Below we give a summary of the results of the project. An easy-to-read brochure with the full visit reports is available from the IVTIP Secretariat
In Vitro Testing Review is a pilot study which demonstrates the importance of a technology transfer programme.
The In Vitro Testing Review team members have looked at five projects in the area of In Vitro Testing funded under the European Commission's Framework Programme 4.
The projects were:
Industry has very real interest in the results of this venture. This was shown by the large response (38) on a questionnaire, sent to 190 European companies with an active interest in in vitro testing. It was also shown by the willingness of some ten industries to meet in person with the consultants involved in this study for an in-depth, personal briefing on the results of the study.
Overall, industry needs tests that are validated or near validation. Although none of the projects reviewed resulted in tests ready for validation (one is very near though), many of the results generated in the projects were of general interest to industry, in particular the biopharmaceutical industry. Below we review the results generated in the five projects reviewed and also indicate which results have a direct industrial potential.
skin reconstructed with immortalised humankeratinocytes, fibroblasts, endothelial cells
cell lines transfected by construction expressing promoter driven reporter gene.
irritation markers included:
HSP 70 for keratinocytes
IL-1ß for all cells
ICAM-1 fibroblasts and endothelial cells
PTX3 fibroblasts and endothelial cells
reproducibility, expression stability proved problematic for several constructs
engineered cells do not differentiate like normal epidermal cells.
SV40 transformed Wi26 fibroblast lines.
PINCO retrovirus vector offers hope of more stable constructions.
Green Fluorescent Protein-transfected fibroblastic and endothelial cell lines
synthetic RNAs as standards for RT-PCR for cytokine, matrix macromolecules and enzyme expression
Highly sensitive synthetic RNA standards for quantitative RT-PCR for IL-6, matrix metalloproteinases and their inhibitors (which are probes for inflammatory conditions, viral disease, cancer and immunological disorders).
The reporter genes developed in this project may have wider applications in cell and molecular biology
If you are interested in one or more elements of this project, please contact the coordinator mentioned below or the IVTIP Secretariat:
Prof. Charles Maurice Lapière
Université de Liège
Tour de Pathologie, B23
BE - 4000 SART-TILMAN
Tel: 3241662455
Fax: 3241662457
Email: lctb@ulg.ac.be
develop stable, immortal differentiated cell lines stably expressing function specific genes of source tissues,or:
one or few functions rarely seen in the existing in vitro systems.
target tissues liver, kidney, lung, cartilage and intestine.
primary objective modify balance between proliferation, differentiation and cell death without losing linkage/communication.
Low efficiency of transfection and retroviral infection in primary culture due to: - spontaneous apoptosis - low rate of proliferation - low retroviral titers
Low expression of cellular genes of interest (c-myc, MEK, cyclin D1...)
Inefficient plasmid constructs (pRetro-off)
identification and/or characterisation of genes involved in proliferation, differentiation, apoptosis (c-myc, MEK/ERK, cyclins, CDKs, CKls, Akt, bc12 gene family, caspases)
G1 cell cycle arrest of differentiated cells in vitro
spontaneous apoptosis of normal cells in primary culture
characterisation of survival signals antagonising apoptotic pathways
specific regulations of balance of proliferation, differentiation, apoptosis characterising different tissues
reversibility of oncogene-induced alterations of proliferation, differentiation, apoptosis equilibrium
Control of cytokine expression. Identification of cell membrane protein implicated in switching on and off differentiation processes in many tissues. The protein is being cloned and studies on its expression should generate important conclusions. The protein seems to be a major player in controlling cytokine expression.
Potential for treating psoriasis. In Dr Evans group transgenic mice were able to demonstrate switchable skin cell proliferation which modelled human psoriasis. It is conceivable that a human therapeutic approach could result.
liver stem cells indicates strong potential for in vitro culture which differentiates in a very liver-like manner with similar biochemical flexibility. An extra-corporeal liver for treating acute intoxication or other liver damage pending regeneration or transplant is under consideration.
Two cell lines from cells expressing cellular genes:
Osteoblastic line (gene: Telomerase, transfection of human primary cultures )
Hepatic cell line (gene: bc12, derived from transgenic mice)
19 cell lines from SV40 T or t antigen expressing cells;
Six cell lines are currently used for in vitro tests:
T5-CYP - Drug metabolism - Toxicology in Vitro (1999)
T9A4 - Drug metabolism - Publication in preparation
HBG - Drug metabolism - Hepatology, submitted
HCEC - Inflammation, Toxicity, oxidative stress - Gastroenterology, submitted
PKSV-PR- Chemotherapy, drug transport - Cell Biol Toxicol (1999)
Induction of mucociliary differentiation using an air-liquid interface culture model
Regulation of mucociliary differentiation by retinoids and cytokines
Application: in vitro toxicity tests of Diesel exhaust particles (Am J Physiol, 1999)
Induction of late differentiation markers in caco-2 cells by extracellular matrix
Application: studies on glucido-lipidic metabolism
If you are interested in one or more elements of this projects, please contact the IVTIP Secretariat or the project coordinator:
Dr. Christiane Guillouzo
Institut National de la Santé et de la Recherche Medicale
Rue Henri le Guillou
FR - 35033 RENNES CEDEX
Tel: 3399547401
Fax: 3399540137
Email: christiane.guillouzou@rennes.inserm.fr
exchange and adaptation of protocols;
definition of culture conditions; purification of Langerhans cells from human skin;
characterisation of Langerhans progenitor cells. Protocol for integration of Langerhans cells into reconstructed epidermis;
characterisation of Langerhans cells in human reconstructed epidermis,
exploration of the model as an immuno-pharmaco-toxicological tool;
prevalidation of the model for toxicological applications
impossible to reconstitute Langerhans cells into "skin"
reproducibility of Langerhans cell integration following differentiation of progenitor cells (solved?)
