In vitro studies imply that BHRF1 is dispensable for both virus replication and transformation. However, the fact that BHRF1 is highly conserved not only in all EBV isolates studied to date but also in the analogous viruses Herpesvirus papio and Herpesvirus pan that infect baboons and chimpanzees respectively, suggests BHRF1 may play an important role in vivo.Epstein-Barr virus (EBV) latently infects about 90% of the human population and is associated with benign and malignant diseases of lymphoid and epithelial origin. BHRF1, an early lytic cycle antigen, is an apoptosis suppressing member of the Bcl-2 family. Herpesvirus papio BHRF1 has been shown to function in an analogous manner to EBV BHRF1 in response to DNA damaging agents in human keratinocytes. In this study we show that the heterologous expression of the previously uncharacterised Herpesvirus pan BHRF1 in the human Burkitt's lymphoma cell line Ramos-BL provides similar anti-apoptotic functions to that of EBV BHRF1 in response to apoptosis triggered by serum withdrawal, etoposide treatment and ultraviolet (UV) radiation. We also map the amino acid changes onto the recently solved structure of the EBV BHRF1 and reveal that these changes are unlikely to alter the 3D structure of the protein.These findings show that the functional conservation of BHRF1 extends to a lymphoid background, suggesting that the primate virus proteins interact with cellular proteins that are themselves highly conserved across the higher primates. Further weight is added to this suggestion when we show that the difference in amino acid sequences map to regions on the 3D structure of EBV BHRF1 that are unlikely to change the conformation of the protein. Apoptosis is a genetically programmed form of cell death employed to regulate cell number, spatial organisation and remove infected and damaged cell populations that may compromise the integrity of the organism . AberranIn vivo, EBV is associated with a number of benign and malignant proliferative diseases, such as infectious mononucleosis, Burkitt's lymphoma and nasopharyngeal carcinoma [in vitro leads to outgrowth of apoptosis resistant lymphoblastoid cell lines (LCLs), which phenotypically resemble activated B-cells. Although B cells can produce progeny virus, epithelial cells are also known to be permissive for viral replication. Studies have shown that EBV has at least two mechanisms of suppressing apoptosis. During latent infection latent membrane protein 1 (LMP1) is the key mediator of protection. LMP1, a CD40 mimic, up-regulates cellular Bcl-2 [Many viruses have evolved multiple strategies to counteract host-mediated apoptosis in order to facilitate infection, replication or persistence ,4. Epstearcinoma ,7. Latenar Bcl-2  and A20 ar Bcl-2  via the ar Bcl-2 ,11 and tar Bcl-2 ,13 whichar Bcl-2 , EBV andin vitro [in vivo remains unclear. BHRF1 is thought to act in lytic cycle to delay apoptosis during replication in terminally differentiating epithelium [in vitro [Despite being considered dispensable for EBV replication or cellular transformation in vitro ,16, BHRFin vitro , and itsithelium . Howeverithelium  and lateithelium  and in Ein vitro . Additioin vitro , DNA damin vitro  and cytoin vitro . It is pHerpesvirus papio (H. papio) and Herpesvirus pan (H. pan) infect baboons and chimpanzees respectively. H. papio encodes a BHRF1 homologue that shares 65% amino acid identity (81% similarity) with EBV BHRF1, whilst the H. pan BHRF1 is even more similar at 82% identity and 90% similarity [H. papio BHRF1 has been shown to confer resistance to cisplatin induced apoptosis in human keratinocytes akin to EBV BHRF1 [H. pan BHRF1 exhibits similar anti-apoptotic activity to that of EBV BHRF1 in the human Burkitt's lymphoma cell line Ramos-BL in response to apoptosis induced by either serum withdrawal, etoposide treatment and the previously untested trigger, ultraviolet radiation. These results show that H. pan BHRF1 is biologically active in human B-lymphocytes in vitro suggesting a conserved role for BHRF1 during in vivo infection of B-lymphocytes.Analogues of EBV are found in the higher primate species. milarity . H. papiBV BHRF1 , but thiH. pan BHRF1 and EBV BHRF1 to regulate serum reduction induced apoptosis in the EBV negative Ramos-BL cell line to ascertain whether the genes are functional, as well as structural, homologues. In order to produce expression vectors, the H. pan and EBV BHRF1 reading frames were amplified by PCR from template vectors pHVPanTW4 and pDH222 respectively using primers incorporating XhoI restriction sites. The PCR fragments were introduced into the pCR2.1Topo® vector and the XhoI fragment was released by restriction digestion of the TopoBHRF1 constructs and cloned into the XhoI restriction site of the pcDNA4/HisMax mammalian expression vector (InVitrogen), which produces an N-terminal fusion protein with 6His and Xpress epitopes.As the senior author has previously shown that EBV BHRF1 suppresses apoptosis in response to serum reduction in various BL cell lines , we initH. pan BHRF1 and cloned by limiting dilution in medium containing 150 μg/ml zeocin. There was no obvious difference in the numbers of drug resistant clones that were produced by transfection with the vector or either of the BHRF1 constructs. Drug resistant clones were screened by RT-PCR for BHRF1 expression and approximately 50% of the clones were shown to be expressing detectable BHRF1. The RT-PCR profiles of two clones each of the three transfected populations which were selected for further analysis, are shown in Figure H. pan BHRF1 (E and F), indicative of BHRF1 expression.Ramos-BL cells were electroporated with either control vector, EBV BHRF1 or H. pan clones Pan 4 and Pan 5 and BHRF1 null vector control clones His C12 and His C13 were seeded in medium containing either 10% or 0.1% serum and the percentage of apoptotic cells was determined at 24-hourly intervals, using acridine orange to distinguish between apoptotic and viable cell nuclei. Necrotic nuclei that appeared after several days due to secondary necrosis of cells already lost by apoptosis were excluded from the counts. Apoptotic death was also confirmed by DNA laddering (not shown). We found that when maintained in medium containing 10% serum  induces apoptosis within hours. The EBV and H. pan BHRF1s afforded significant protection against the induction of apoptosis by all three triggers, compared to the vector controls. There was no statistical difference between the EBV and H. pan BHRF1s, (except for clone EBV1B at 96 hours in 0.1% serum), suggesting that H. pan BHRF1 is indeed a true functional homologue of EBV BHRF1. The difference between clone EBV1B and the other BHRF1 clones at 96 hours in low serum could possibly be attributed to slightly higher levels of BHRF-1 expression in this clone, although it is difficult to quantify absolute expression levels from the fluorescence images shown in Figure In this study we present evidence that the BHRF1 protein encoded by poptosis  and delain vitro . The priin vitro ,25 and hWhen one looks more closely at the regions of the protein that differ between the human and chimpanzee viruses Figure , it is pH. pan) and EBV BHRF1 in a human lymphoid cell background. Both proteins protect against apoptosis induced by two previously described triggers and also a new trigger, UV radiation, against which BHRF1 has never been reported to provide protection. Comparison of the EBV and H. pan proteins at the 3D structural level reveals that none of the changes is likely to significantly alter the structure of the protein.Our results are the first demonstration of functional homology between a primate BHRF1 , 55°C (30 sec) and 72°C (60 sec). The PCR products were introduced by Topo TA cloning into pCR2.1-Topo® (Invitrogen) and the XhoI fragments were released by digestion with XhoI and cloned into the Xho1 site of pcDNA4/HisMaxC (Invitrogen). The plasmids were sequenced to verify that the sequences had joined in frame and contained no PCR induced errors.The 573 bp coding sequences of EBV BHRF1 and m pDH222  and pHVPm pDH222  respecti6 Ramos-BL cells were independently transfected with 20 μg endotoxin-free EBV BHRF1, H. panBHRF-1 or vector control constructs (pEBV9, pPan4 and pcDNA4/HisMax respectively) by electroporation in a BioRaD Gene Pulser II at 0.45 kV, 125 μF. The cells were seeded in 96-well plates at a density of 5 × 104 cells/ well in RPMI 1640 supplemented with 10% FetalClone III calf serum (HyClone) 2 mM L-glutamine (Sigma) 0.4% gentamycin (Sigma) and 200 μg/ml Zeocin (Invitrogen). Wells showing outgrowth of drug resistant cells were cloned by limiting dilution and clones were expanded into 2 ml cultures and maintained in medium containing 150 μg/ml Zeocin. All cultures were fed twice weekly by replacing 90% of the culture with fresh medium.6 × 106 cells using the RNA Safekit (Q Biogene) according to manufacturers instructions. Reverse transcription of 100 ng RNA with oligo dT primer using the Improm II kit (Promega) in a total volume of 20 μl according to manufacturer's instructions was followed by PCR (parameters as above) using 5 μl of the template cDNA and 50 pmols each of the forward and reverse primers CTGTACGACGATGACGATAAG and GTGTCTTCCTCTGGAGATA respectively, to amplify a 655 bp product.Total RNA was purified from 5 × 10Cells were suspended in PBS and spotted onto multiwell glass slides and air dried for 60 minutes. Slides were fixed for 20 minutes in methanol at -20°C followed by 5 minutes in acetone at -20°C. The cell spots were rehydrated in 20% normal rabbit serum (in PBS) for 20 minutes then incubated with a 1 in 500 dilution of the monoclonal anti-poly histidine antibody (Clone HIS-1, Sigma-Aldrich) for 1.5 hours at room temp. After three 5 minute washes with PBS, the cells were incubated with a 1 in 50 dilution of FITC goat anti-mouse IgG (Biomeda) for 1 hour at room temp. Thereafter the slides were washed thrice in PBS, a drop of DABCO was added and coverslips were placed over the cell spots. The cells were viewed with by fluorescence microscopy and photographed with a Leica digital camera using a 1.27 sec exposure time and 15× magnification.6 cells (BHRF1 or vector clones) were seeded in either medium containing 10% serum or 0.1% serum and the percentage of viable versus apoptotic cells was determined by acridine orange flourescence at 24 hour intervals. For the etoposide assays, 0.5 × 106 cells (washed 3 times) were seeded in either routine medium containing DMSO  or medium containing 200 ng/ml etoposide (diluted from a 1 mg/ml solution in DMSO) and the % of apoptotic cells versus viable cells was determined every 24 hours by acridine orange fluorescence. For the UV induction assays, 1 × 106 cells in 10 ml of medium were placed in a Petri dish and exposed to 30 Jm-2 UV. The cells were then pelleted by centrifugation and resuspended at 2 × 105cells/ml in medium and returned to culture flasks.For the serum reduction assay cells were washed 3 times in PBS and then 0.5 × 10Each clone was tested in triplicate and each experiment was repeated 3 times.10 μl acridine orange (100 μg/ml) was added to 100 μl of cell culture containing the individual clones. At least 200 cells were counted using a haemocytometer at 15 × magnification and the number of viable versus apoptotic cells in the population noted.H. pan – Herpesvirus pan, PBS phosphate buffered saline, PCR – polymerase chain reaction, UV – ultraviolet, v-Bcl-2 – viral Bcl-2 homologueEBV – Epstein Barr virus, MH did the RT-PCR and UV assay, TW constructed the expression vectors and carried out some of the statistical analyses and SAH produced the transfected clones, carried out the serum withdrawal and etoposide assays, some of the statistical analyses and did the structural comparisons.


