. there are now several leading rabbits and humans” cornea in vitro induced by surfactants Draize eye test. This led to cosmetics publications between 1984 and 1987 techniques that could comprise a and industrial chemicals compared industry funding to research are acknowledged as providing the The BCOP test’s advantages include battery to fully replace the rabbit eye EU expert group on with ocular irritancy in vivo. Toxicol. alternative methods, followed by essential groundwork and inspiration simplicity of operation, quantifiable irritancy test, including physico- Lett. 24:157-162. eye irritation [7] amendments to the European Union for the BCOP test [4, 5]. and relevant endpoints, suitability for chemical, cytotoxicity and Cosmetics Directive that introduced identifying moderate and severe organotypic methods. It’s been a 6. Gautheron P, Dukic M, Alix D & deadlines by which animal testing for In 1992 the pharmaceutical industry irritants, applicability to many long journey since those first Sina JF (1992). Bovine corneal opacity cosmetics would be banned. became interested in the Muir assay. chemicals and products, developments in the 1970s and ’80s, and permeability test: an in vitro assay Gautheron and colleagues from preservation of three-dimensional but the goal is clearly in sight. of ocular irritancy. Fundament. Appl. The Draize eye test has Merck Sharp and Dohme developed it tissue structure and avoidance of Toxicol. 18:442-449. acknowledged failings, including its further, adding an extra endpoint: animal suffering. There are References poor reproducibility and species corneal permeability changes as limitations: it performs less well with 7. ECVAM (2002). Local toxicity: differences between rabbits and determined by retention of fluorescein solids and alcohols, tends to 1. Eskes C, Bessou S, Bruner L et al acute dermal and ocular effects. In: humans [1]. Dr Hadwen Trust was one [6]. underestimate some chemicals, is (2005). Eye irritation. In: Alternative Alternative (Non-Animal) Methods for of the first-ever funders to support not as sensitive in distinguishing mild (Non-Animal) Methods for Chemicals Testing: Current Status and research to replace the test, with a This was followed by an international irritants, and uses a bovine — not a Cosmetics Testing: Current Status Future Prospects. ATLA 30 (Suppl. project at the then Hazleton evaluation of the BCOP test. human — tissue. and Future Prospects. ATLA 33 1):35-47. Laboratories in 1975 [2]. The study Supported by the European (Suppl. 1):47-81. demonstrated the feasibility of distin- Commission, twelve laboratories Neither the BCOP assay nor any

5 Dr Hadwen Trust Science Review 2005 Dr Hadwen Trust Science Review 2005 6 HWAEPROJECTS SHOWCASE PROJECTS SHOWCASE Viral villains in dementia

DR HADWEN TRUST RESEARCH STUDENTSHIP & EQUIPMENT GRANT 1996 – 2000 Herpes simplex type 1 virus in Alzheimer’s disease Grants awarded to Professor R Itzhaki and Dr M Wozniak, Molecular Neurobiology Laboratory, University of Manchester

reactivating periodically in some APOE-ε4 carriers more frequently people to cause cold sores. Rarely, exhibit dementia and peripheral herpes simplex viruses can cause neuropathy. encephalitis affecting the same regions of the brain as AD, and The Dr Hadwen Trust’s grants causing memory loss. enabled the training of PhD student Matthew Wozniak in the highly At a time when transgenic animals sensitive analytical techniques are increasingly used to investigate required for this research, which need AD, Ruth Itzhaki chose instead to extreme care against contamination. study post-mortem human brain Matthew applied these techniques to specimens. Her work with human search for the presence of other tissue demonstrated for the first time herpes viruses in post-mortem human that HSV1 could reside in the central brains. nervous system and that, combined with a specific genotype, it is strongly DNA of human herpes virus 6 (HHV6) associated with AD. was found in a high proportion of AD brains, and cytomegalovirus (CMV) in www.photos.com Analysis of human brain specimens many vascular dementia brains [2, 3]. Brain scans help diagnose dementia, but not before damage has Whether either virus is a cause or a with an ultra-sensitive polymerase been done chain reaction technique established consequence is still uncertain, that the DNA of HSV1 was present in although studies elsewhere suggest a

