12.2
Repopulating immunodeficient mice with human cord blood stem cells
D. Bernard, M. Peakman & A. Hayday
Peter Gorer Department of Immunobiology, King’s College, 2nd Floor New Guy’s House, Guy’s Hospital, London SE1 9RT, United Kingdom Rodents have been widely used to study mammalian immune system because of their short reproduction period and the relative ease of generating inbred mutants, knock-out and transgenic strains. How- ever, it is seldom possible to extrapolate directly observations from
rodent models directly into clinical practice. Thus, mice in which human progenitors develop and differentiate to establish a humanized immune system would constitute a major improvement of the model for functional studies. A number of such promising models have been described where immunodeficient mice have been reconstituted with human immune cells or human hematopoietic stem cells at different stages of differentiation. Here we describe progress with the isolation of highly purified CD34+ human cord blood stem cells suitable for intrahepatic injection into conditioned newborn immunodeficient mice. Thede novodevelopmental competence of such cellsin vivowill be investigated using flow cytometry and molecular approaches.
Session 13: Visualising the immune response
IS56
Proteomic and lipidomic approaches to autoimmune disease W. H. Robinson
GRECC MC 154R, Palo Alto VA, 3801 Miranda Ave, Palo Alto, CA 94304, USA
Autoimmune diseases affect 3% of the world population, yet the diagnosis and classification of autoimmune diseases remain based on clinical examination combined with traditional laboratory tests and imaging studies. We have developed protein and lipid microarray technologies to characterize the specificity of autoimmune responses and to identify novel biosignatures to diagnose, classify, and guide therapeutic decision making in patients with autoimmune disease.
We developed and validated myelin protein and myelin lipid mic- roarrays to profile the autoimmune responses in murine and human multiple sclerosis. Employing statistical algorithms we discovered unexpected extensive spreading of autoimmune responses in mul- tiple sclerosis to attack multiple components of the myelin sheath, including both proteins and lipids. Data will be presented demon- strating use of proteomic and lipidomic biosignatures to diagnosis, to identify rodents and humans at risk to develop severe disease, and to monitor responses to therapy. Further, we are developing antigen-specific tolerizing therapeutics based on our proteomic and lipidomic findings, providing the potential to accelerate the dis- covery to market cycle for a pharmaceutical agent by many years.
Data will be presented demonstrating tolerization of established murine multiple sclerosis with both protein and lipid microarray identified targets. Our results suggest the intriguing possibility that profiles of serum proteins such as autoantibodies, determined using protein and lipid array technologies, can be used to develop disease- specific, and even patient-specific, therapies.
IS57
Using the eye to image the immune response J. T. Rosenbaum
Oregon Health and Science University, 3181 SW Sam Jackson Park Rd L467AD, Portland, OR 97239, USA
The eye is especially useful in studies to image the immune response because the clear cornea allows visualization of internal vasculature without resorting to surgical trauma and the eye is a known target of immunological diseases such as uveitis. Using an epifluorescent microscope, time lapse imaging, image stabilization software, and several mouse models of uveitis including one secondary to locally injected endotoxin and an antigen specific model with DO11.10
fluorescently labelled ovalbumin-specific splenocytes, we have ob- served: i) an apparent random walk by neutrophils which infiltrate the iris, ii) a surprising immobility of antigen presenting cells within the iris and iii) T cell migration which is antigen specific, sometimes directed toward the APC, and results in prolonged contact between the APC and T cell at the site of inflammation.
13.1
Assessing dynamic responses in thousands of individual leuco- cytes simultaneously: a cell population array imager
K. Farrell-Dillon,* O. V. Salata,* S. J. Payne, S. P. Youngà&
S. V. Hunt*
*Lecturer in Immunology, University of Oxford Mail, Dunn School of Pathology, South Parks Road, Oxford OX1 3RE, United Kingdom, Department of Engineering Science, Oxford University, Parks Road, Oxford OX1 3PJ, United Kingdom,àDivision of Immunity and Infection, Medical School, Birmingham University, Birmingham B15 2TT, United Kingdom
Primary leucocytes make heterogeneous 2nd-messenger responses to external stimuli, e.g. Ca2+oscillations of differing periodicities, which result in complex patterns of gene expression. To classify kinetic response patterns, especially of the rarer classes, surveillance of large numbers of cells is needed. With conventional video-imaging, few cells can be simultaneously observed, insufficient for accurate classification.