Drosophila.Cocaine addiction wreaks profound changes on the brain, hijacking reward circuits and depressing inhibitory loops to the point that drug seeking and taking become central drivers of behavior. Lying at the core of these behavioral changes are molecular ones; at its most basic level, addiction alters the sensitivity of neurons. While primates and rats are useful for mapping out the neural complexity of these behavioral manifestations, insights into the molecular basis of drug abuse can be garnered more easily from simpler models, such as the fruitfly, The reigning model of cocaine's effects on the brain has highlighted its ability to block reuptake of dopamine by cells of a brain region called the nucleus accumbens. But numerous experiments show this is not the whole story. Ulrike Heberlein and colleagues describe their discovery of a new gene that modulates sensitivity to cocaine within the cells of the fruitfly's internal clock. They further show that the cells' role in regulating cocaine sensitivity is distinct from its function as a timekeeper.Drosophila is loss of “negative geotaxis,” or wall climbing, in response to startle. Using this behavior to screen 400 different mutants, the researchers identified seven with an increased response to cocaine, and for two of these, the disrupted gene was the same, Lmo. The Lmo protein, whose levels were reduced by the mutations, is known to regulate certain transcription factors during development. Despite this, no developmental defects were detected in the loss-of-function mutants that might explain the cocaine effect. The researchers also found that a third mutation in the same gene, previously associated with disruption in wing formation, increased levels of the Lmo protein, and decreased response to cocaine. Thus, Lmo appears to play a central role in regulating cocaine sensitivity.One known effect of cocaine on Lmo is found throughout the body, it is enriched in the brain, and by expressing normal Lmo in oversensitive mutants, Heberlein and colleagues discovered that its cocaine-related effects were localized to the ventral lateral neurons (LNvs). Comprising about ten cells per hemisphere, these neurons provide the fly with an internal clock, driving circadian activities even in the absence of light. Not surprisingly, Lmo mutants had weaker circadian rhythms than normal flies.While v is a small peptide called PDF, and PDF mutation causes circadian disruptions. It does not, however, alter cocaine sensitivity. Furthermore, completely obliterating the LNv, or blocking its ability to fire, disrupted circadian rhythmicity but reduced cocaine sensitivity, rather than increasing it. These results indicate than the LNv normally enhances sensitivity to cocaine, a function enhanced further by Lmo mutants, and does so independently of circadian regulation.But is increased cocaine sensitivity a simple consequence of a broken clock? Apparently not. To date, the only known output of the LNLmo's role in modulating cocaine sensitivity. Drawing on recent evidence that a subset of LNv cells possess dopamine receptors, they suggest that Lmo expression normally regulates the density of these receptors on LNv cells. Loss of Lmo would raise the number of receptors, thereby increasing the sensitivity to cocaine. A key prediction from their findings is that the LNv has another output, as yet undetected, in parallel with PDF that mediates responsiveness to cocaine.Based on their results, Heberlein and colleagues propose a possible model for Lmo-related proteins are found in key areas of mammalian brains, these results may have important implications for understanding innate differences in sensitivity to cocaine in humans, and potentially provide targets for development of drugs to treat or prevent addiction.Because


We will disseminate our evidence base, GIS models, and documentation through the project's Web server. The models will be human-readable in different ways to inform NHS GIS implementers, and it will be possible to also use them to generate the necessary template databases  for running the modelled applications. Our products and experience in this project will be transferable to address other national health topics based on the same principles. Our ultimate goal is to provide the NHS with practical, vendor-neutral, modular workflow models, and ready-to-use, evidence-based frameworks for developing successful GIS business plans and implementing GIS to address various health issues. NHS organisations adopting such frameworks will achieve a common understanding of spatial data and processes, which will enable them to efficiently and effectively share, compare, and integrate their data silos and results for more informed planning and better outcomes.EB-GIS4HEALTH UK aims at building a UK-oriented foundation evidence base and modular conceptual models for GIS applications and programmes in health and healthcare to improve the currently poor GIS state of affairs within the NHS; help the NHS understand and harness the importance of spatial information in the health sector in order to better respond to national health plans, priorities, and requirements; and also foster the much-needed NHS-academia GIS collaboration. The project will focus on diabetes and dental care, which together account for about 11% of the annual NHS budget, and are thus important topics where GIS can help optimising resource utilisation and outcomes. Virtual e-focus groups will ensure all UK/NHS health GIS stakeholders are represented. The models will be built using Protégé ontology editor  Geocoding (street address matching or assignment of latitude and longitude) will be the basis for data linkage and analysis in the 21st century. The versatility of GIS supports the exploration of spatial relationships, patterns, and trends that otherwise would go unnoticed." – US Healthy People 2010 Objectives (item 23-3 – )"Geography plays a major role in understanding the dynamics of health, and the causes and spread of disease . The claToday's health planners aim at developing health policy and services that address geographic and social inequalities in health, and therefore should benefit from evidence-based approaches that can be used to investigate spatial aspects of health policy and practice, and evaluate geographic equity (or inequity) in health service provision .According to the US Federal Geographic Data Committee (FGDC), geographic location is a key feature of 80–90% of all government data . The samUnlike other resources like employees or funds, spatial data do not suffer any wear and tear from repeated use. On the contrary, reusing data increases the possibilities for improving the content quality of data collections and gaining new insights by linking and exploring the relationships between the different datasets that makeup the big picture .This implies the need to develop applications and not just focus on data. An overemphasis on data acquisition, without health sector-linked applications, will not provide any momentum for further development. Visualisation, modelling and analysing activities will be the focus of value-added services in the coming years ,5. Metho), a step reflecting the importance and growing use of GIS in health and healthcare research and practices.In 2003, the US National Library of Medicine added the term "Geographic Information Systems" (GIS) to its controlled vocabulary thesaurus known as MeSH . The inGIS offer a very rich toolbox of methods and technologies that goes far beyond the mere production of simple maps . From a community health perspective, GIS could potentially act as powerful evidence-based practice tools for early problem detection and solving. When properly used, GIS can be used to: inform and educate ; empower decision-making at all levels; help in planning and tweaking clinically and cost-effective actions, in predicting outcomes before making any financial commitments and ascribing priorities in a climate of finite resources; change practices; and continually monitor and analyse performance and changes, as well as sentinel events.Traditionally, two broad types of GIS applications can be distinguished which also reflect the two traditions in health geography , namely health outcomes and epidemiology applications, and healthcare delivery applications. There are also studies at the interface (overlap) between epidemiological and healthcare delivery applications, for example in relation to healthcare commissioning and needs assessment ,7,8.). GIS are also explicitly included in the National Electronic Disease Surveillance System (NEDSS) specifications and systems architecture of the US Centres for Disease Control and Prevention (CDC – ).The use of GIS in the UK National Health Service (NHS) can still be considered as an emerging technology . Despite all their potentials, GIS remain very much under-utilised in the UK NHS in mostly low-level, non-strategic tasks, and in a largely fragmented and uncoordinated way. Spatial data and GIS are still not mentioned in any main UK health information strategy or policy document ,8. In stAlthough multiple novel spatial statistical and GIS methods are potentially available, we still need to unambiguously determine which method(s) and data specifically should be used by practitioners for each specific health condition of interest, and whether the proposed methods are cost-effective and scalable in the context of UK/NHS settings .Moreover, several researchers have highlighted a gap between academic health-related applications of GIS and their everyday use within the NHS. Research undertaken in academia has certainly highlighted the benefits of spatial statistics and GIS approaches in mapping disease and in healthcare planning, but still needs to respond to NHS needs on the ground ,8. On thIn a recent major review paper by this author, the reasons behind the under-utilisation of spatial information and GIS in the NHS, as well as the causes of the gap between academic GIS research and the current everyday use of GIS in the NHS were investigated, and remedial recommendations were made . The reaHow could GIS be beneficial in optimal ways in the context of UK/NHS settings and needs? Any answer(s) or proposed solutions must be based on the best current evidence in the health GIS field.This author proposes to build, consolidate and disseminate a comprehensive evidence base for GIS applications in health and healthcare in the context of UK/NHS settings, and an associated set of evidence-based GIS programme and application models (EB-GIS4HEALTH UK). We will learn from national projects running in the US  and elsewhere, while ensuring that EB-GIS4HEALTH UK's output properly fits the UK health and healthcare agenda.