www.photos.com causal role. In contrast, herpes the brains of a high proportion of has revealed that APOE-ε2 is risk References elderly people, both with and without simplex virus 2 is only present in a factor for HSE [5]. The particular Age brings increasing susceptibility to dementia, but other factors are AD. This does not refute a viral role in low proportion of brains, and does APOE allele that confers susceptibility 1. Itzhaki RF, Lin W-R, Shang D et involved too AD, as infection can have varying not appear to be involved in AD. to severe damage, or protection from al (1997). Herpes simplex virus type 1 effects in people, probably reflecting it, in a specific disease might be in brain and risk of Alzheimer’s a difference in genetic factors. If, as postulated, HSV1 reactivates in disease. Lancet 349:241-244. Indeed, it was then discovered that the central nervous system, causing determined by cell type as well as by Some 25 million people the pathogen involved [6]. the virus confers a strong risk of AD mild encephalitis, one would expect 2. Lin W-R, Wozniak MA, Wilcock succumb to dementia worldwide, when in the brain of carriers of the to find antibodies to the virus in GK et al (2002). Cytomegalovirus is inflicting a heavy cost in terms of cerebrospinal fluid. Matthew used The work has demonstrated the type 4 allele of the gene for present in a very high proportion of human suffering and the health ε ELISA to successfully detect rationale and utility of studying human apolipoprotein E (ApoE- 4). This was brains from vascular dementia economy. The causes of dementia are antibodies to HSV1 in cerebrospinal “Our research uses cell the first report of an environmental autopsy and biopsy specimens for patients. Neurobiol. Dis. 9:82-87. largely unknown and there are no agent acting with an inherited factor fluid of elderly normal and AD investigating the major risk factors in effective treatments. With an in a neuropsychiatric disease [1]. patients, providing evidence that the cultures and post- Alzheimer’s and other diseases, rather 3. Lin W-R, Wozniak MA, Cooper increasingly elderly population, virus had been active in the brain — than animals. In the future the RJ et al (2002). Herpesviruses in the methods of prevention and treatment One explanation is that latent HSV1 in possibly recurrently [4]. mortem human tissue. It research will be extended to HSV1- brain and Alzheimer’s disease. J. are urgently being sought. the central nervous system may be infected cell cultures to follow Pathol. 197:395-402. has yielded invaluable A number of other intriguing lines of periodically reactivated (a type of mild pathogenic mechanisms and test Ruth Itzhaki’s and Matthew Wozniak’s encephalitis) during episodes of research were pursued. Post-mortem anti-viral agents for the possible 4. Wozniak MA, Shipley SJ, information, and the ground-breaking research on post- stress or immunosuppression, brains of Down’s syndrome patients, treatment or prevention of AD. Combrinck M et al (2005). Productive mortem human brain tissue has causing cumulative damage. This who eventually develop dementia, more we obtain, the less herpes simplex virus in brain of shown that a virus plays a major role effect may be greater in carriers of were examined for the presence of elderly normal subjects and in the development of the main type APOE-ε4, than in people with other herpes viruses. HSV1, HSV2 and Professor Ruth Itzhaki, who co-wrote need there would be for Alzheimer’s disease patients. J. Med. of dementia, Alzheimer’s disease. APOE alleles. Apolipoprotein E has CMV were only detected with low this report, is in the Faculty of Life animal experimentation several major functions in the body, frequency, but HHV6 was found in 96 Sciences at Manchester University and Virol. 75:300-306. The idea of a viral involvement in including the transport of lipids to per cent of Down’s syndrome brains, has won awards from The Lancet, the — currently much used Alzheimer’s disease (AD) arose cells and cell repair. compared to only 52 per cent of Wellcome Trust and the Olympus 5. Lin W-R, Wozniak MA, Esiri MM et al (2001). Herpes simplex for AD studies” because several common viruses (e.g. normal brains. Foundation. Dr Matthew Wozniak is measles) can cause serious The APOE-ε4 allele is also a risk pursuing post-doctoral research at encephalitis: involvement of apolipoprotein E genotype. J. Neurol. neurological disease many years after factor for developing cold sores: Herpes simplex encephalitis (HSE), a Manchester University into Ruth Itzhaki Neurosurg. Psychiatry 70:117-119. initial infection. Herpes simplex virus further evidence that the combination rare but severe neurological disease neurovirology and Alzheimer’s type 1 (HSV1) was a possible of HSV1 and APOE-ε4 is damaging in caused by HSV1, also involves a disease. 6. Dobson CB, Wozniak MA & candidate, as it is extremely common, the nervous system. Some strikingly genetic susceptibility related to the Itzhaki RF (2003). Do infectious residing in a latent form in the similar findings have come from a APOE gene. Examination of the DNA Contact agents play a role in dementia? peripheral nervous system, and study of AIDS patients that shows of HSE patients and normal controls [email protected] Trends Microbiol. 11:312-317.

7 Dr Hadwen Trust Science Review 2005 Dr Hadwen Trust Science Review 2005 8 WORK IN PROGRESS 10 et in et al Methods 90:5662- 26:13-17. 26:347-355. who co-wrote this who co-wrote 412:150-157. NeuroImage NeuroImage Gazzaniga MS, Senior C & www.allp.com/drug_dev.htm Nature (2005). The missing link: analogous (2001). Neurophysiological

6.Adjamian P Hall SD & The chemistry of cognition. In: (in press). in Mind. Russel T (eds.). Publ. MIT Press, Cambridge, USA. 4. OA, Behar KL Rothman DL, Petroff & Mattson RH (1993). Localized 1H of gamma- NMR measurements aminobutyric acid in human brain Acad. Sci. USA Natl. vivo. Proc. 5666. 5. Jensen O, Goel O, Kopell N (2005). On the human sensorimotor- and sources cortex beta rhythm: modeling. Dr Stephen Hall, article, has published magnetoen- cephalography (MEG) studies of the visual cortex and of migraine visual aura, as well as a new technique for analysing MEG data. The research reputation has an international group based on 16 years of experience in this field. References 1. Corp. Alliance Pharmaceutical website: 2.A Hillebrand Hall SD, Holliday IE, et al human and primate cortical gamma oscillations. 3.Augath M Logothetis NK, Pauls J, al investigation of the basis of the fMRI signal.

Institute, Aston Institute, Neurosciences Research Neurosciences neuronal electrical activity, and activity, electrical neuronal indicates that power in the ongoing rhythms of electrical as activity is radically altered diazepam is absorbed into the to the delivered and circulation brain. that demonstrate Early results change in the is a profound there (15 - 25 Hz) beta frequency activity in the sensory motor cortex (see illustration), as might be expected [5]. Using this of the brain can method, regions diazepam has be identified where on neuronal maximal effect activity. This combination of MEG and MRS into a pharmaco-imaging method [6] is a novel approach potential which possesses great — for drug development as well of primate as the replacement experiments. The Aston group’s complementary imaging the char- techniques could refine acterisation of therapeutic agents, as well as give valuable information on very low doses of drugs which could be used to Alzheimer's disease epilepsy, treat and many common psychiatric disorders. Dr Hadwen Trust Science Review 2005 Trust Dr Hadwen concentration data from MRS to concentration data from of give a complete profile changes oscillatory neurochemical drugs. by specific triggered profile this neuronal Additionally, may be combined with cognitive or behavioural data to better understand the mechanism by are which mental processes modulated by specific drugs. being The imaging techniques are of used to follow the effects drugs, in particular those that butyric acid gamma-amino affect in the (GABA) neurotransmitters brain. GABA is an inhibitory neu- which reduces rotransmitter, excitation. At the neuronal is moment, the Aston research focused on the well-characterised [4] and widely used psychotropic The drug drug diazepam (Valium). is known to influence electrical activity in the brain by enhancing the size of GABA synaptic electrical activity and also influences the overall electrical by electroen- patterns recorded cephalography (EEG). the level of MRS can measure GABA in the visual cortex and so of diazepam follow the effects administration. MEG has enabled the passive observation of the magnetic fields generated by Beta (15 – 25 Hz) power increase in the sensory motor cortex after diazepam administra- saturating at 40 - 50 mins, consistent with tion, showing increase over one hour, expected drug uptake (modified from [6])

