WO2003106665A1 - Procede de culture in vitro du virus vhc - Google Patents
Procede de culture in vitro du virus vhc Download PDFInfo
- Publication number
- WO2003106665A1 WO2003106665A1 PCT/FR2003/001820 FR0301820W WO03106665A1 WO 2003106665 A1 WO2003106665 A1 WO 2003106665A1 FR 0301820 W FR0301820 W FR 0301820W WO 03106665 A1 WO03106665 A1 WO 03106665A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- cells
- lipoproteins
- medium
- synthesis
- virus
- Prior art date
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Classifications
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- C—CHEMISTRY; METALLURGY
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- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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- A—HUMAN NECESSITIES
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- C—CHEMISTRY; METALLURGY
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/24011—Flaviviridae
- C12N2770/24211—Hepacivirus, e.g. hepatitis C virus, hepatitis G virus
- C12N2770/24251—Methods of production or purification of viral material
Definitions
- the present invention relates to a new method of in vitro culture of the hepatitis C virus.
- Hepatitis C is the main cause of transfusion-acquired hepatitis. Hepatitis C can also be transmitted by other percutaneous routes, for example by injecting drugs intravenously. The risk of contamination of health professionals is also not negligible.
- Hepatitis C is distinguished from other forms of liver disease associated with viruses, such as hepatitis A, B or D.
- Hepatitis C virus (HCV) infections are often chronic, resulting in diseases of the liver.
- liver such as hepatitis, cirrhosis and carcinoma in a large number of cases.
- HCV hepatotropic virus isolated using molecular biology techniques. The viral genome sequences were cloned without the viral particles being viewed.
- virus in the present application any particle containing RNA of the HCV virus.
- the two terms will also be used interchangeably.
- HCV is a positive single-stranded RNA virus, approximately 9.5 kb, which replicates with a copy of complementary RNA and the translation product of which is a single polyprotein of approximately 3,000 amino acids.
- the 5 'end of the HCV genome corresponds to an untranslated region adjacent to the genes which code for structural proteins, the core protein of the nucleocapsid and the two envelope glycoproteins, E1 and E2.
- the 5 'untranslated region and the core gene are relatively well conserved in the various genotypes, but the E2 envelope proteins are encoded by a hypervariable region different from one isolate to another isolate.
- the 3 'end of the HCV genome contains genes that code for non-structural proteins (NS) and for a well-conserved 3' non-coding region.
- NS non-structural proteins
- HCV Because of its genomic organization and its supposed mode of replication, HCV has been classified into a new genus in the family of Flaviviridae, hepaci viruses.
- HCV virus refers to any viral species, including strains pathogenic to humans, attenuated strains and defective strains derived from said strains. Indeed, it is known that RNA viruses have a high rate of spontaneous mutations. There may therefore be multiple strains which may be more or less virulent. It is within the ability of those skilled in the art to identify such strains, for example by homology of nucleic and / or peptide sequences with respect to a reference strain and / or by identifying a strain or an isolate with respect to to morphological and / or immunological criteria.
- diagnostic immunoassays have been performed to detect antibodies to HCV proteins in patient sera.
- Synthesis of cDNA by reverse transcription of viral RNA and amplification by PCR. have also been used to detect the HCV genome, as an indirect measure of a potentially infectious virus in the sera of chronically infected humans or those of experimentally infected chimpanzees.
- hybridization techniques with a DNA probe have also been developed.
- Patent application WO01 / 09289 describes a method of in vitro culture of viruses, such as HCV, from at least a fraction of LVPs obtained from serum or plasma from an infected patient, using cells which have an endocytosis pathway relayed by at least one lipoprotein receptor and modulated by an activating agent. This process is especially concerned with promoting the entry of fractions into cultured cells.
- replication of the virus remains limited and must therefore be optimized.