[A systematic and critical review and consolidation are needed of the evidence for GIS for specific preventable, mitigable and treatable health conditions. A good model is the CDC "Guide to Community Preventive Services" . Topics . However, it should be noted that this particular handbook was conceived with the US healthcare system in mind and is thus more suited to US settings, though still useful as a model to follow.We propose to address a subset of the topics in the CDC Community Guide  during the three-year period of our project by conducting a focused review of GIS literature on the chosen topics, and then categorising the "nature of the scientific evidence" documenting whether GIS add any value to our understanding and management of the reviewed topics and/or the evidence that it would be feasible and cost-effective for the respective UK NHS and public health programmes tackling the reviewed topics to adopt GIS. Areas requiring further research will be also highlighted. A good example that comes to mind in this context is the 73-page "GIS for cancer" handbook titled "Using Geographic Information Systems Technology in the Collection, Analysis, and Presentation of Cancer Registry Data: A Handbook of Basic Practices" that was published by the North American Association of Central Cancer Registries In reviewing GIS literature for the above mentioned purposes, this author appreciates the fact that the set of definitions and criteria for reviewing evidence as used in the CDC Community Guide is not directly usable for reviewing currently available GIS literature due to the nature of the latter; a modified set of definitions and criteria will first need to be developed. Furthermore, we will organise virtual e-focus groups that bring together UK programme administrators, practitioners and the public to complement the expected gaps and deficiencies in current GIS literature.The desired GIS information outputs and ways of using them within the NHS will be determined for both diabetes and dental care. Datasets (inputs) and the appropriate processing methods required to reach the desired outputs will be identified driven by the best available evidence (from the literature and e-focus groups), and vendor-neutral GIS programme and application models or ontologies will be created accordingly. Any limitations of these applications and any associated possible data/analysis problems or errors will be also highlighted, along with techniques for recognising and reducing their negative impact on result interpretation and any drawn conclusions.We will also launch EB-GIS4HEALTH UK Web server to disseminate our results and reach out to the wide NHS audience and the public.et al, a substantial proportion of respondents from health authorities (90%) and trusts (74%) stated that a dedicated Web site giving advice on GIS matters for NHS organisations would be helpful in promoting and disseminating good practice examples of GIS use in healthcare [In a recent study by Higgs althcare . EB-GIS4d trusts 4% statedalthcare ,10.EB-GIS4HEALTH UK will establish links between real-world NHS practice and the growing body of health and healthcare GIS research produced by the academia and research communities to ensure quality GIS practice and innovative applications are developed and implemented in the UK health service.By putting strong emphasis on real-world, practical GIS scenarios in a UK context, and by being based on the current best evidence, EB-GIS4HEALTH UK will also provide a much needed contribution to future national GIS training and raising awareness campaigns. Such evidence-based training and raising awareness activities have been strongly recommended by different researchers to improve the current poor GIS state of affairs within the NHS .Raising awareness activities are also vital given the need to build business cases for the development of GIS within NHS organisations and to show the capabilities and "business benefits" of GIS to directors  Figure . EB-GIS4EB-GIS4HEALTH UK will provide semantically and procedurally consistent health and healthcare GIS application frameworks. This will help individual NHS organisations adopting such frameworks achieve a common understanding of data and processes (shared semantics), which in turn will enable them to efficiently and effectively share, compare, and integrate their data silos and results at local, regional and national levels for more informed planning, national comparisons, joined-up working, and better outcomes.EB-GIS4HEALTH UK also forms the basis of our vision of a next-generation intelligent tools specifically designed for the UK public health and NHS, and seamlessly weaved into everyday workflows and decision-making processes to enable users to focus and spend the larger part of their work time on what they want to achieve rather than on learning and overcoming the limitations of tools they are supposed to use to achieve their goals. These tools are part of our wider vision of a "national spatial data and information infrastructure and associated multivariate, GIS-enabled health surveillance services processing real-time or near-real-time data streams rather than just retrospective data" mentioned above and in .Such tools must be able to convey reasonable conclusions (rather than just bunches of facts on thematic, coloured maps). The ideal tools also need to be fault-tolerant and capable of analysing and presenting assembled data in ways that facilitate only appropriate interpretations of integrated data. This can be achieved by using some form of user friendly, "intelligent", goal-oriented health GIS wizards based on robust statistical and epidemiological methods, so that only valid results and maps are produced, even when users attempt to select inappropriate settings for a particular analysis. The tools are also best designed and built to work in modular and nested fashions, so that they may be reused, linked and combined in different ways as needed to serve different scenarios and compound situations with little or no modifications (of the tools) .This vision definitely calls for a sound evidence base and comprehensive conceptual blueprints (the proposed EB-GIS4HEALTH UK evidence-based models) to drive the envisaged tools and wizards.An incremental approach has been widely recommended in the literature for programmes with a national vision like EB-GIS4HEALTH UK. Rather than addressing all the spectrum of health and healthcare topics that are amenable to GIS processing in one project, we selected only two health conditions to start with. Diabetes and dental care were chosen because of their importance and the huge burden they place on the NHS, and the facts that they affect various age and socio-economic groups and involve a wide spectrum of preventive and curative interventions, which make it possible to apply the GIS models we are proposing to develop for these two topics to many other health and healthcare topics with little modification.Diabetes has a significant impact on the UK health and social services. Around five per cent of total NHS resources and up to ten per cent of hospital in-patient resources are used for the care of people with diabetes . It has ). Of this latter sum, £35 million will be allocated to enable PCTs to improve access, choice and quality for patients, and £30 million will be directed to information technology to integrate dentistry within the national information technology programme. Regarding dental care, the cost of General Dental Services to the government in the year to March 2002 was £1.12 billion  . Under tProfiling service areas and needs assessment, dental healthcare commissioning, and improving/ensuring equitable access to dental services are all areas where GIS can make a positive difference. GIS have significant potential in examining spatial patterns in dental health, and in analysing patterns of registration and utilisation of dental services for different sectors of the community. GIS can reveal gaps in dental health provision, and thus help target resources and programmes to particular areas of needs. GIS can be also used to analyse the composition and spatial distribution of the dental workforce, and to inform the development and monitor the execution of programmes for attracting dental care professionals to work in under-served areas ,17,18., could also benefit from EB-GIS4HEALTH UK as a foundation resource to provide evidence-based training programmes to NHS staff in "GIS for health and healthcare" and foster the much-needed NHS-academia collaboration. NHSU currently does not have programmes covering this important area. As an aside, one might add that when the Director of the Medical Informatics programme that the US CDC runs for its new staff conducted a poll of 40 students on the area they most wanted more information about, top of their list was GIS ; and (3) the UK citizenry and communities who will be empowered to become more active partners in their healthcare, and will also benefit on the long run from improved health services and outcomes as a result of the introduction of well-founded spatial information management within the NHS through EB-GIS4HEALTH UK and other synergistic/follow-on projects in the future.These include: (1) the NHS and public health services in England (and the UK) – EB-GIS4HEALTH UK aims at adding the missing spatial information dimension to NHS organisations, and informing the development of successful GIS business plans for the health conditions under consideration; (2) the recently established NHSU, the corporate university for the NHS with Special Health Authority status – EB-GIS4HEALTH UK aims at building a foundation evidence base and conceptual models for GIS applications in health and healthcare in the context of UK/NHS settings to (i) improve the currently poor GIS state of affairs within the NHS; (ii) help the NHS understand and harness the importance of spatial information in the health sector in order to better respond to national health plans, priorities, requirements, and NSFs ; and also (iii) foster the much-needed NHS-academia GIS collaboration.1. Organise e-focus groups of representatives of all stakeholders of UK/NHS health GIS to inform the development of all EB-GIS4HEALTH UK products.2. Review the literature and current UK Public Health/NHS data flows and practices of relevance, and build an evidence base of GIS applications in diabetes and dental care (the two topics chosen for this project) in the context of UK/NHS settings.3. Build modular GIS programme and application models for diabetes and dental care tailored to UK/NHS settings (driven by the evidence identified through objectives 1 and 2).4. Run a Web server to disseminate the project's evidence base and resultant GIS programme and application models for diabetes and dental care to the wide NHS audience and the public.5. Conduct a small-scale formative evaluation of EB-GIS4HEALTH UK server use and potential impact on NHS resource utilisation and improving health outcomes.We will gain insight about current Public Health/NHS (in England) data assets, flows and processes of relevance to EB-GIS4HEALTH UK through our NHS collaborators, and by identifying key contacts and organising virtual e-focus groups (see below). This insight will help us come up with a set of "localised" (UK oriented) questions that we will have to answer through our literature review and further e-focus group rounds., Transactions in GIS , International Journal of Geographical Information Science , CDC Public Health GIS News and Information , ESRI's HealthyGIS and ArcUser Online ( and ), etc. We will also hand search a range of textbooks on GIS applications