accurately predict the outcome of accurately predict humans is a particular drug in to say the least. problematic, BarnesGareth and colleagues at Research the Neurosciences models Institute intend to provide linked of human neurochemistry to the electrical behaviour of brain cells located in the cortex. In Dr Hadwen Trust to do so, order funding was obtained to make use of non-invasive imaging at Aston: techniques, pioneered magnetoencephalography (MEG) and magnetic resonance (MRS) [2]. spectroscopy MEG and MRS have which complementary strengths information detailed permit more to be collected than can be attained by either method used of MEG generates images singly. at electrical activity the brain’s high spatial and temporal but cannot provide resolution, anatomical or neurochemical information; MRS shows the neuro- distribution of targeted transmitters and the various within the metabolites produced brain. and colleagues have used Gareth methods to neuroimaging establish non-invasive human of findings which other correlates have obtained by researchers means of invasive primate [3]. recordings work being In the current fellow undertaken by research Stephen Hall, MEG is used to identify the electrical activity of the cortex over time following drug administration, and is combined with chemical Jane Thomas Jane Dr Hadwen Trust Science Review 2005 Trust Dr Hadwen Current drug development Current upon animal is heavily dependent Only 20 per cent of research. drugs tested on animals succeed in human clinical trials and reach the market [1], suggesting that only frequently animal models are of human approximations physiology. This distinction is perhaps most evident in the field of neuropsy- as mental chopharmacology, health conditions such as particularly difficult are depression to characterise in animals. either the use of Consequently, physiological or behavioural elicited in animals to responses least” Stephen Hall physiological or “The use of either drug in humans is elicited in animals to accurately predict the behavioural responses problematic, to say the outcome of a particular DR HADWEN TRUST FELLOWSHIP RESEARCH – 2007 2004 Neuropharmacology: towards directed non-invasive human drug testing Dr G Barnes, Research Furlong and Dr S Hall, Neurosciences Dr A Hillebrand, Dr P University Institute, Aston Drug picture– a clearer action Aston has the only whole-head MEG system in the country

WORK IN PROGRESS 9 WORK IN PROGRESS 12 et al et al is a in vitro 204(S1):2A. 62:3289-3297. J. Pathol. 201:562-572. Cancer Res. Dr Debbie Holliday [email protected]

J. Pathol. Jane Thomas Jane 2. Jones JL, Shaw JA, Pringle JH & RAWalker (2003). Primary breast myoepithelial cells exert an invasion- cancer cells on breast effect suppressor of MMP via paracrine down-regulation and tumour in fibroblasts expression cells. 3. RA Holliday DL, Shaw JA, Walker & Jones JL contribution (2004). Stromal cancer invasion: intrinsic to breast to invasion relate genetic differences ability. promoting (2002). Altered Tenascin-C isoform (2002). Altered Tenascin-C in invasive and pre-invasive expression cancer. breast 4. H, Shaw JA Wan Mulligan KT, models to study breast cancer models to study breast progression. Contact References 1. RA Adams M, Jones JL, Walker of S100A9 in the role The (in press). of normal effect tumour suppressor myoepithelial cells. Poster breast at the Pathology Society of presentation Newcastle, Britain and Ireland, Great 2005. and the Royal London NHS Trust to and the Royal London NHS Trust of the microenviron- investigate the role cancer of breast ment in the control progression. Fellow funded post-doctoral Research working on the by the Trust, of development and refinement , who wrote this , who wrote phenotype, form DCIS-like structures in co-cultures with tumour cells (see illustration) and exhibit a tumour- suppressor activity in keeping with This primary myoepithelial cells [4]. indicates that these cell lines are likely to be a useful component of our model are now directed at system. Our efforts further refining these complex models, including the three major cell populations, both as primary and immortalised lines, and to validate these model systems against primary tissues. believe these models will provide We powerful tools for the investigation of basic cell biological mechanisms in both normal and diseased breast, as well as evaluation of new pharmaco- logical agents, such as the small- molecule tyrosine kinase inhibitors, that may target DCIS, and the analysis of chemopreventative agents in early stage disease. As well as replacing use of animals, these models are likely to be superior to animal models because they more closely represent human disease and recapitulate the complex intercellular interactions that may be important in modifying tumour cell behaviour. Professor Louise Jones Pathology of Breast article, is Professor at Queen Mary’s Schools of Medicine Barts. She holds grants and Dentistry, Cancer Campaign, MRC the Breast from of St. Bartholomew’s and the Trustees in vivo Dr Hadwen Trust Science Review 2005 Trust Dr Hadwen Debbie Holliday encourages breast cells to act ‘in concert’ Debbie Holliday encourages breast cells to These different cell populations have These different been pre-labelled with vital dyes and incorporated into three-dimensional cultures. date, we have generated models of To tumour cells with myoepithelial cells, in which acinar structures resembling breast glandular units are formed; and are tumour cells with fibroblasts. We now generating models incorporating all three cell populations. The use of primary cell populations allows important investigations into donor-donor variability which may influence tumour progression. For example, we have shown that donors vary in fibroblasts from different their ability to promote tumour invasion and this relates in part to genetic polymorphisms regulating matrix met- alloproteinase expression [3]. it is also important to However, develop systems without the variability do To donors. introduced by different this, we are collaborating with Professor Mike O’Hare at the Ludwig Institute to generate immortalised breast fibroblast and myoepithelial cell lines, which will then allow targeted manipulation of individual molecules to on tumour investigate their effect invasion and growth. Early experiments using an immortalised myoepithelial cell line have demonstrated that the cells largely maintain their models human In vitro in vitro to invasive disease would not to invasive disease would complexity of DCIS, thus in-situ mechanisms that lead to progression mechanisms that lead of for more tailored only provide a means could also therapy for patients, but provide novel predictive and therapeutic targets. Many studies on breast cancer progression use mouse models, such model, or Tag as the SV40 large xenograft systems, implanting transformed mammary cells or human DCIS into nude mice. generally sacrifice complexity for use of human cells and greater ability to The directly manipulate the system. primary aim of this project is to develop and refine models which more closely reflect the in vivo replacing the need for animal model systems. One of the major considerations in generating a physiologically relevant model is the re-creation of the microenvironment. It has become evident that the behaviour both of normal and neoplastic cells is profoundly influenced by the host microenvironment. and others have demonstrated the We importance of tumour interactions with the stroma in promoting tumour cell invasion, and we have shown that alterations in the breast stroma occur around DCIS lesions, prior to invasion — suggesting that such changes may have a role in the initiation of the invasive process [1]. Furthermore, myoepithelial cells have a central modulatory role in the breast with the potential to suppress tumour cell Thus any proliferation and invasion [2]. meaningful model of DCIS must cell populations. contain these different achieve this, we have refined To methods of separating luminal, myoepithelial and fibroblast cell populations from normal and malignant breast tissue. With the development of defined media, we have now achieved short-term culture of these primary cell populations without the use of fetal calf serum. in-situ Dr Hadwen Trust Science Review 2005 Trust Dr Hadwen is the commonest Immortalised myoepithelial cells (green) form a layer around breast tumour cells in vitro (all nuclei red), reca- pitulating DCIS lesions in vivo (left)