- Patent application WO02 / 10353 describes complexes consisting of LVPs associated with human immunoglobulins, of density less than or equal to 1.063 g / ml, and containing mainly the RNA of the HCV virus, contrary to known data (Hijikata et al., 1993, J. Viral., 1953-1958). Indeed, Hijikata et al. showed that a high infectivity in the chimpanzee was found in the presence of particles of density less than 1.06 g / ml so that there could not be human immunoglobulins, very dense, in this type of particles low density. These complexes constitute a particular form of the HCV virus which is very infectious.
- This patent application also describes a method of in vitro culture of the HCV virus from the LVP / immunoglobulin complexes, using cells comprising on their surface at least one type of receptor for the Fc fragment of immunoglobulins or one type of receptor having the capacity to bind immunoglobulins.
- this process promotes the entry of the complexes into the culture cells but it does not allow an abundant multiplication of the virus.
- the present inventors have now found a new method for in vitro culture of the HCV virus which makes it possible to solve the above drawback, namely that it uses cells capable of both bringing in the particles containing the HCV RNA and improve ensure replication and production.
- - are globally spherical unit particles; they are therefore not agglomerates of virions linked to normal lipoproteins, as had been suggested in the prior art, - contain apolipoproteins B and E and triglycerides: they therefore have a lipoprotein structure, and in particular of the VLDL or chylomicron type, and
- RNA of the virus contain the capsid and the RNA of the virus: they therefore differ from normal lipoproteins found in humans and constitute an unconventional form of the virus.
- LVPs are hybrid particles, i.e. lipoproteins containing viral components, replication can be unexpectedly improved by using cells capable of synthesizing lipoproteins.
- the subject of the present invention is a method of in vitro culture of the HCV virus comprising the steps consisting in bringing into contact particles containing the hepatitis C virus RNA with cells having the capacity to synthesize and secrete lipoproteins. , in an appropriate culture medium promoting the synthesis and secretion of lipoproteins, and in harvesting the virus thus obtained.
- particles containing the HCV RNA is meant viral particles, such as in particular in the form of an LVP / immunoglobulin complex or present in the serum or the plasma of patients detected positive for HCV and containing such particles, and any inoculum containing viral RNA, regardless of the mode of preparation of viral RNA in said inoculum.
- Apolipoprotein B is a human protein associated with the membrane of the endoplasmic reticulum and which initiates the assembly of VLDLs, the introduction of triglyceride in the presence of the microsomal triglyceride transfer protein, as well as the assembly of VLDL in the lumen of the endoplasmic reticulum.
- This apolipoprotein is delivered in two forms, apo B 100 and apo B 48, and is synthesized in the liver and the intestine.
- Apolipoprotein E can be synthesized by numerous cells and in particular by hepatocytes. Furthermore, it can be acquired by VLDLs in the blood stream.
- the cells used in the method of the invention are all cells having the capacity to synthesize and secrete lipoproteins.
- These cells can be either primary cells or cell lines.
- cell line refers to established lines, immortalized spontaneously or by manipulation. In practice, to carry out a viral culture of interest, it is necessary to have permissive cells easily maintained in culture.
- the cell line is therefore preferably an established cell line or which results from immortalization by different methods.
- a cell line for example an immortalized B cell line, for example with the Epstein-Barr virus
- cells having the capacity to synthesize and secrete lipoproteins used in the process of the invention is understood to mean cells having spontaneously this capacity and cells having acquired this capacity after induction in a culture medium promoting the differentiation or redifferentiation of cells or after transfection of genes for the synthesis of apolipoprotein apo B and of the microsomal protein for the transfer of triglycerides MTP.
- Examples of cells having the ability to synthesize and secrete lipoproteins suitable for the purposes of the invention include enterocyte-type intestinal epithelium cells, brain cells and liver cells. According to one embodiment, the cells used are cells which have acquired the capacity to synthesize and secrete lipoproteins after induction in a medium promoting the differentiation or redifferentiation of cells.
- liver cells have lost their ability to produce lipoproteins or other cells, such as the cells of the intestinal epithelium, have an ability that can be improved.