in health and healthcare.We will formulate a search strategy to locate potential resources for our evidence base. This will also cover the so-called grey literature. Our search strategy will span MEDLINE/PubMed and other journal resources not listed in PubMed, e.g., Cartography and Geographic Information Science ; the US CDC National Public Health Performance Standards Programme (NPHPSP – ); the US Primary Care Service Area Project (PCSA – ) and the related Dartmouth Atlas project ; the "Mapping A Shared Vision of Hope: The American Indian/Alaska Native (AI/AN) GIS Diabetes Atlas", which was developed to assist in the analysis, design, and evaluation of AI/AN diabetes intervention and prevention programmes ; Georgia Medical Care Foundation Diabetes Quality Indicators project ; and CDC's data systems for oral health .We will also review the extensive online documentation of flagship UK and foreign projects and initiatives of relevance to our proposed research. These include, among others, the UK Department of Health NSFs We will review studies that used GIS or spatial methods in diabetes and dental care , practitioners and representatives of the public to (1) inform the project about NHS settings; (2) define and refine the key questions that decision makers would want to be able to answer with GIS for diabetes and dental care, and think explicitly about what data and methods should be used to answer those questions; (3) discuss the reviewed literature and guide us to any relevant literature we may have missed for our evidence base; (4) complement our review of the evidence by the group members' own experiences and consensus of opinions, especially in areas where the literature is lacking; and (5) provide iterative feedback on EB-GIS4HEALTH UK models, as these are developed and refined. The virtual e-focus groups will ensure that representatives of all stakeholders of UK/NHS health GIS are involved in formulating EB-GIS4HEALTH UK products, a very important ingredient of success ,20.The e-focus groups will take place online using threaded discussion technology on EB-GIS4HEALTH UK Web server. Simple audio-conferencing over the Internet, e-mail/e-mail list, and/or telephone may be also used if necessary. The project's team will moderate/facilitate and guide/coordinate the discussions by adopting a kind of Delphi process and/or "cluster analysis" approach (where group members list their ideas, opinions and own experiences, then these are sorted and compiled into "themes" by the moderators and presented back to the group, and further rounds of knowledge distillation and aggregation are carried out as appropriate).et al's set of categories they have recently used in a small-scale GIS literature review, with the following top classes/sub-classes among others: data collection method(s) (e.g., field survey, disease surveillance system input, etc.); spatial data analysis method(s) ; study scale (e.g., country/state, city/town, suburb/district, etc.); resolution/geographic unit of analysis (areas, points, others); study purpose ; GIS software used; bias of study design/limitations; etc. . The metadatabase will include fields to record the strength of evidence and bottom-line summaries of the reviewed articles. EB-GIS4HEALTH UK models and their documentation will have cross-links to the underpinning evidence in the online metadatabase.A metadatabase of reviewed resources classified according to topics and applications will be created and populated. This will form EB-GIS4HEALTH UK searchable online evidence base (see below). The design of resource records in this metadatabase will be guided by the spirit of the matrix method for conducting and organising literature reviews . A classSuitable input from the virtual e-focus groups will be also included in the evidence base. Resources and findings of insufficient evidence quality will still be documented in the evidence base , but will not be used in developing EB-GIS4HEALTH UK models.. EB-GIS4HEALTH UK will take the concepts and methodologies of these projects one step further by developing evidence-based, vendor-neutral modular GIS programme and application models rather than mere data models or vendor-specific solutions.We will develop a general conceptual framework for EB-GIS4HEALTH UK models as outlined below, guided by Briggs' indicators methodology for environmental health hazard mapping ,23, the EB-GIS4HEALTH UK will feature two interrelated types of models: application models and programme models Figure .An application model comprises (1) data and/or input from other models to be processed; (2) processing methods and tools ; (3) desired information outputs and output visualisation methods; as well as (4) all valid interpretations/inferences that can be made from the model's output and ways of using them within the NHS. The best current evidence  will be used to formulate all EB-GIS4HEALTH UK models, and will form an integral part of their documentation.For any single broad health/healthcare topic covered in EB-GIS4HEALTH UK , modelled applications can include all or some of the following (among other possibilities): (1) monitoring and measuring NHS performance ; (2) surveillance/monitoring/early detection of health and disease patterns and trends in populations; (3) profiling target populations and health service catchment areas; (4) selection of appropriate target groups for public health interventions, optimising these interventions to match target group profiles, and measuring intervention success; and (5) needs assessment and optimising healthcare system planning and responses, including new healthcare facility siting and improving hospital bed availability.EB-GIS4HEALTH UK models will take into account the influence of non-medical determinants (e.g., income, occupation, and environment) on population health status, qualitatively relate these determinants to health outcomes, and consider their implications on healthcare service planning and delivery ,20. For A programme model is also an ontology that addresses a single broad health/healthcare topic, and comprises at least two application models linked together and interacting with each other in predetermined and purposeful ways towards a broader goal than that covered by a single application model. Application models may have uses in more than one programme (either unchanged or with slight modifications to suit different programmes, e.g., different input datasets)., a free ontology modelling tool from Stanford Medical Informatics with which this author has long experience [, including a Java-based Web application for sharing Protégé ontologies over the Web . Protégé is not alien to geographic information science; last year (2003) for example, a geographic information metadata (ISO 19115) ontology was developed at Drexel University, US, using Protégé  will be built using Protégé perience ,26. ProtA model's ontology/documentation will specify the model's version/date and release history, the programme(s) (for application models)/topic(s)/sub-topic(s) to which the model relates, the model's rationale and role, any alternative or related models/model sets (programmes), links to the underpinning evidence (from the literature/the project's evidence base), and a listing of all agencies/NHS bodies involved in the model's process(es) (and their exact roles).Data involved in the modelled applications will be identified and assessed regarding availability, quality, characteristics, and constraints in terms of the model in question. Whenever this is possible, EB-GIS4HEALTH UK models will strive to refer to and model readily available data already collected for other purposes, since implementing new data collection processes can have prohibitive costs, and healthcare workers have repeatedly demonstrated poor compliance with additional data collection and administrative tasks .For data that are not available but are "required" we will investigate and suggest the most efficient and effective way(s) for collecting and maintaining such datasets. During the course of the project, we will be identifying and communicating with the custodians of various datasets in the UK health sector and related sectors, as appropriate, to check the availability of required datasets to the UK NHS, note any constraints related to their release and use (e.g., privacy issues), and flag any need to review policies related to the release of such data. The results of all these data-related investigations will be included in the documentation of EB-GIS4HEALTH UK models.only valid analyses and visualisations of data.The spatial and related methods involved in processing the model's input to produce the desired outputs will be described in detail in the model. A reusable toolbox of "generic" methods will be identified to make this process easier for all the models we develop, e.g., "pattern spotters and testers" and "relationship seekers and provers" . All metThe model's information output(s), any units of measurements used in presenting outputs, and any specified output visualisation methods will be also described in detail.The area across which the model can be used  will be determined. Finally, the ways in which the model's output may be interpreted (in relation to the topic(s)/sub-topic(s) it covers) and linked to other EB-GIS4HEALTH UK models/programmes will be described. This includes determining what inferences can be made from apparent trends or patterns in the model's output and how such inferences can be used within the NHS, and any constraints on the interpretation of this output, due for example to data limitations or complexities in the relationships implied by the model.Data and methodological problems and limitations are not uncommon and a wide range of them has been well documented in the literature and must be anticipated and cared for . TechniqFor a programme model, the component application models will be listed and their relationships and interactions within the programme also documented Figure .EB-GIS4HEALTH UK knowledge-based models (ontologies) in Protégé will not only be human-readable and visualisable in a variety of ways (to inform NHS GIS service developers and implementers), but it will be also possible to use the models to generate the necessary template databases for running the modelled applications. Furthermore, the resulting ontologies could form the basis for the development of goal-oriented, user friendly and fault tolerant health GIS wizards and solutions in the future using software agents technology ,29, sincThe goal for EB-GIS4HEALTH UK application and programme models is to provide practical, vendor-neutral modular workflow models and ready-to-use, evidence-based frameworks for implementing GIS to address various health and healthcare topics within the NHS. NHS organisations adopting such frameworks will be able achieve a common understanding of data and processes (shared semantics), which in turn will enable them to efficiently and effectively share, compare, and integrate their data silos and results at all levels.We will launch EB-GIS4HEALTH UK public Web server (with it's own domain name) to disseminate our evidence base, GIS models and associated