Breast cancer cancer affecting women in the Western cancer affecting world, with an estimated 15,000 new cases each year in the United Kingdom and a one-in-nine likelihood of a woman developing the disease during her lifetime. Understanding the factors that control normal breast function and the mechanisms leading to development and progression of malignancy is likely way of to be the most effective identifying appropriate new therapies. Whilst the series of events leading to invasive breast cancer is not fully established, a pre-invasive lesion — known as ductal carcinoma (DCIS) — in which neoplastic cells lie within a glandular structure but have not yet acquired the ability to invade, is recognised as a principal precursor of invasive carcinoma. With the advent of screening and improved imaging, DCIS now accounts for 20 – 40 per cent of breast cancers. Untreated, DCIS will progress to invasive disease in 25 – 30 per cent of to predict patients, however it is difficult in individual cases which lesions will progress and therefore require more Understanding the aggressive therapy. JL Jones/D Holliday/Barts Jones/D JL behaviour” Louise Jones because they more modifying tumour cell disease and recapitulate the complex intercellular closely represent human of animals, these models “As well as replacing use are likely to be superior… DR HADWEN TRUST – 2006 2004 ASSOCIATE RESEARCH interactions… important in Developing three-dimensional models of normal multicellular breast and pre-invasive breast to replace cancer animal models St. Bartholomew’s Biology Laboratory, Tumour and Dr D Holliday, L Jones Professor Hospital, London Cancer cells in concert cells Cancer

WORK IN PROGRESS 11 OKI PROGRESS IN WORK Vaccine safety in vitro PROGRESS IN WORK

DR HADWEN TRUST RESEARCH GRANT 2003 – 2006

Identification of gene markers for in vitro toxicity control test for pertussis vaccines Dr D Xing, National Institute for Biological Standards and Control, Potters Bar, Hertfordshire

(MWGT) is a general toxicity test used for whole cell vaccines, and the Histamine Sensitisation Test (HIST), a lethal test in mice, is used to detect specific toxicity of pertussis toxin. The precise mechanisms underlying these tests and the toxicological correlations between humans and animals are not clear.

Pertussis toxin (PT) in its detoxified form is a major component in pertussis vaccine. Monitoring chemically

detoxified PT for residual toxicity and www.photos.com reversion to toxicity is an essential part Whooping cough causes 400,000 fatalities each year, mainly in developing countries of the safety evaluation of acellular pertussis vaccines and is required by regulatory authorities. At present, the HIST is the only assay accepted by the The lethal mouse test is difficult to standardise and a priority for World Health Organization and the of toxicity, and aid in the development of neuroblastoma cell for neurotoxicity; and This is the first-ever study of gene replacement [Corbel & Xing] European Pharmacopoeia for in vitro tests for the control of pertussis human umbilical vein endothelial expression of the NL20 and HUVEC determining specific pertussis toxin vaccines. (HUVEC) primary cells for vascular cell lines after pertussis toxin activity in the final vaccine formulations. permeability. treatment, using microarray “If successful, the in vitro Whooping cough is a highly Dorothy Xing says, “At present, techniques. So far, the research has Initial meticulous cytotoxicity assays assay would avoid animal contagious disease caused by In the HIST, groups of mice are injected considerable variations in HIST identified a number of genes of Bordetella pertussis. Worldwide this with doses of the test vaccine and then performance have been observed. were conducted to determine suitable interest that, according to their tests for toxicity in bacterial agent causes some 20 - 40 challenged with histamine; the number Invalid assays normally lead to repeat concentrations of pertussis toxin and functions, are potentially implicated in million cases of pertussis and an of mice dying within 24 hours is tests and large numbers of animals toxoid (detoxified PT) preparations, and toxicological responses. pertussis vaccines and estimated 200,000 - 400,000 fatalities recorded. Large variations in test being used by manufacturers and optimum incubation times. These each year, mainly in developing performance have been observed and control laboratories. According to our parameters have so far been used in pertussis-containing countries. Vaccination is an effective demonstrated to be dependent upon plan, if the in vitro assay were preliminary DNA microarray experiments means of preventing life-threatening such variables as: animal strain, successful, it would avoid the use of with HUVEC and NL20 cells. combination vaccines. It whooping cough, and pertussis vaccine number and age, injection route and animals in testing for toxicity in pertussis coverage worldwide is currently about challenge route. This variability vaccines and pertussis-containing So far, results from both cell types show should also be more 80 per cent. frequently leads to repeat testing and combination vaccines. It should also be that the expression of more than 100 large numbers of mice are used. more reproducible and obviate the need genes change in response to PT Selected publications reproducible and obviate The National Institute for Biological for frequent repeat tests”. treatment, either upregulated or Standards and Control (NIBSC) plays The Dr Hadwen Trust is supporting downregulated. Using genespring Corbel MJ & Xing DKL (2004). Toxicity the need for frequent an important national and international Dorothy Xing’s work to develop in vitro Preliminary investigations with software to cluster genes based on their and potency evaluation of pertussis molecular function showed that some vaccines. Expert Rev. Vaccines 3:89-101. repeat tests” role in evaluating and ensuring the assays that will be more accurate, more microarray technology have shown that safety of vaccines. Dorothy Xing’s humane and replace the routine animal gene expression changes induced by genes have identified roles while others Dorothy Xing research at NIBSC aims to improve the tests. A three-year Trust grant was pertussis vaccine could be classified are of unknown function. Xing D, Canthaboo C, Douglas-Bardsley quality control and safety testing of awarded to fund research into changes into a number of functional categories. A et al (2002). Developments in pertussis vaccines. in gene expression in selected human These include cytokines and their The gene expression profiles for these reduction and replacement of in vivo cell lines using microarrays, with the receptors, growth factors, transcription two types of cells lead to speculation toxicity and potency tests for pertussis Dr Dorothy Xing, who co-wrote this There are two main types of pertussis aim of identifying gene markers of factors and other immune system that PT could have an important role in vaccines. Dev. Biol. (Basel) 111:57-68. report, is a Principal Scientist in the vaccine, inactivated whole cell vaccine toxicity. proteins. The majority of genes the induction of cell-mediated immunity Division of Bacteriology at the NIBSC in and a newer and increasingly used identified were newly associated with (e.g. galectin 3, small inducible cytokine Prior S, Xing D, Auda G & Corbel M Hertfordshire. She has a background in acellular vaccine consisting of The latest DNA microarray technology pertussis vaccine treatment, and subfamily 20, Thy-1 cell-surface antigen (2002). Evaluation of the toxicity of microbiology and pharmacy, and her components of B. pertussis. Either type allows the study of global gene indicated that further investigation was and CD63 genes). PT could also play a recombinant Bordetella pertussis recent publications focus on of pertussis vaccine is used in expression in cells. Gene expression warranted. part in the induction of vascular adenylate cyclase toxin preparations. In: improvements in the development, stan- combination with diphtheria and tetanus changes induced by a given toxin permeability (e.g. platelet-derived Advancing Science and Elimination of dardisation and safety testing of toxoids, and sometimes other antigens. should be reflective of its toxicity. Thus, Dr Xing proposes investigating a range growth factor, vascular endothelial the Use of Laboratory Animals for pertussis vaccines. comparing the gene expression profiles of different human cell types including: a growth factor-C), and the effect may be Development and Control of Vaccines Quality control of pertussis vaccines of toxin-treated cells against control T-cell line (Jurkat cells) for the immune related to the nervous system (glial and Hormones. Brown F, Hendriksen C, involves animal testing of each batch of (non-treated cells) may allow the identi- system; NL20 bronchus epithelial cell fibrillary acidic protein, chlorine channel Sesardic D (eds.). Dev. Biol. (Basel) vaccine. The mouse weight gain test fication of genes as predictive markers for the site of infection; SHSY5Y 3, cholinergic receptor). 111:119-129.