We have designed and built a cell population array imager, CPAI, which records responses of thousands of cells simultaneously. Cells are arrayed within a shallow flow chamber into a tightly packed ultra- microwells, individually addressed by a separate optical fibre. Illu- minated by a strong diode light source, the whole-cell fluorescence intensity of cells loaded with the Ca2+-sensitive reporter Fluo4 is dig- itally captured in an image stack taken at intervals of a few seconds. A neural-network-based algorithm searches for similarities in kinetic patterns. Data will be presented on calcium responses in Jurkat and primary rat lymphocytes which demonstrates the diversity of signalling in leucocyte populations. Funded by Wellcome Trust: See www.cpai.eu
13.2
Rapid detection and characterization of immune responses using label-free biacore immunoassays
F. Sundberg & R. Karlsson
Biacore AB, Rapsgatan 7, Uppsala, 75450, Sweden
We demonstrate how label-free protein interaction analysis facilitates efficient development of biotherapeutics and vaccines by providing rapid detection and information-rich characterization of immune Session 13: Visualising the immune response
2007 The Authors Journal compilation2007 Blackwell Publishing Ltd,Immunology,120, Supplement 1, 3 49 46
responses in pre-clinical development and clinical trials. Using this approach, immune responses are readily identified using label-free assays in which serum antibodies are detected by binding to the bio- therapeutic, which is immobilized on the sensor chip surface. Multiple immobilized proteins can also be analyzed simultaneously in this way, providing additional information on specificity and selectivity of binding. Following binding of serum antibodies to the biotherapeutic, several procedures can be easily employed to rapidly provide a more thorough characterization of these patient immune responses. Isotype or subclass-specific reagents can be used in series, for example to define and monitor the Ig composition of responses. Real-time detection also enables monitoring of antibody dissociation rates and hence, relative affinities among different responses, providing a measure of thera- peutically relevant binding properties. Crucially, this real-time analysis also enables detection of low/medium affinity antibodies with rapid binding kinetics, which are otherwise difficult to detect, but may be clinically important in efficacy and safety issues.
13.3
Malaria pigment impairs the T cell clustering associated with immune priming despite normal signal 1 from dendritic cells O. R. Millington,* V. B. Gibson,* C. M. Rush,* B. H. Zinselmeyer,*
R. S. Phillips, P. Garside* & J. M. Brewer*
*Centre for Biophotonics, Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow G4 0NR, United Kingdom, Division of Infection and Immunity, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow G12 8TA, United Kingdom
The interaction between antigen-presenting dendritic cells (DCs) and T cells is an essential step in the induction of an immune response.
However, during malaria infection, dendritic cell function is impaired and immune responses against parasite and heterologous antigens fail.
In this study, we used multiphoton microscopy to demonstrate that while antigen presentation (signal 1) intensity remains unaltered, the four-dimensional interactions between T cells and DCs are inhibited by the parasite pigment hemozoin and by malaria infection bothin vitro andin vivo. This alteration in cell behaviour is associated with sup- pression of functional T cell responses, explaining why immunity is reduced during malaria infection.
13.4
A novel method to visualise the interaction between schistosome larvae and cells of the innate immune response
R. A. Paveley, S. Ferret-Bernard & A. P. Mountford
Department of Biology (Area5), University of York, York YO10 5YW, United Kingdom
Infection of skin byS. mansonicercariae causes an acute inflammatory reaction and activation of cells of the innate immune response. Using a
novel technique, live cercariae were labelled with the fluorescent tracers, CFSE, or FarRed DDAO-SE. Fluorescent dyes bound predominantly to parasite molecules located within the pre- and post- acetabular glands which are then released upon transformation.
Labelled material released by parasites was taken up by co-cultured thioglycollate-elicited macrophages as determined by flow cytometry and confocal microscopy. Uptake was greatest in cells expressing high levels of MHCII, CD40 and CD86 indicating their role as putative antigen presenting cells. Uptake of parasite material was also accom- panied by the secretion of IL-12p40, IL-6 and IL-10. However, addition of cytochalasin D (disrupts actin rearrangement), or mannan (block receptors of mannose), both inhibit uptake of labelled parasitic material and the secretion of specific cytokines. This suggests that phagocytic and mannose reactive receptors are required for the uptake of released schistosome molecules by innate immune cells.
13.5
3D imaging of lymphocytes in intact mouse carotid artery P. Maffia,* B. H. Zinselmeyer, A Ialenti,* S. Kennedy,à A. H. Baker,§I. B. McInnes,–J. M. Brewer & P. Garside
*Department of Experimental Pharmacology, University of Naples Federico II, via D. Montesano,49, Napoli, 80131, Italy, Centre for Biophotonics, Strathclyde Institute for Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow G4 ONR, United Kingdom,
àDepartment of Physiology and Pharmacology, Strathclyde Institute for Biomedical Sciences, University of Strathclyde, 27 Taylor Street, Glasgow G4 ONR, United Kingdom,§Division of Cardiovascular and Medical Sciences, Glasgow Biomedical Research Centre, 120 University Place, Glasgow G12 8TA, United Kingdom,–Division of Immunology, Infection and Inflammation, Glasgow Biomedical Research Centre, 120 University Place, Glasgow G12 8TA, United Kingdom
For evaluation of atherosclerotic processes and their relationship to immune activation, imaging of 3D structures in intact vascular tissues and functional aspects of the diseased artery are required. Multi- photon laser-scanning microscopy allows to penetrate directly into tissues at sufficient depths to image tissuesin situin the absence of significant phototoxicity. By moving the focal point axially, a stack of optical sections at various depths can subsequently be viewed as a movie. Thus, two-photon microscopy potentially allows combined functional and structural studies and can therefore be suitable for investigating immune response in diseased vessels. We show the entire structure of an isolated intact ApoE -/-mouse carotid artery, identifying, for the first time in three dimension, homed fluorescently tagged adoptively transferred lymphocytes, mainly localized in the adventitia. Results in line with recent data indicating an important role of the adventitia as a site of localization of immune cells.
Consequently, this system could be a powerful tool to study immune cells behaviour in artery disease.
Session 13: Visualising the immune response
2007 The Authors
Journal compilation2007 Blackwell Publishing Ltd,Immunology,120, Supplement 1, 3 49
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