documentation/metadata, as well as other project documentation and recommendations. The server will also host the virtual e-focus groups. The server will run one of the many free content management and discussion board platforms available today to manage and make searchable all of the project's online components.We will advertise EB-GIS4HEALTH UK online service among target groups (NHS and academia) and the general public, and conduct a small-scale formative evaluation of its use and user-perceived utility of the project using an online user questionnaire, in addition to analysis of EB-GIS4HEALTH UK server transaction logs. This formative evaluation study will be carried during the third year of the project  with the goal to inform and guide any further development of EB-GIS4HEALTH UK or similar projects. The study will evaluate the potential impacts of EB-GIS4HEALTH UK on NHS resource utilisation and improving health outcomes, among other things, by surveying participants opinions. Evaluation results will be also available from EB-GIS4HEALTH UK public Web server.Research into ontology-driven geographic information systems (ODGIS) is very rapidly growing due to the many potential and unique advantages that ODGIS promise -36. We a and ). We conduct many complex analyses in health and healthcare to answer much more complicated questions (compared to other sectors), and there are many problematic issues surrounding our analyses like, for example, data confidentiality and Jacquez's famous "gee whiz" effect [This author also advises the use of Tomlinson's methodology to plan for the successful deployment of GIS within the NHS, but only at a later stage ,37,38. T.Besides being one of the main project beneficiaries, the UK citizenry (and indeed anyone connected to the World Wide Web) will have full access to EB-GIS4HEALTH UK public reports and fully documented products on the project's public Web server. Representatives of the public will be also involved in the project's e-focus groups (see above). Lay audience short summaries of the project's progress and expected benefits in a jargon-free language will be also published on EB-GIS4HEALTH UK public Web server (see Table  and ), but this has never resulted in any coherent national strategic adoption of GIS within the NHS. This is due to the fact that a successful national strategic implementation of GIS in the health sector is dependent upon many other very important ingredients besides the acquisition of hardware and software systems, and core digital geo-datasets [Most major GIS vendors and solution providers have been targeting the UK health sector for many years now (e.g., datasets . This auin this country (England). All national health GIS stakeholders must be involved in this process, including representatives of the general public (the ultimate beneficiaries). EB-GIS4HEALTH UK is just the start towards this goal. An incremental approach has been widely recommended in the literature for programmes with a national vision like EB-GIS4HEALTH UK. The experience gained at the end of this project will hopefully be transferable to further develop the covered topics , and for addressing other national health and healthcare topics not initially covered in this project, based on the same principles.Healthcare systems work differently around the world and this has knock-on effect on health GIS. When it comes to UK health GIS, we still need to go back to the design board, and to link/integrate what research has to offer into workflow models and "recipes" that are both usable and useful in everyday practice per se our ultimate goals. The actual purpose of this design/modelling exercise and its ultimate goals are optimising NHS resource utilisation and improving health outcomes by paving the way to the incorporation of the missing spatial information dimension in NHS organisations.It must be emphasised that the evidence base and conceptual GIS workflow models we are proposing to build in this project are not This paper presented an overview of a project we are proposing to carry out here in England, where the author is based. However, the same concepts, principles and approaches described in this paper can be also applied in other countries, especially where financial constraints are not a major issue. The task might not be as easy as replacing "UK" in this proposal with another country name, but the author believes that the paper has provided enough details and set an example to enable "quick starting" similar health GIS foundation projects in other countries.Our proposal also fits very well into the spirit of Mark Musen's philosophical paper on medical informatics as an academic discipline, in which he argued that "informatics involves the construction of ontologies that define the concepts relevant to different aspects of human experience and the elucidation of problem-solving methods that can solve specific computational tasks" .A detailed breakdown of human and other resources required to complete this project with a timeline of the main EB-GIS4HEALTH UK execution tasks distributed over a suggested three-year duration of the project are not provided in this manuscript, but are directly available from the author.).A slightly abridged version of this proposal has been submitted to, and very favourably reviewed by the UK Medical Research Council (MRC) during 2004, and received a final ALPHA-C banding , health, geography, public health, policy and practice, epidemiology, etc. It is therefore important to bear in mind that if the proposal is judged by criteria generally applicable to only one or two of these specialist areas, it might be found deficient and it would be quite inappropriate to do this".


Although feeding deficits have been reported in snakes and lizards following vomeronasal system disruption, no deficit has been previously reported in a mammal. We tested gray short-tailed opossums with items from four different food categories prior to occluding access to the vomeronasal organ. Preoperatively, opossums preferred meat to fruit or vegetables. Following occlusion of the nasopalatine canal, but not after control treatment, opossums failed to demonstrate food preferences. The vomeronasal system (VNS) is a nasal chemosensory system usually considered primarily a pheromone-detecting system -4 for re for re4 In snakes and lizards, in addition to its role in pheromone detection, the VNS is important for feeding behavior -7. To daMonodelphis domestica, are members of the Didelphidae or American opossums. The didelphids are the most ancient marsupial family and thought to be the origin of all other New World and Australian marsupials [M. domestica was recently introduced into laboratories [Gray short-tailed opossums, rsupials ,9. M. doratories  and has,ratories -13, theyad libitum.Six male and seven female gray short-tailed opossums, 12–16 months old, were used as test subjects in this study. The opossums were progeny of animals originally purchased from the Southwest Foundation for Biomedical Research (San Antonio, Texas, USA) and were offspring of different parents. The opossums were housed individually in plastic cages (42 × 21 × 20 cm) with wood shavings as bedding, and provided with cylindrical plastic containers for nesting. The animals had dry fox food  and water available To determine which foods to use in the formal experiment, a preliminary test was conducted in which 24 different foods, representing meats, fruits and vegetables were presented to the opossums. Each opossum was presented with one food per day, selected at random, and its consumption of the food noted. The animals were allowed three minutes to investigate and eat the food, which was placed on an 8 × 20 cm cardboard tray at the front of the home cage. Each animal received two trials with each food. Of the 24 foods tested 16 were selected for the formal experiment based on the animals' observed preferences.3) of food from each food category, was placed in a dish.During testing the opossums were simultaneously presented with four foods one from each of the following food groups: fruits , meats , processed vegetables (Raisin Bran, Cheerios, whole wheat bread, bagel) and unprocessed vegetables (corn, peppers, carrots, broccoli). The animals were tested daily with one of 16 food combinations (4 foods × 4 categories). Four sterile Petri dishes, each 3.5 × 1.0 cm, were placed equidistant from each other and glued to an 8 × 20 cm cardboard tray. One piece  placed at a distance of 3 m from the test cage. Trays, dishes and unconsumed food were discarded after each trial. After testing, food troughs containing fox food were replaced in the animals' home cages. Fresh disposable gloves were used when handling dishes, trays and foods to prevent transfer of food odors.Videotapes of food trials were analyzed using a videocassette recorder (JVC SR-VS30U), BTV Pro 5.4.1 and "Videoanalyzer" software .For control surgery and occlusion of the nasopalatine canal, opossums were lightly anesthetized with Ketaset (0.25 cc/100 g, i.m.) and atropine sulfate (.05 cc/100 g, i.m.) Access to the vomeronasal organ (VNO) was blocked with gel foam and Crazy Glue™ (Elmer's Products, Inc., Columbus, OH) applied to the roof of the mouth, covering the opening to the nasopalatine canal. Cresyl violet crystals were added to the Crazy Glue to facilitate visualization of the block. Opossums were visually checked daily to insure that the VNO block remained in place. Control surgery consisted of placing Crazy Glue on the roof of the mouth to either side of the nasopalatine canal. Opossums were allowed two days to recover before postoperative testing.The VNO block remained in place for only one test day in all thirteen opossums. Therefore, pre-post operative and control comparisons were only made for trials identical in composition (foods tested) and order (position on the tray) to the first postoperative test day. Control postoperative trials were run as described above. All 13 animals were tested under both control and experimental  conditions, with the order of surgical treatment randomized.The first food selected and consumed on each trial was identified as the animal's food choice. Statistical analyses utilized a chi-square test of the frequencies of choices of each food type for pre-operative, control and experimental trials. Figure Preoperatively, opossums preferred meats to fruits and fruits to processed and unprocessed vegetables Figure . The mos2 = 10.08, df = 3, p < .02), postoperatively, no significant difference in food category preference was observed (χ2 = 0.85, df = 3, p > .05). During control trials the opossums continued to demonstrate significant food preferences (χ2 = 9.46, df = 3, p < .05,). Meat was again, the most preferred food category (Figure Whereas the preoperative comparison trials on the thirteen opossums revealed significant preferences among the food categories (χOne deficiency of this study is that, because the opossums removed the glue blocks after the first day, we were unable to verify that the nasopalatine occlusion had, indeed, prevented access to the vomeronasal organ. However, we had previously  demonstrThis study suggests that without a functional VNS, the food preferences of gray short-tailed opossums are significantly impaired. Previous studies on the VNS of mammals have not addressed the issue of changes in feeding behavior. It remains to be seen whether other mammals, newly introduced into the laboratory, or bred over many generations in the laboratory, also demonstrate a food preference deficit when deprived of a functional VNS. Studies comparing wild populations with laboratory-reared populations might contribute information resolving this issue.It is not surprising that food preferences might be influenced by vomeronasal stimulation since the vomeronasal organ of many vertebrates, including opossums, is directly accessible from the oral cavity  via the MH conceived of and designed the experiment and wrote the manuscriptYD conducted the experiment, extracted the data from the videotapes and analyzed the data statisticallyIZ supervised YD in the conduct of the experiment and instructed YD in the experimental procedures and data analysis