13 Dr Hadwen Trust Science Review 2005 Dr Hadwen Trust Science Review 2005 14 NTEGRAPEVINE THE ON ON THE GRAPEVINE ON THE News and Views News and Views

Replacement: the Nuffield view Vaccine tests report

Information Reference It's 335 pages long, divided into five sections totalling fifteen chapters, and In March 2005, the Associate Parliamentary Group for Animal Welfare (APGAW) has five appendices. This hefty volume — the latest publication from the published its report on the use of animals in vaccine testing. APGAW, a long- APGAW (2005). The Use of Animals in 1. Nuffield Council on Bioethics Nuffield Council on Bioethics — looks at the ethics (and a lot of the science) standing parliamentary group, established a working party to investigate the Vaccine Testing for Humans. Publ. (2005). The Ethics of Research Using testing of human vaccines because of the animal suffering and the large numbers of animal use in laboratories [1]. Associate Parliamentary Group for Animals. Available on the Council of animals involved. Animal Welfare, March 2005. Available website: www.nuffieldbioethics.org The Council has a doughty reputation for examining complex and often The working party is highly critical of the lack of research into the development of from the APGAW website: contentious subjects, past reports having dealt with genetically modified alternatives (i.e. the 3Rs) to animal testing, and the slowness in implementing www.apgaw.org crops, genetics and human behaviour, and ethical standards for healthcare alternatives that become available. research. The report recommends that a UK This latest report was produced by a working party government minister should be given comprising experts from academia, industry and animal specific responsibility for championing protection. Whilst in some aspects of the ethics of alternative methods for vaccine testing, and animal experiments the report was not unanimous, it that ambitious targets with realistic dates makes a major contribution to this important area, should be set, with sufficient funding to including stressing the need for reduction of suffering to achieve them. The group suggests that government should also make specific be placed at the top of the agenda. efforts to expedite the development, validation and regulatory acceptance of It was agreed that the need to find non-animal alternatives. replacement methods cannot be overstated, and it was questioned why more alternatives are not widely In Europe, a co-ordinated rolling programme available. A variety of ways of addressing this need needs to be developed to identify and were suggested, including recommendations for a validate non-animal alternatives to replace thorough analysis of the scientific barriers to animal tests in the European Pharmacopoeia replacements and that published papers should contain (EP). The process of updating EP more information on how the 3Rs were applied in the monographs is extremely complex and time- work described. The ethical review process should play consuming, and should be reviewed so as to a more active role in promoting the 3Rs. accelerate the incorporation of alternative methods and the deletion of obsolete animal tests. The Trust supports the report's emphasis on replacement, especially its role both in research and

APGAW’s working party clearly had www.photos.com best practice — after all, we have been saying this for evidence that Britain might be responsible thirty-five years. for some duplicative animal testing of vaccine batches, “possibly in breach of Alternatives to vaccine tests on animals

Keith Weller/ARS article 114” of the Medicines Directive 2001/83/EC. Several recommendations aim — the government should do more to ensure that duplicate animal testing is avoided, such as increasing transparency From multiwell plates to microchips — Biosensors — a marriage of biology and and improving mutual acceptance of data. in vitro technology can replace animals microelectronics Smarter drug development A biosensor comprises a recognition system, for example biological References and information molecules, coupled to microelectronics, and facilitates real-time detection at Accelerator mass spectrometry (AMS) is one of the most precise bioanalytical methods available. It can be used to measure the presence of drugs and 1. Combes RD, Berridge T, Connelly J References low concentrations of a vast range of substances, including biomarkers (pages 19 - 20). Biosensors have potential to replace various animal metabolites in volunteers in the low picogram and femtogram ranges. Such et al (2003). Early microdose drug sensitivity makes it possible to conduct safe human studies of experimental drugs studies in human volunteers can 1. Metz B, Jiskoot W, Hennink experiments too, as our interview with Professor Sir Patrick Bateson at tiny microdose levels, very early in the development process [1]. minimise animal testing: Proceedings WE et al (2003). Physicochemical suggests (pages 21 - 22). of a workshop organised by Volunteers and immunochemical techniques in Research and Testing. Eur. J. Pharm. predict the quality of diphtheria Early human microdose studies make for improved selection of drug candidates. Recent progress in cell culture and microfabrication technologies has This reduces the likelihood of later candidate failure, by early elimination of Sci. 19:1-11. toxoid vaccines. Vaccine 12:156- contributed to the development of biosensors for characterising many 167. developmental drugs with poor human pharmacokinetics or metabolism, thereby substances, such as drugs and toxins, paving the way for testing without minimising animal testing. Many useless animal tests can be forestalled and the 2. Garner RC (2005). Less is more: the human microdosing concept. Drug 2. Park TH & Shuler ML (2003). using animals (pages 3 - 4). likely success of phase I trials is improved. Discov. Today 10:449-451. Integration of cell culture and microfabrication technology. In vaccine production the use of animals to assess potency and safety A recent trial by the Consortium for Resourcing and Evaluating AMS Microdosing 3. Xceleron website: Biotechnol. Prog. 19:243-253. could be replaced by biosensors to monitor the biochemical characteristics demonstrated the effectiveness of microdose studies in predicting human pharma- www.xceleron.co.uk then click on of the vaccine. Such an approach has been reported for testing safety and cokinetics at therapeutic doses. Despite selecting drugs whose human pharmaco- “Conference” for copies of 3. Otto AM, Brischwein M, kinetics were difficult to predict, results showed 70 per cent correspondence potency of diphtheria toxoid vaccine [1]. Microscale human cell culture presentations. Motrescu E et al (2004). Chips systems using, for example, sensitive pH transduction techniques, show between the microdoses and pharmacological doses [2]. instead of mice: cells on promise as assays for drugs and disease biomarkers [2]. Biosensor bioelectronic sensor-chips as an A conference called Optimizing Drug Development for Success — What Needs to microchips using human cell lines to detect basic metabolic and electrical alternative to animal experiments. be Done? was held in Summer 2005 by Xceleron, the UK company largely ALTEX 21 (Suppl. 3):70-76. parameters could lead to in vitro methods of testing candidate drugs [3]. responsible for developing human microdosing. Speakers included Professor Michael Rawlins, Professor Malcolm Rowland, Dr Chris Milne and Dr Carl Peck [3]. 15 Dr Hadwen Trust Science Review 2005 Dr Hadwen Trust Science Review 2005 16 ON THE GRAPEVINE ON THE News and Views News and Views GRAPEVINE ON THE