- the cells suitable for the purposes of the invention are the cells of the intestinal epithelium, and in particular the cells
- Caco-2 (Van Greevenbroeck, M.M.J., et al., 2000, Atherosclerosis, 25-31).
- the cells of the intestinal epithelium can be previously cultured for 3 weeks with DMEM medium supplemented with 10% fetal calf serum, in boxes covered with collagen or under semi-permeable membranes.
- liver cells examples include hepatocytes (Moshage, H., et al., 1992, Journal of Hepatology, 15, 404-413) and hepatocarcinomas such as Hep G2 cells (Gherardi, E ., et al., 1992, Journal of Cell Science, 103, 531-539).
- the cells used in the method of the invention are hepatocarcinoma cells.
- these cells are Hep G2 cells.
- lipids such as fatty acids, preferably C 18 or C 20 , for example oleate (Luchoomun, J., et al., 1999, The Journal of Biological Chemistry, Vol
- oleate in the medium for redifferentiation of hepatocarcinomas constitutes a particular embodiment of the invention.
- the modified DMEM medium consists, in addition to DMEM (Gibco BRL) and an agent inducing lipoprotein synthesis, 1% HEPES (Gibco), 1% glutamine (Gibco ), gentamycin
- Another type of cell which can be used in the process of the invention consists of cells obtained after transfection of genes for the synthesis of apolipoproteins apo B and optionally apo E and of the microsomal protein for the transfer of triglycerides.
- transfected insect cell lines as described by Gretch, D.G., et al. in The Journal of Biological
- the particles containing the HCV RNA when they are brought into contact with cells having the capacity to synthesize and secrete lipoproteins, enter these cells either via the lipoprotein receptors of said cells, of the LDL receptor type, as in the case of LVP for example, or by internalization by transfection according to techniques known to those skilled in the art, as in the case of preparations of viral RNA for example.
- the culture medium used in the process of the invention is such that it promotes the synthesis and the secretion of lipoproteins, preferably of VLDL or chylomicron type.
- a suitable medium retained is a modified DMEM medium supplemented with agents promoting the metabolism of the cell in culture, as well as with at least one agent inducing the synthesis of lipoproteins.
- dexamethasone and insulin agents promoting the metabolism of the cell.
- a preferred modified DMEM medium is as defined above and supplemented with dexamethasone and insulin.
- agents inducing the synthesis of lipoproteins mention may be made of different lipids such as fatty acids, preferably C 18 or C 0 , for example Poleate, phospholipids such as lysophosphatidylcholine, as well as 22 or 25 OH -cholesterol.
- the use of the oleate as an agent inducing the synthesis of lipoproteins, optionally in combination with 22 or 25 OH-cholesterol, constitutes another particular embodiment of the invention.
- the virus thus cultivated by the method of the invention can be harvested by various methods such as centrifugation, for example on a density gradient, and immunoprecipitation using anti-apo B and / or anti-apo E antibodies.
- the invention also relates to a diagnostic composition
- a diagnostic composition comprising at least the viral particles obtained according to the method of the invention or one of its components as a source of antigen.
- the invention also relates to a method for screening and / or selecting at least one antiviral molecule, comprising the step of bringing said antiviral molecule into contact in the culture medium during the culture method of the invention.
- the selection of the antiviral molecules is carried out by techniques well known to those skilled in the art such as the reduction in the number of intracellular viral RNA and / or of infectious viral particles secreted in the supernatant in the presence of variable concentrations of the various inhibitors.
- inhibitors can be nucleotide or nucleoside analogs, inhibitors of viral proteases or other molecules interfering with the functions of other viral proteins.
- the invention also opens up other therapeutic perspectives in that it makes it possible to develop a therapeutic composition capable of influencing in a qualitative and / or quantitative manner the propagation and the in vivo replication of HCV, which is characterized in that it comprises inter alia an agent capable of modulating, repressing or inhibiting the synthesis of lipoproteins, such as for example an inhibitor of the microsomal protein for the transfer of triglycerides.