reconstitution of "skin" is documented
reaction to UV or DNFB mimics natural skin reaction with migration of Langerhans cells.
UV "sunblockers" inhibit Langerhans cell migration as in natural skin
successful transfer of protocols between laboratories
L´Oréal is a global leader in skin care products and as such directly concerned with application of model
willingness to license this technology (patent protected) to other companies
almost ready to go to ECVAM for validation
If you are interested in one or more elements of this projects, please contact the IVTIP Secretariat or the project coordinator:
Dr. Rainer Schmidt
L´Oréal s.a.
Rue du General Roguet, 90
FR - 92583 CLICHY CEDEX
Tel: 33147567713
Fax: 33147567965
Email:rschmidt@recherche.loreal.com
in vitro culture systems for:
spermatogenic and supporting germ cells
early gametic cells
selected genotoxics for in vitro protocols to assess:
culture progression
markers of genotoxicity
changes in specific gene expression
DNA damage and cytological markers;
comparative analysis in vitro/in vivo. Assessment of fertility and early embryonic damage.
transfer of protocols between groups has proved difficult
transgenic mice models for homologous recombination studies need refinement.
in vitro measurements were more sensitive than the reproductive effects seen in vivo.
cDNA probes for defined genetic markers and quantification of toxicological stress
transgenic mice germ cells carry reporter gene constructs for potential pharmaco-toxicological agents
Results and their potential for industrial applications:
Testicular Sertoli cells (primary culture and 15P1 cells)
Primordial Germ Cells (PGCs)
Spermatogonia
Foetal oocytes
plus co-cultures of seminiferous tubules
mono-ethylhexylphthalate (MEHP)
adriamycin (ADR)
N-ethyl-N-nitrosourea (ENU).
If you are interested in one or more elements of this project, please contact the IVTIP Secretariat for the full report withe results or contact the project coordinator:
Prof. Jesus del Mazo
Consejo superior de Investigaciones
Cientificas
Velazquez 144
ES - 28006 MADRID
Tel: 3415644562
Fax: 3415627518
Email: jdelmazo@cib.csic.es
characterisation of plasma protein and cellular peptide targets for drug conjugation;
antigen presenting cell response to drugs and drug-derived peptides
cytokine expression response of drug-specific human T-cells
derivation cell clones from drug-allergic patients
optimal generation and activation of drug-specific T-cells
development in vitro IgE drug test.
uncertain that B cell switching to IgE production has been demonstrated. IgE clones established in vivo may have produced observed "switching"
not clear that ß-lactam antibiotics stimulate specific IgE corresponding to IgM epitope recognition. Maybe a low level of stimulation of random IgE clones
Antigen generation by Benzyl Penicillin (BP) on serum proteins and white blood cells was characterised in terms of kinetics and concentration-dependence
cytokines represent an important functional target for drug modification
BP conjugates to cell surfaces of white blood cells and dendritic cells (DC). Leads to functional differences in mixed Lymphocyte responses to haptenated DC
BP-responsive T cells from allergic and non-allergic donors characterised by surface marker and cytokine expression
designer composite peptides incorporating BP-haptenated self and a recall T cell epitope demonstrated primary B cell antibody responses and switching to IgE anti-BP in vitro
Transcription factor activation in T and B cells was characterised in primary in vitro antibody responses and class switching to IgE
in vitro diagnostic test for antigen susceptibility - of particular interest to physicians prescribing ß-lactam antibiotics such as penicillin
new approach for generating non-allergenic ß-lactams by rational drug design
potential for developing new immunomodulating drugs
possible company spin-off from research programme
If you are interested in one or more elements of this study or want to receive the full review report, please contact the IVTIP Secretariat or the project coordinator:
Dr. John Coleman
Department of Pharmacology & Therapeutics
University of liverpool
PO Box: 147
GB - L69 3BX LIVERPOOL
Tel: 441517945551
Fax: 441517945540
Email:Coleman@liv.ac.uk
The study received a grant from the European Commission under its Framework Programme 5, Theme 1, Quality of Life and Management of Living Resources, Key Action 3, the Cell Factory.
Project nr. QLK3 - 1999 - 30005