The use of peptide libraries for the identification and characterization of T cell antigen peptide epitopes and mimotopes has been hampered by the need to form complexes between the peptides and an appropriate MHC molecule in order to construct a complete T cell ligand. We have developed a baculovirus-based peptide library method in which the sequence encoding the peptide is embedded within the genes for the MHC molecule in the viral DNA, such that insect cells infected with virus encoding a library of different peptides each displays a unique peptide–MHC complex on its surface. We have fished in such a library with two different fluorescent soluble T cell receptors (TCRs), one highly peptide specific and the other broadly allo-MHC specific and hypothesized to be much less focused on the peptide portion of the ligand. A single peptide sequence was selected by the former αβTCR that, not unexpectedly, was highly related to the immunizing peptide. As hypothesized, the other αβTCR selected a large family of peptides, related only by a similarity to the immunizing peptide at the p5 position. These findings have implications for the relative importance of peptide and MHC in TCR ligand recognition. This display method has broad applications in T cell epitope identification and manipulation and should be useful in general in studying interactions between complex proteins. A baculovirus expression library -- encoding peptides in the context of MHC molecules -- has been developed to identify and characterize unique peptide-MHC complexes that bind specific T cell receptors The identification of peptide epitopes associated with particular αβ T cell receptors (αβTCRs) is often still a bottleneck in studying T cells and their antigenic targets in, for example, autoimmunity, hypersensitivity, and cancer. A direct genetic or biochemical attack on this problem can be successful, especially with class I major histocompatibility complex (MHCI)-presented peptides. For example, tumor  and tranThe reward for the labor involved in identifying peptide epitopes directly can often be the identification of the protein source of the peptide, especially as the sequencing of the genomes of many organisms approaches completion. However, in many situations, rather than identifying this precise peptide epitope, it is sufficient to identify a peptide “mimotope.” Mimotopes can be defined as peptides that are different in sequence from the actual peptide recognized in vivo, but that are nevertheless capable of binding to the appropriate MHC molecule to form a ligand that can be recognized by the αβTCR in question. These peptides can be very useful for studying the T cell in vitro, for altering the immunological state of the T cell in vivo , for vacMimotopes can sometimes be identified in randomized peptide libraries that can be screened for presentation by a particular MHC molecule to the relevant T cell . Thus fab. The peptides in the library varied in five peptide amino acids known to be surface exposed and predicted to lie within the footprint of αβTCR interaction.In other applications, another powerful library method has been sequential enrichment/expansion of a displayed library of protein–peptide variants by direct ligand–receptor binding, e.g., using bacterial phage or yeast  and the lack of synchrony in viral infection and expression. To focus on cells bearing a particular level of IAb, we stained the cells simultaneously with the fluorescent αβTCR reagents and with an anti-IAb monoclonal antibody (mAb) that did not interfere with αβTCR binding. In this case, there was a direct correlation between the amount of surface IAb-p3K expressed by an individual insect cell and the amount of αβTCR bound (b-p3K, binding the αβTCRs uniformly (Insect cells displaying IAeneously A, probabCR bound B with ceniformly C. Therefb-p3K molecule and 99% encoded a control molecule (an αβTCR β chain). The infected cells were stained with fluorescent YAe-62 αβTCR and analyzed by flow cytometry. Although a distinct population of brightly fluorescent cells was not seen, the 1% of the cells with the brightest fluorescence were sorted, as were an equal number of cells that were very dully fluorescent   in-frame with the IAb signal peptide and with a 3′ termination codon  compensated for the absence of the asparagine sidechain.When bound to IA APC see B. Perhapb–peptide combination that bound the YAe-62 αβTCR. It is likely that many more would be identified if more clones were analyzed. Five sequences were found multiple times. Not unexpectedly, these were among those that bound the YAe-62 αβTCR most strongly. There was a 100-fold range in the intensity of αβTCR binding to the different IAb–peptide combinations, ranging from about 4-fold to 400-fold binding above that seen with a negative control peptide. One obvious property of these peptides stands out. There appeared to be a very strong selection for a basic amino acid at position 5. In 16 of 20 of the peptides, a lysine, arginine, or histidine was found at position 5, matching the lysine found in p3K. As a control, we sequenced random clones picked either from the original E. coli construction of the library (17 clones) or from the baculovirus library that expressed IAb–peptide well, but did not bind either αβTCR (11 clones). The frequencies of basic amino acids at p5 in these sequences were only 12% and 9%, respectively (data not shown).Consistent with the hypothesis that the αβTCR of YAe-62 would be more peptide promiscuous than that of B3K-06, we found 20 different peptide sequences among the analyzed clones that produced an IAb-binding peptides that were not bound by the YAe-62 αβTCR, but only two of 17 (11.8%) of the random E. coli plasmids. The amino acid in this position is only partially exposed on the surface and can contribute significantly to peptide–MHC interaction  of the YAe-62 αβTCR-selected peptides and three of 11 (27.2%) of the IAeraction . After action  at this position. Perhaps this indicates that, in general, larger sidechains can be inhibitory at this position, but again more data would be required to test this idea.b–peptide combinations that bound the YAe-62 αβTCR most strongly were also the ones that were able to induce IL-2 production from YAe-62. Among these, a number with the very highest apparent affinities stimulated YAe-62 better than did p3K. However, there was not a direct correlation between apparent affinity and the level of IL-2 production; i.e., several peptides that yielded complexes with IAb with about the same apparent affinity for the αβTCR nevertheless stimulated very different levels of IL-2 production from YAe-62. This may be related to the phenomenon of altered peptide ligands  for the αβTCR was already known. However, this method should be useful as well in identifying mimotopes for αβTCRs whose peptide epitope is not known, provided that suitable peptide anchor residues for MHC binding are known. One limitation of this display method as presented here is the size of the peptide library. The bottlenecks caused by the preparation of the library in an ligation  or in viligation . In prelligating  a randomb as the displayed MHCII molecule carrying the peptide library. However, using the same strategy, we have successfully displayed numerous other MHCII molecules, such as murine IEk and human DR4, DR52c, and DP2 (data not shown). While the leucine zippers that we included in this construct are not strictly required for expression of IAb, they have helped considerably in expression of some of these other MHCII molecules. Moreover, we (d, with a β2m-tethered peptide from HIV gp120 (data not shown). Given that baculovirus has been such a successful expression system for many different types of complex eukaryotic proteins that express or assemble poorly in E. coli, this method may have broad applications to other receptor–ligand systems.We have developed this method using IAover, we  and otheover, we  have shob–peptide combinations. Thus, depending on whether one was interested in high- or low-affinity ligands for the αβTCR, one could enrich for peptides with these properties directly during the screening of the library. Such an approach has been used with antibody , p3K (FEAQKAKANKAVD), and various alanine-substituted variants of p3K were synthesized in the Molecular Resource Center of the National Jewish Medical and Research Center (Denver, Colorado, United States), as were all oligonucleotides used in PCR and DNA sequencing. Automated DNA sequencing was also performed in this facility.b-p3K-reactive T cell hybridoma B3K-06 was produced from C57BL/6 mice as previously described . The IAescribed . The IAbLB-15.13  was usedb β gene (ΔIAβ) and the invariant chain (ΔIi) and that carried a transgene for the IAb β gene that was modified to insert sequence encoding pEα and a flexible linker between the signal peptide and the N-terminus of the β chain. These mice were immunized intravenously with 3 × 106 dendritic cells from ΔIAβ/ΔIi C57BL/6 mice. These cells had been retrovirally transduced  addition region were synthesized by PCR using baculovirus DNA as template. The fragments were used to construct an insect cell expression vector (pTIE1) on a pTZ18R  backbone with the hr5 enhancer at the 5′-end, followed by the IE1 promoter, a large multiple cloning site , and the IE1 poly(A) addition region. The complete sequence of the pTIE1 vector has been deposited in GenBank (see Supporting Information). DNA fragments encoding mouse ICAM and B7.1 were cloned between the XhoI and NotI sites of the multiple cloning site. Sf9 cells were transfected with a combination of the plasmids by the standard calcium phosphate method and cells expressing both molecules on their surfaces were cloned without selection at limiting dilution to establish the line Sf9-ICAM/B7.1.5) were added to microtiter wells containing either (1) saturating immobilized peptide–MHC, (2) 10 μg/ml peptide plus 105 LB-15.13 cells, (3) 5 × 104 Sf9-ICAM/B7.1 insect cells infected 3 d previously with baculovirus encoding a displayed peptide–MHC, (4) 106 spleen cells from IAb-pEα single peptide mice, or (5) 106 spleen cells from various knockout or MHC congenic mice. After