- agent capable of modulating, repressing or inhibiting the synthesis of lipoproteins means any molecule making it possible, respectively, to control, reduce or suppress the propagation and replication in vivo of HCV.
- agents can be selected by screening from a pool of molecules having recognized pharmacological activity on lipid metabolisms.
- FIG. 1 represents a graph showing the influence of culture conditions (modified DMEM medium plus oleate compared to the standard medium) of Hep G2 cells on the secretion of the HCV viral particles, secretion evaluated by the quantification of the viral RNA in the culture supernatant as a function of time,
- FIG. 2 represents a graph showing the influence of the addition of oleate in the modified DMEM culture medium on the secretion of the viral particles evaluated by the quantification of HCV RNA in the culture supernatant as a function of time
- FIG. 3 shows a graph showing the production of the HCV virus by Caco-2 cells, after differentiation for 3 weeks of culture, said production being demonstrated by the number of viral RNA in the culture supernatant as a function of time .
- the low density fraction (LDL) freed of the purified LVPs was diluted, as well as the purified LVPs in PBS and were visualized using an electron microscope (JEOL device, Laennec Joint Imaging Center, Lyon, France) after floating drops of the sample on 200 mesh copper grids coated with a Formvar support film (Electron Microscopy Science, PA) for 3 min at room temperature, colored for 3 min by flotation on a medium of photungstic acid at 4% (mass / vol), buffered to pH 7.2 with NaOH, then dried.
- JEOL device Laennec Joint Imaging Center, Lyon, France
- the LDL fraction consisted of particles of homogeneous spherical structure, with an average diameter of 25 nm, in agreement with normal LDL. In contrast, the purified LVPs were unusually large spherical structures, with an average diameter of 100 nm.
- the total cholesterol, phospholipid and triglyceride concentrations were determined using the Cholesterol RTU, Phospholipids Enzymatic PAP 150 and Triglyceride Enzymatic PAP 150 kits (bioMérieux, Marcy l'Etoile, France) according to the manufacturer's recommendations, and in establishing standard curves. b) Determination of the apo B concentration of the LVPs
- the apolipoprotein apoB concentration in the purified LVPs was determined using an ELISA assay. To do this, 96-well flat bottom ELISA plates (Maxisorb; Nunc) were coated with 100 ⁇ l of human anti-apo B monoclonal antibody.
- the LVPs which are found in the two fractions of different density, namely low and very low density, contain more triglyceride per apo B molecule than normal lipoproteins of the same fractions.
- the LVPs contain many lipids, - the lipid concentration of the LVP is different from that of normal lipoproteins; this therefore excludes any contamination, and
- - LVP contains apo B.
- PLC / PFR / 5 (ATCC CRL 8024) (Alexander Cells) (Human Hepatoma) in 96-well plates (Maxisorb, Nunc?)
- DMEM culture medium Gibco, BRL
- 10% "of fetal calf serum Biowhittaker, Emerainville, France
- 2mM HEPES Gibco / BRL
- 1% non-essential amino acids Gibco / BRL
- 50 IU of penicillin / streptamycin (Gibco / BRL) / ml at 37 ° C.
- the apo B receptor binding sites have been blocked with monoclonal antibodies directed against the apo B receptor binding site (4G3 and 5 B 11; Ottawa Heart Institute Research Corporation, Ottawa, Ontario Canada ) and on the other hand the binding sites to the apo E receptors with monoclonal antibodies 1D7 (Ottawa Heart Institute Research Corporation).
- the PLC cells were washed three times with PBS and incubated for 3 h with purified LVP. The cells were washed, then they were harvested in 350 ⁇ l of lysis buffer from the Rneasy kit (Qiagen) and the RNA was extracted as indicated above.