Primate research inquiry Asthma and the common cold

Asthma attacks are often triggered by rhinovirus infection — The use of primates in biological and medical research is being cause of the common cold. Using primary bronchial epithelial examined by a study group set up by the Academy of Medical cells from asthmatic and healthy control subjects, researchers at Sciences, the Royal Society, the Medical Research Council and Synairgen in Southampton have found evidence that interferon- the Wellcome Trust. beta might hold the key to understanding the virus-triggering of asthmatic attacks [1]. In setting the group’s remit, the four organisations referred to the need to adopt a more strategic approach to research on Interferon-beta usually causes apoptosis in cells invaded by primates, and to take a fresh and independent look at the viruses such as those initiating respiratory infections. Therefore in scientific basis of primate research. situations where interferon-beta is lacking, infection can be difficult to hold in check and may stimulate an asthma attack. The study group is chaired by Sir David Weatherall, and members with expertise in animal behaviour, drug development, In vitro, bronchial epithelial cells from asthmatics produced much less than normal interferon-beta in response to viral infection. ethics and other fields have been drawn from academia, industry And in infected asthmatic cells in culture, exogenous interferon- and public life [1]. None of the members are known for having beta induced apoptosis and reduced virus replication. This special interest and expertise in animal protection or in the demonstrates a causal link between deficient interferon-beta, replacement of animal experiments. impaired apoptosis and increased virus replication.

Even so, Lord May, President of the Royal Society, said the Synairgen is a small spun-out company at Southampton group would take “…a hard look at whether advances in science University, with a major focus on the development of advanced in now, or in the future, will mean that alternative techniques can vitro cell models. Professor Stephen Holgate, a leading answer our questions just as effectively”. researcher in respiratory disease and one of Synairgen’s founders, says about the group’s human tissue models that “The www.photos.com The Dr Hadwen Trust has presented written and oral evidence to technique is much closer to human proof-of-principle than animal studies”. Virally triggered asthma attacks might be preventable

BUAV the group on the potential to replace primate experiments with non-animal alternatives [2]. The report of the inquiry may be Clinical trials to assess interferon-beta’s effectiveness in preventing infection-associated asthma attacks are Research on primates is under the spotlight published as soon as Spring 2006. planned for later this year. Buoyed by this success, Synairgen intends to provide its cell model assays as a service to other companies. Information References 1. The Non-Human Primate Study Group website: www.nhpstudy.com e-mail: [email protected] Tel/Fax: 020 7839 6300. 1. Wark PA, Johnston SL, Bucchieri F et al (2005). Asthmatic bronchial epithelial cells have a deficient innate 2. Submission by the Dr Hadwen Trust for Humane Research to the Non-Human Primate Study Group, May 2005. Available immune response to infection with rhinovirus. J. Exp. Med. 201:937-947. on our website www.drhadwentrust.org.uk

Biosimulation network of excellence NC3Rs — replacement centre stage? Developing new drugs is getting increasingly complex, as companies wade through mountains of biological information. The government’s National Centre for Replacement, Refinement and Reduction of Animals in Research (NC3Rs) didn’t exactly hit the ground running when it was launched in 2004, but it is starting to get into its stride. According to a consortium of 26 academic groups, 10 companies and four regulatory agencies in the European Union (EU), current methods of drug development “…suffer from the lack of an effective means to evaluate, combine and accumulate Criticised for an over-emphasis on refinement and reduction while lacking expertise in the replacement of animal biological knowledge” [1]. experiments, the NC3Rs has not yet redressed this imbalance. Stakeholder consultation was boosted by a The consortium, led by Dr Erik Mosekild of the Danish Technical University, has received a grant of 10.7 million euros from workshop in January 2005, where speakers — including the Trust’s Gill Langley — set out their organisations’ views the EU, for strengthening expertise in biosimulation to help address the problem. Biosimulation uses computer models to on the 3Rs and how the Centre might establish its priorities and measure success. This was followed in February by examine biological function and predict the action of drugs, and the grant will establish a network of excellence in this field. a workshop for the NC3Rs’ Board members on the use of primates in research and toxicology. This should help develop computational models that provide a dynamic and more quantitative description of the relevant No visible progress has been made in reinforcing the NC3Rs’ independence from the Medical Research Council, biological, pathological and pharmacokinetic processes. As a Novo Nordisk spokesperson explained: “We hope that in the where it is located both physically and, some fear, philosophically. future biosimulation will enable us to develop drugs faster and cheaper while at the same time relatively reducing the number of experimental animals and human trials” [2]. An increase in its budget enabled the Centre to announce, in July 2005, £1 million-worth of new research grants, with a notably greater emphasis on replacement than previously. This is welcomed by the Trust, although according Among several British partners in the project are Dr Amin Rostami-Hodjegan of Sheffield University, a Dr Hadwen Trust to the NC3Rs, it is not a policy change but simply reflects the quality of the applications received. grant-holder (2002 – 2004) developing computer models to predict drug clearance in humans on the basis of in vitro — rather than animal — data [3].