overnight incubation the culture supernatants were assayed for IL-2 as previously described  at a molar ratio of 2.5:1 (biotin:antibody) for 4 h at room temperature. The reaction was quenched with 0.1 M lysine and the product dialyzed extensively against PBS. The resulting derivative contained about one biotin per molecule of mAb. The biotinylated mAb was complexed in excess with AlexaFlour647–streptavidin (Molecular Probes, Eugene, Oregon, United States). The complex was separated from the free biotin–antibody using Superdex-200 size exclusion chromatography (Pharmacia). In preliminary experiments, the amount of soluble αβTCR required to saturate an aliquot of a large single batch of this reagent was determined. To prepare the multivalent αβTCR, the appropriate amount of soluble αβTCR was mixed with an aliquot of the fluorescent anti-Cα reagent overnight. For staining for flow cytometry, this mix was used without further purification. Each 100 μl sample contained approximately 2 μg of the fluorescent reagent plus 105 Sf9 insect cells. This mixture was incubated at 27°C for 1–2 h. The cells were then washed for analysis. The advantages of this method for preparing fluorescent multimers over using direct enzymatic biotinylation . For sorting, a MoFlo instrument was used (Dako/Cytomation, Glostrup, Denmark).b on the surface of baculovirus-infected insect cells, modifications were made as described in b-p3K (b-binding peptides. The constructions were incorporated into baculovirus by homologous recombination using the BaculoGold system (PharMingen).For displaying IAin b-p3K . Other vE. coli transfer plasmid to replace the portion encoding p3K with sequence encoding enhanced GFP, flanked by sites for the restriction enzymes SbfI and CeuI. A PCR fragment was produced as described in b-binding peptide randomized at positions p2, p3, p5, p7, and p8, but identical to p3K at other positions. This sequence was flanked by sites for the restriction enzymes PstI and BstXI, such that the cohesive ends generated by these enzymes were compatible with those generated by SbfI and CeuI in the GFP-containing plasmid. Cloning the restricted fragment into this site regenerated a covalent peptide in-frame with the signal peptide and flexible linker of the IAb β chain . An estimated 3 × 104 to 10 × 104 independent transformants were obtained that were used to make a mixed plasmid preparation. This mixture was incorporated into baculovirus by homologous recombination as above. In order to assure a high efficiency of conversion of plasmids to virus, 1.5 × 107 Sf9 cells were cotransfected with 6 μg of the plasmid mixture and 1.5 μg of BaculoGold DNA.As described in hain see E. After http://www.ncbi.nlm.nih.gov/Genbank/) accession numbers for the sequences described in this paper are B7.1 (AJ278965), baculovirus DNA (L22858), ICAM (X52264), and pTIE1 vector (AY522575).The GenBank (


Real-time PCR is becoming a common tool for detecting and quantifying expression profiling of selected genes. Cytokines mRNA quantification is widely used in immunological research to dissect the early steps of immune responses or pathophysiological pathways. It is also growing to be of clinical relevancy to immuno-monitoring and evaluation of the disease status of patients. The techniques currently used for "absolute quantification" of cytokine mRNA are based on a DNA standard curve and do not take into account the critical impact of RT efficiency.in vitro stimulation of human PBMC by lipopolysaccharide (LPS) or Staphylococcus aureus strain Cowan (SAC). Results obtained show that CyProQuant-PCR is powerful enough to precociously detect slight cytokine induction. Finally, having demonstrated the reproducibility of the method, it was applied to malaria patients and asymptomatic controls for the quantification of TGF-β1 transcripts and showed an increased capacity of cells from malaria patients to accumulate TGF-β1 mRNA in response to LPS.To overcome this pitfall, we designed a strategy using external RNA as standard in the RT-PCR. Use of synthetic RNA standards, by comparison with the corresponding DNA standard, showed significant variations in the yield of retro-transcription depending the target amplified and the experiment. We then developed primers to be used under one single experimental condition for the specific amplification of human IL-1β, IL-4, IL-10, IL-12p40, IL-13, IL-15, IL-18, IFN-γ, MIF, TGF-β1 and TNF-α mRNA. We showed that the beta-2 microglobulin (β2-MG) gene was suitable for data normalisation since the level of β2-MG transcripts in naïve PBMC varied less than 5 times between individuals and was not affected by LPS or PHA stimulation. The technique, we named CyProQuant-PCR (Cytokine Profiling Quantitative PCR) was validated using a kinetic measurement of cytokine transcripts under The real-time RT-PCR technique based on a RNA standard curve, CyProQuant-PCR, outlined here, allows for a genuine absolute quantification and a simultaneous analysis of a large panel of human cytokine mRNA. It represents a potent and attractive tool for immunomonitoring, lending itself readily to automation and with a high throughput. This opens the possibility of an easy and reliable cytokine profiling for clinical applications. Cytokines are a family of low-molecular weight proteins secreted by various cell types, with pleiotropic functions and constitute a tightly regulated network that plays a central role in the immune system. Cytokines, classified into different groups such as interleukins (IL), interferons (IFN), colony-stimulating factors (CSF), tumour necrosis factors (TNF), tumour growth factors (TGF) and chemokines are implicated in the differentiation, proliferation, migration and effector functions of immune cells. Interacting one with the others, they have polarizing effects on the target cells and are pivotal in tuning immune responses . TherefoCytokines are often produced locally so that the concentration of circulating cytokines in the plasma is usually low. Their half-life and turnover may vary complicating the delineation of informative cytokine profiles. Although transcription of messenger RNA is not strictly correlated to protein secretion and activity, detection of cytokine RNA by real time PCR is now considered a reference technique for analysis of small-size samples with high sensitivity . It can The already available techniques, which offer a so-called "absolute quantification" of the target cytokine mRNA, achieve quantification by reference to an external standard curve based on serial dilutions of a known amount of the corresponding cDNA . MoreoveIn the present study, we first show that the efficiency of the RT step depends on the target mRNA and on the experiments and that these variations have critical impact on the reliability of mRNA quantification. To overcome this, we describe here CyProQuant-PCR, a new technique for absolute measurement of cytokine mRNA based on an external RNA standard curve. Primer pairs have been designed for allowing amplification of a set of cytokine mRNA using the same conditions both in terms of thermocycling parameters and master mix components, a prerequisite for multiple cytokine mRNA measurements with high throughput.in vitro stimulated PBMC and its intra and inter-experimental variability were assessed. Finally, CyProQuant-PCR was used to quantify TGF-β1 transcripts in small blood samples from children with acute Plasmodium falciparum malaria.In the present paper, we describe i) the construction of the synthetic RNA standard, ii) the primer pairs specific for the following human cytokines IL-1β, IL-4, IL-10, IL-12p40, IL-13, IL-15, IL-18, IFN-γ, and for the tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein, zeta polypeptide (YWHAZ), the β2-microglobulin (β2-MG) and the ubiquitin-C (UBC) to be used as internal standards and iii) the conditions for efficient real time amplification of multiple cytokine specific mRNA. The technique was validated using Primer pairs were designed from published genomic sequences using Primer Express software (Applied Biosystems), except for the UBC and the YWHAZ genes for which the primers had already been described . When poT) and the maximum signal-to-noise fluorescence ratio (ΔRn) while minimising non-specific amplification (data not shown). Among the final concentrations of 50, 300 and 900 nM tested for each primer, the optimal final concentration was set at 900 nM for every primer. The primer pairs are shown in Table Primer concentrations were optimised to determine the minimum primer concentrations giving the lowest threshold cycle . The molecular mass of each RNA standard was calculated on the basis of its sequence and solutions ranging from 101 to 1012 copies of standard RNA were made.To generate the standard RNA corresponding to each target sequence, gene specific primers were fused in their 5' end to the RNA polymerase T7 promoter sequence. The PCR performed with these modified primers pairs lead to a larger PCR product with the T7 promoter sequences upstream and downstream from the specific amplicon. The T) against the logarithmic value of the starting RNA copy number for each dilution. Figure These serial diluted solutions were reverse transcribed and the cDNA amplified in duplicate to generate a standard curve by plotting the threshold cycle . The slope of the standard curves generated by these three ranges as well as the corresponding efficiency are shown in Table CyProQuant-PCR was compared to the classical approach based on a DNA standard curve for the quantification of TNF-α, IL-1β and IFN-γ transcripts. DNA templates for RNA transcription of TNF-α, IL-1β and IFN-γ were used to generate a range of concentration from 10 to 10In order to show that these intergenic differences were not due to interassay variation, we compared amplification efficiencies of TNF-α, β2-MG, MIF and UBC from i) a single cellular RNA sample, ii) a pooled RNA sample containing standard RNA for each of the targets and iii) a pooled DNA sample corresponding to the cDNA obtained from the pooled RNA sample containing standard RNA for each of the targets. Results obtained are summarised in Table Altogether, these data demonstrate that RT efficiency displays intergenic variations that are not due to interassay differences.RT efficiency has critical impact on transcript quantification as shown in Table Taken together, these results support the use of RNA as external standard for reliable and reproducible quantification of transcripts.Since RT-PCR efficiency varies, absolute mRNA quantification can only be reliably obtained if the external RNA standard and the cellular RNA are retro-transcribed and amplified with the same efficiency. This was secured by comparing the standard curves obtained after amplification of a range of 10X serial dilution of cellular RNA and β2-MG external RNA standard. The slopes obtained were of -3,307 and -3,305 for the cellular RNA and for the external RNA standard, respectively. This corresponds to efficiencies of 100,6% and 100,7% respectively (data not shown).in vitro conditions. Moreover, based on the approach recently described by Pachot et al. [The choice of a stable expressed endogenous gene