- Blocking the recognition of LVP by blocking the recognition sites of apo B and E indicates that they contain apolipoproteins and highlights the lipoprotein structure of LVP.
- the presence of the capsid was demonstrated by delipidating the purified LVPs as follows: the LVPs were incubated for 30 min while stirring gently, in a solution of 85%> ether-15% butanol. The presence of the capsid was visualized using an electron microscope (JEOL device, Center Commun d'Imagerie de Laennec, Lyon, France) as indicated in point 1.1 above.
- the presence of the core protein of the HCV virus was confirmed by Western blot by contacting the delipidated LVPs as described above, with anti-HCV core protein monoclonal antibodies (19D9D6; Jolivet-Reynaud, CP, et al. , 1998, J. Med. Virol., 56, 300-309) and secondary antibodies labeled with 10 nm gold and visualization using the grids indicated above by immunodetection after negative staining by flotation on l uranyl acetate 3%.
- Hep G2 cells were cultured either in a medium consisting of DMEM and 10% fetal calf serum (standard medium), as in patent application WO01 / 09289, or in a modified DMEM medium, c ie containing DMEM supplemented with PHEPES 1%, glutamine 1%, gentamycin 0.25 mg / ml, PUloser-G 1.5%, Forskoline 5.10 "6 M, PMA 1.6.10 "7 M, retinol acetate 5.6 IU / ml, sodium butyrate 0.5.10 " M, niacidamide 10 " M, polybrene 2.10 " 6 g / ml, sodium selenite 2.9.10 “8 M and triiodo-L-thyronine sodium l.lO " 9 M.
- the medium was aspirated and the cells were incubated in the presence of the virus, at the rate of 500,000 HCV RNA per well, for 6 h using DMEM supplemented with 0.2% BSA.
- the cells were then washed with PBS and cultured either in standard medium or in modified medium, as indicated above, but also supplemented with hexamethasone and insulin and a mixture of oleate and BSA 0.15 mM oleate.
- Hep G2 cells were first cultivated for 24 h in a modified medium as described in Example 2 above, and they were added to the wells of the Falcon 24 well culture plates at a rate of 150 000 cells per well.
- the cells were then incubated in the presence of the virus, at a rate of 400,000 HCV RNA per well, for 6 h in DMEM medium supplemented with 0.2% BSA alone or with 0.1 ⁇ M of 25 OH cholesterol. The cells were then washed as indicated in Example 2 above and cultured in a modified medium.
- Caco-2 cells are differentiated on semi-permeable membranes (Transwell, Costar) contained in 24-well plates, for 3 weeks in standard medium (DMEM supplemented with 10% fetal calf serum).
- the cells thus prepared were incubated with the viral particles at the rate of 200,000 HCV RNA / well for 6 h.
- the cells were washed and cultured in DMEM medium supplemented with 10% o fetal calf serum and 0.15 mM oleate-taurocholate.
- FIG. 3 highlights the culture of the HCV virus by cells of the intestinal epithelium in a medium which promotes the synthesis and secretion of lipoproteins.