The six replacement projects range from generating transgenic cell lines for use in reporter gene assays as References and information alternatives to animal tests for drug metabolism and toxicity; to the development of a human cell-based wound- healing assay in vitro. The latter NC3Rs grant was awarded to Dr Hadwen Trust grant-holder Phil Stephens at 1. The EU research and development website (CORDIS): http://ica.cordis.lu/search then enter “Biosim”. Cardiff University. 2. Anon. (2004). EU biosimulation project gets underway. Scrip 17 December 2004. Information 3. Proctor NJ, Tucker GT & Rostami-Hodjegan A (2004). Predicting drug clearance from recombinantly expressed CYPs: 1. Workshop reports and other information available on the NC3Rs website: www.nc3rs.org.uk intersystem extrapolation factors. Xenobiotica 34:151-178.

17 Dr Hadwen Trust Science Review 2005 Dr Hadwen Trust Science Review 2005 18 REVIEWS REVIEWS Combined tactics: the new epidemiology

Chemical toxicology Integrating molecular techniques into conventional The predictivity of animal toxicity tests is limited by species variations and inherent uncertainties. epidemiology is yielding a panoply of Conventional epidemiology, lacking individual exciting new human data, with potential to exposure indicators and being prone to confounding, replace some experiments on animals. has sometimes produced conflicting data. Thus for many chemicals the risks to human health remain Classical epidemiological research is unclear. essentially a ‘black box’ approach. Exposure to causes of ill-health is Organochlorine compounds have been linked with correlated with outcome in populations, colorectal cancer, but occupational studies have given but without detailed exploration of the mixed results. A recent case-control study overcame biological mechanisms, or the individual the classical limitations by studying a population using data that could firmly prove cause and serum levels of organochlorines as a marker for effect. individual exposure [7].

Consequently, population research has Several organochlorines were measured in blood often been supplemented with animal samples from colorectal cancer patients and controls. experiments aimed at providing the Point mutations in K-ras and p53 genes were missing evidence. These experiments assessed in tissue samples, and the expression of p53 cause suffering and, as animal models are protein was measured by immunohistochemical often imperfect [1, 2], are far from ideal. methods. Some polychlorinated biphenyls were associated with higher risk of colorectal cancer and Today, much sharper tools borrowed from with mutations in K-ras and p53 genes. The results www.photos.com molecular biology are prising open the strongly indicate a causal link. Combined tactics — genetic approaches in epidemiology ‘human black box’. In another example, the study population comprised workers occupationally exposed to cobalt and Gene genie out of the bottle tungsten [8]. Exhaled breath condensate provided a means to assess individual doses in the lungs, and www.photos.com levels of malondialdehyde were a biomarker for Dramatic strides have been made in genetic epidemiology by cross-fertilisation between disciplines. The first gene to be Test-tube methods provide the missing link pulmonary oxidative stress. Results suggested that associated with asthma was recently identified as ADAM 33 on chromosome 20. The hunt started with a large genome- exhaled breath condensate will be useful for integrating biomonitoring and health surveillance procedures among workers. wide screen involving UK and US families with asthma [3]. Analysis of genetic linkage provided a statistical view of the co- inheritance of DNA, to see if a particular chromosomal region contained a gene related to the disease. This was followed by Recent technical developments (such as fluorescence in situ hybridisation painting of chromosomes) are transforming fine mapping of the likely chromosomal region, and led to identification of the gene by positional cloning. Further work will population-level assays for chromosomal aberrations and micronuclei. These sensitive biomarkers of exposure to focus on the mechanisms by which ADAM 33 contributes to bronchial hyperreactivity. genotoxic substances have proved their worth in studies of occupational and environmental hazards, and prevention policies can be expected to benefit. Identical-twin studies reveal information about the impact of nature and nurture, and the roles of specific genes, in common diseases. The St Thomas’ UK Twin Registry, the largest in the world, contains clinical and genome-scan data for several Traditional epidemiology has already underpinned many life-saving public health policies. The new molecular toolbox is thousand twins and has boosted research into cardiovascular, musculoskeletal, metabolic, dermatological and ophthalmo- dramatically improving epidemiology’s ability to clarify links between exposure and effect, whether in terms of disease, logical diseases. treatments or toxicity, in the species of interest — as well as replacing experiments on animals. Combined tactics have recently been used to probe how variations in DNA sequences might affect the regulation of References baseline gene expression. Microarray analysis of immortalised peripheral blood lymphocytes from 14 families, combined with genome-wide linkage analysis, showed for the first time that levels of gene expression are amenable to analysis in the 1. Linazasoro G (2004). Recent failures of new potential symptomatic treatments for Parkinson’s Disease: causes and same way that other traits are [4]. This opens the way to a new and important suite of human genetic studies. solutions. Movement Dis. 19:743-754.

Tissue microarrays, developed in 1997, use thin sections from a paraffin block injected with tiny tissue cores; one section 2. Sloviter RS (2005). The neurobiology of temporal lobe epilepsy: too much information, not enough knowledge. C. R. can contain tissues from hundreds of patients. Tissue microarrays are being used particularly for gene marker validation Biologies 328:143-153. and also for multi-marker discovery [5]. The technique’s power is that it allows links to be forged between gene expression, pathology and detailed clinical data. 3. Holgate ST, Davies DE, Rorke S et al (2004). ADAM 33 and its association with airway remodeling and hyperresponsive- ness in asthma. Clin. Rev. Allergy Immunol. 27:23-34. Hunting for biomarkers 4. Morley M, Molony CM, Weber TM et al (2004). Genetic analysis of genome-wide variation in human gene expression. Identifying new human biomarkers for population studies is a fast-moving field. Biomarkers can reveal exposure (e.g. to Nature 430:743-747. chemicals or cancer-causing viruses); assess dose or internal dose (e.g. formation of DNA adducts); identify altered structure or function (such as chromosomal aberrations or organ toxicity); and predict individual prognoses (e.g. from 5. Harding A (2005). Tissue microarrays go mainstream. The Scientist, 11 April, 26-28. polymorphisms in drug-metabolism genes). 6. Brindle JT, Antti H, Holmes E et al (2002). Rapid and noninvasive diagnosis of the presence and severity of coronary heart disease using 1H-NMR-based metabonomics. Nat. Med. 8:1439-1444. Ideally, biomarkers are measured non-invasively in the blood, breath or urine of volunteers. An intriguing example is metabonomics, a systems approach to examining hundreds or thousands of metabolites in tissues or body fluids. A recent 7. Howsam M, Grimalt JO, Guino E et al (2004). Organochlorine exposure and colorectal cancer risk. Environ. Health study of the severity of coronary heart disease used serum from human volunteers [6]. Nuclear magnetic resonance Perspect. 112:1460-1466. spectroscopy of serum samples, combined with novel pattern-recognition techniques, produced a highly sensitive and specific ‘fingerprint’ of biochemical changes characteristic of severe blockage of the coronary arteries. Further analysis of 8. Goldoni M, Catalani S, De Palma G et al (2004). Exhaled breath condensate as a suitable matrix to assess lung dose and the molecular basis of the spectral differences should provide insights into underlying biological mechanisms. effects in workers exposed to cobalt and tungsten. Environ. Health Perspect. 112:1293-1298.