to be used as an internal standard is a prerequisite for accurate RT-PCR expression profiling. This has to be adapted to the clinical situation and the tissue of origin of the samples. Since our purpose was to establish a technique to be used with peripheral blood leukocytes, we tested three genes reported in stable amounts in leukocytes: the β2-MG, the UBC and the YWHAZ . We compt et al. , we asset et al. . β2-MG w® technology developed by Applied Biosystems is considered as the "gold standard", we compared CyProQuant-PCR to TaqMan® for the quantification of TNF-α transcripts in isolated monocytes stimulated for 6 hours with LPS. Total RNA was extracted from 106 cells and 10X serial dilutions were prepared and reverse transcribed. The resulting cDNA were amplified in duplicate on the same plate in the same thermocycling conditions using either the TaqMan® commercial kit (Applied Biosystems) or CyProQuant-PCR primers with SYBR Green PCR master mix (Applied Biosystems). Figure ® in terms of sensitivity and even more efficient: 105% (slope -3,198) for CyProQuant-PCR versus 80 % (slope -3,908) for TaqMan®.Since TaqManTo validate our approach, we measured the levels of TNF-α and MIF transcripts and secreted proteins by CyProQuant-PCR and ELISA respectively. PBMC from healthy individuals were stimulated by LPS or SAC for 3, 9 and 18 hours. Figure To validate the panel of primers designed for CyProQuant-PCR, we assessed the early cytokine response as well as the kinetics of expression for PBMC from two healthy donors under stimulation with LPS or SAC. Figure Before the application to clinical samples, the experimental reproducibility of CyProQuant-PCR was evaluated using a range of TGF-β1 and β2-MG RNA standards. Inter-experimental variability was assessed from eight independent experiments. The coefficients of variation were satisfactory whatever the starting copy number of RNA standard, ranging from 0,39 to 1,07% and from 0,91 to 1,2% for TGF-β1 and β2-MG respectively Table . TGF-β1 ® commercial kit, which represents the "gold standard", CyProQuant-PCR was as sensitive but less expensive and more flexible. Indeed, for a given gene, the design of the probe might be quite difficult and, with judicious selection of primer pairs, comparable sensitivity can be achieved with the use of SYBR Green [In this paper we have described a new technique, CyProQuant-PCR, for absolute quantitative profiling of human cytokine mRNA using real time RT-PCR. Although real time PCR is now becoming a popular technique, it still requires improvement for proper quantification. All the techniques for absolute quantification available so far use a DNA standard curve assuming that RT efficiency is constant and approaches 100% . We showBR Green .in vitro transcribed RNA to specific RNases [A major reason for not using an RNA standard curve is its poor stability due to its sensitivity to RNase degradation. In our hands, we did not find any detectable degradation when keeping the standard in concentrated aliquots (stock solution at 1000 μg/mL) in RNase-free water at minus 80°C and avoiding freeze-thawing cycles. However, for a greater stability, we incorporated modified dNTP, 2'-Fluoro-dCTP and 2'-Fluoro-dUTP, which have been reported to decrease the sensitivity of the c RNases ,22, and The methodology described here was easily and successfully applied to the quantification of several cytokine genes. The quantification is reliable on 7 to 8 logs with a sensitivity ranging from 1000 to 100 copies depending the cytokine. It was first used to determine the magnitude and the kinetics of early induction of cytokines mRNA upon PBMC stimulation using bacterial derived materials. This showed that CyProQuant-PCR is powerful enough to detect early on modest cytokine induction such as IL-4. Such a tool is useful to decipher the kinetics of cytokine response involved in physiopathological pathways but also as a read-out to measure minor immune responses to specific antigens .We finally applied CyProQuant-PCR to measure the level of TGF-β1 transcripts in asymptomatic controls and malaria patients after LPS stimulation. Interestingly, we observed that cells from malaria patients have a significant higher capacity to respond to LPS compared to controls. The increased TGF-β1 transcript accumulation by patients' cells, suggests that an inadequate production of TGF-β1 might play a role in malaria pathogenesis as already proposed . Further® which is as sensitive as the TaqMan® technique. Because the parameters of amplification are identical for all the cytokines developed, CyProQuant-PCR is readily automatable notably for 384-well plates and might allow multiple cytokine profiling of samples of very limited size at a relatively high throughput. CyProQuant-PCR opens the possibility to use cytokine mRNA measurements in clinical studies not only for an increased knowledge but also to help clinicians in patients' stratification and treatment decision.In conclusion, we provide here CyProQuant-PCR, a simple technique for genuine absolute quantification of cytokine mRNA using SYBR Green+ human serum (French Blood Bank) at 2.106 cells/mL and either used directly for RNA extraction or cultured in duplicate with or without LPS (10 ng/mL, E. Coli O111: B7, Sigma, L'Isle d'Abeau Chesnes, France) or SAC . After incubation, cells were washed with PBS and re-suspended in RNA-PLUS (Q-Biogene, Illkirch, France) for RNA isolation.Blood was collected from healthy donors at the French Blood Bank. Peripheral blood mononuclear cells (PBMC) were isolated by density separation over Ficoll Hypaque and washed two times in RPMI 1640 (Gibco BRL, Invitrogen, Cergy Pontoise, France). Cells were re-suspended in RPMI 1640 (Gibco BRL, Invitrogen, Cergy Pontoise, France) supplemented with 2 mM glutamine (Gibco BRL, Invitrogen, Cergy Pontoise, France) and 10% ABet al. [E. Coli O111: B7, Sigma, L'Isle d'Abeau Chesnes, France).Twenty children admitted during the high malaria transmission season of 2001 to the emergency room at the Department of Child Health, Korle-Bu Teaching Hospital, Ghana were included. Five asymptomatic controls matched to patients for age, residence location and time of sample collection were enrolled. The general inclusion and exclusion criteria were as described by Kurtzhals et al. . ParentsRNA was extracted following supplier's instructions, re-suspended in 60 μL of RNase-free water (Ambion, Huntingdon, UK) and quantified spectrophotometrically at 260 nm.E. Coli O111: B7, Sigma, L'Isle d'Abeau Chesnes, France) and PHA-L (10 μg/mL, Sigma-Aldrich, Lyon, France) was used. Analysis of the amplicons was assessed by 4% agarose gel electrophoresis and dissociation curve studies using Dissociation Curve Software (Applied Biosystems, Foster City, CA, USA). PCR products were cloned into pCR2.1 vector using Original TA cloning kit (InVitrogen, Cergy Pontoise, France) and sequenced (Genome Express, Meylan, France).Oligonucleotide primers were synthesized at Eurogentec (Saraing, Belgium). To validate primers, a pool of cDNA from healthy human PBMC stimulated for 6 and 12 hours with LPS (10 ng/mL, 12 copies of standard DNA /3,85 μL were made in Tris-EDTA buffer (Ambion, Huntingdon, UK), split in single-use aliquots and stored at -80°C in safe-lock tubes. External DNA standard range was made extemporaneously by 1:10 serial dilutions in water.PCR products generated by each primer pairs were column-purified (Nucleospin, Macherey-Nagel, Hoerdt, France) and quantified spectrophotometrically at 260 nm. The molecular weight of the standard DNA was calculated by N*487-[(N-1)*175] were N is the number of bases composing the standard DNA. Stock solution of 10in vitro transcribed (MegaShortScript, Ambion, Huntingdon, UK). The standard RNA generated was purified (MegaClear, Ambion, Huntingdon, UK) and loaded on a 4% agarose gel for electrophoresis. The concentration of the standard RNA was determined spectrophotometrically at 260 nm. The molecular weight of the transcript was calculated by N*500-[(N-1)*175] were N is the number of bases composing the standard RNA. Stock solution of 1012 copies of standard RNA /3,85 μL were made in RNA storage solution (Ambion, Huntingdon, UK), split in single-use aliquots and stored at -80°C in safe-lock tubes. External RNA standard range was made extemporaneously by 1:10 serial dilutions in water.Gene specific primers were fused on their 5' end to the sequence of the RNA polymerase T7 promoter to generate modified primers. These primers were used to amplify a gene specific PCR product flanked by transcription initiation sites. Five hundred nanograms of this construct were For CyProQuant-PCR assays, 100 ng of total cellular RNA from PBMC and serial dilution of external RNA standard were reverse transcribed simultaneously in a parallel procedure using Reverse Transcription TaqMan reagents (Applied Biosystems, Foster City, CA, USA) on a MasterCycler Gradient (Eppendorf, Le Pecq, France). The final volumes were set at 100 μL for the cellular RNA samples and 50 μL for the external RNA standard range. The thermocycling parameters were as follows: 25°C, 10 min.; 48°C, 60 min. and 95°C, 5 min. cDNA were immediately used for PCR amplification.T) against the logarithmic values of the starting RNA standard copy number.Reverse-transcribed standard RNA and cellular RNA were amplified simultaneously on the same PCR plate on an ABI Prism 7700 (Applied Biosystems, Foster City, CA, USA). An aliquot of 5 μL of the RT reaction was amplified in duplicate in a final volume of 30 μL of SYBR Green PCR Master mix (Applied Biosystems, Foster City, CA, USA). Thermocycling conditions were 50°C for 2 min., 95°C for 10 min. and 40 cycles of [95°C/15 sec.; 60°C, 1 min]. The sample target RNA copy numbers were calculated using SDS 1.9 Software (Applied Biosystems, Foster City, CA, USA). The baseline fluorescence was set manually to correct for differences in initial cDNA concentration and the threshold was positioned at a fluorescence level that was 10 times higher than the background signal. Target mRNA copy numbers in cellular samples were calculated based on a standard curve generated by SDS 1.9 Software (Applied Biosystems, Foster City, CA, USA) by plotting cycles at threshold (CTNF-α and MIF secreted proteins were quantified by sandwich ELISA following supplier's instructions (Bio-Source, Clinisciences, Montrouge, France and R&D, Lille, France respectively). Results are expressed as pg/mL for one million living cells.Tests for significance were done using Stata software (Stata Corporation, College Station, Texas, 77845 USA) by Kruskal-Wallis rank test.Results disclosed in this manuscript have been protected in French patent application FR0408645.in vitro stimulation experiments. DJ gave technical assistance. SL helped in RNA extraction of malaria samples. JCB introduced PB to molecular biology techniques and provided critical advices. BDA designed and conducted the study that yielded the malaria samples. OMP revised the manuscript and supported the work. CB conceived the strategy and coordinated the study. PB and CB drafted the manuscript. All authors read and approved the final manuscript.PB developed the entire technique. IV did the