19 Dr Hadwen Trust Science Review 2005 Dr Hadwen Trust Science Review 2005 20 IN THEIR OWN WORDS 22 in Design for a 30:31-39. cells via Proc. Roy. Soc. Roy. Proc. is Professor of is Professor Fresenius J. Anal. Fresenius Curr. Opin. Curr. in silico J. Biosci. 32:617-623. 168:110-111. 16:350-355. ATLA in the Dr Hadwen Trust's Vet. J. Vet. A to Medical Approach Model 369:23-29. tissues for toxicological and , Vintage, 2000. , Vintage, 272:671-677. vitro biomedical screening. Chem. 4. I & Nielson J (2005). Borodina genomes to From 3.A Thielecke H, Mack — (2001). Biohybrid microarrays A & Robitzki impedimetric biosensors with 3D metabolic networks. Biotechnol. Ethology at the University of Ethology at the University Biological Cambridge. He was and of the Royal Society Secretary King’s College, Cambridge. of Provost of the President He is currently London. His Zoological Society of the are interests main research of development and evolution but he has written behaviour, and in research extensively on animals that led the was author of the report to ban the hunting of National Trust land. He is co- deer with dogs on its author with Paul Martin of Life Selected publications Gluckman PD, Hanson MA, Spencer HG & Bateson P (2005). Environmental influences during development and their later consequences for health and disease: implications for the interpreta- tion of empirical studies. B. Bateson P of the (2005). The return whole organism. Sir Patrick Bateson FRS Sir Patrick Bateson P (2004). Do animals suffer like us? — the assessment of animal welfare. References and information 1. See Research Science Review 2004. 2. Fentem J, Chamberlain M & Sangster B (2004). The feasibility of animal testing for assessing replacing consumer safety: a suggested future direction. How could the learnedHow could the moresocieties facilitate rapid replacement of animal experiments? Royal As things stand, the its website a Society has put on use of statement about the from Apart animals in research. of their researchers reminding told that they are responsibilities, they must, when appropriate, animals. attempt to replace Many learned societies, publishing important international journals as they do, can raise world wide by insisting standards all that, internationally, submitting papers researchers comply with the strict guidelines as such and legal requirements, those operating in the UK.

A model approach to medical research My sense is that too much energy is that too much My sense to attempts to has been devoted on research front-line discredit that uses biological problems animals. people, This has driven many friends, who could be powerful The effort onto the defensive. the should be on demonstrating example, excitement of, for for of screening solving problems biological activity. Finding answers will bring great academic kudos and that is important given the perception that looking for alternatives can It is also likely to damage careers. be highly lucrative — which should be important when seeking private investment for new projects. Dr Hadwen Trust Science Review 2005 Trust Dr Hadwen www.photos.com www.systemsbio to biological problems are to biological problems Finding replacement methods will bring academic kudos, says Pat Bateson The Dr Hadwen Trust has been The Dr Hadwen Trust at the forefront of developing replacement methods for over 35 years. What should it be doing to meet today's challenges? Developing biosensors involves Some of cutting-edge technology. (i.e. the systems approaches as integrated studying organisms and interacting networks of genes, and biochemical proteins See processes. logy.org) also undoubtedly advanced [4]. Experimentalists who are accustomed to varying systemati- cally at most a handful of factors while randomising the others or holding them constant find such a new Training difficult. approaches generation of scientists to use will take time. systems approaches The government has said that non-animal research methods are “advanced” methods with potential to improve medical progress. What do you think? Dr Hadwen Trust Science Review 2005 Trust Dr Hadwen The main problem is the sheer The main problem of possible heterogeneity of solutions to the over-use animals. The chemical engineer who is developing a biosensor is unlikely to have much to say to someone who is developing a full-scale simulation of the mammalian heart. My own is for a virtual centre preference rather than one that is constructed out of bricks and mortar and is populated by a The value of permanent staff. meeting people, in terms of stimulating collaboration and of research, starting up new areas can be obtained by periodic meetings in established centres. conference The new government-backed National Centre for Replacement, Reduction and Refinement has an important What problems role to play. could limit the success of the Centre? Given that many of the animals Given that many of for are used by laboratories of efficacy testing the safety and I think that most new products, focused on should be effort techniques developing automated for assessing biological activity and toxicity [2]. For instance endocrine disrupting chemicals could be detected with biosensors (analytical devices incorporating biological material with a transducer system to read signal) and the the resulting rapid could be extremely process [3]. What might be the most useful be the most What might research areas for non-animal developed? approaches to be Those who attack animal experiments attempt to occupy I do not the moral high ground. think that the ethical case for doing experiments on animals, in suffering namely reducing a humans and animals through understanding of thorough should go biological processes, by default. That said, it is plainly unethical to use animals in without considering and, research trying the appropriate, where alternatives. What are the ethical issues underlying the importance of non-animal approaches to medical questions? I believe that computer simulation I believe that computer to play in has an important role and, by sharpening up research questions, can greatly research the number of animals reduce used [1]. I do not think that such animal modelling can fully replace experiments because reality always needed and checks are the parameter settings used in models have to be guided by the performance of actual biological biology systems. Furthermore continues to surprise us. Besides simulations, I see great in the development of promise automated systems used in testing the biological activity of (see below). new products From your perspective as a perspective as From your highly respected researcher and as former Biological Secretary Society, of the Royal arewhat do you think the strengths of non-animal research methods? Professor Sir Patrick Bateson talks to the Trust about non- Trust talks to the Bateson Patrick Sir Professor ahead alternativesanimal the challenges and

IN THEIR OWN WORDS 21