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Abstract
Description
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Priority Applications (4)
Application Number | Priority Date | Filing Date | Title |
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AU2003260593A AU2003260593A1 (en) | 2002-06-18 | 2003-06-16 | Method for culturing hvc virus in vitro |
JP2004513478A JP2006500918A (ja) | 2002-06-18 | 2003-06-16 | invitroにおけるHCVウイルスの培養方法 |
EP03760045A EP1513930A1 (fr) | 2002-06-18 | 2003-06-16 | Procede de culture in vitro du virus vhc |
US10/515,381 US20050221464A1 (en) | 2002-06-18 | 2003-06-16 | Method for culturing hcv virus in vitro |
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FR02/07600 | 2002-06-18 | ||
FR0207600A FR2840921B1 (fr) | 2002-06-18 | 2002-06-18 | Procede de culture in vitro du virus vhc |
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EP (1) | EP1513930A1 (fr) |
JP (1) | JP2006500918A (fr) |
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US7645732B2 (en) * | 2007-01-24 | 2010-01-12 | Board Of Regents, The University Of Texas System | Treating hepatitis C virus infection |
WO2008124384A2 (fr) * | 2007-04-03 | 2008-10-16 | Aegerion Pharmaceuticals, Inc. | Méthodes de traitement de l'hépatite c |
Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2001009289A1 (fr) | 1999-07-30 | 2001-02-08 | Bio Merieux | Procede de culture in vitro de virus des familles togaviridae et flaviviridae et applications |
WO2002010353A1 (fr) * | 2000-07-31 | 2002-02-07 | Bio Merieux | Complexe de lipo-viro-particules, procede de preparation et applications |
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2002
- 2002-06-18 FR FR0207600A patent/FR2840921B1/fr not_active Expired - Fee Related
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2003
- 2003-06-16 AU AU2003260593A patent/AU2003260593A1/en not_active Abandoned
- 2003-06-16 CN CNB038140926A patent/CN100500836C/zh not_active Expired - Fee Related
- 2003-06-16 EP EP03760045A patent/EP1513930A1/fr not_active Withdrawn
- 2003-06-16 JP JP2004513478A patent/JP2006500918A/ja active Pending
- 2003-06-16 US US10/515,381 patent/US20050221464A1/en not_active Abandoned
- 2003-06-16 WO PCT/FR2003/001820 patent/WO2003106665A1/fr active Application Filing
Patent Citations (2)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2001009289A1 (fr) | 1999-07-30 | 2001-02-08 | Bio Merieux | Procede de culture in vitro de virus des familles togaviridae et flaviviridae et applications |
WO2002010353A1 (fr) * | 2000-07-31 | 2002-02-07 | Bio Merieux | Complexe de lipo-viro-particules, procede de preparation et applications |
Non-Patent Citations (7)
Title |
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ANDRÉ P. ET AL.: "Characterization of Low- and Very-Low-Density Hepatitis RNA-containing particles", J. VIROL., vol. 76, no. 14, July 2002 (2002-07-01), pages 6919 - 6928, XP002243035 * |
DASHTI N. & WOLFBAUER G.: "Secretion of lipids, apolipoproteins and lipoprotiens by human hepatoma cell line, HepG2: effects of oleic acid and insulin", J. LIPID RES., vol. 28, 1987, pages 423 - 436, XP002243038 * |
DASHTI N; WOLFBAUER G, J. LIPID RES., vol. 28, 70319, pages 423 - 436 |
LENTZ K.A. ET AL.: "Development of a more rapid, reduced serum culture system for Caco-2 monolayers and application to the biopharmaceutics classification system", INT. J. PHARMACEUTICS, vol. 200, 2000, pages 41 - 51, XP002243037 * |
MOBERLY ET AL: "Oleic acid stimulation of apolipoprotein B secretion from HepG2 and Caco-2 cells occurs post-transcriptionally", BIOCHIMICA ET BIOPHYSICA ACTA, AMSTERDAM, NL, vol. 1, no. 1042, 1990, pages 70 - 80, XP002073234, ISSN: 0006-3002 * |
VAN GREEVENBROEK M.M.J. ET AL.: "Caco-2 cells secrete two independent classes of lipoproteins with distinct density: effect of the ration of unsaturated to saturated fatty acid", ATHEROSCLEROSIS, vol. 149, 2000, pages 25 - 31, XP002243036 * |
VAN GREEVENBROEK MMJ ET AL., ATHEROSCLEROSIS, vol. 149, 21220, pages 25 - 31 |
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US20050221464A1 (en) | 2005-10-06 |
CN100500836C (zh) | 2009-06-17 |
EP1513930A1 (fr) | 2005-03-16 |
CN1662646A (zh) | 2005-08-31 |
JP2006500918A (ja) | 2006-01-12 |
AU2003260593A8 (en) | 2003-12-31 |
FR2840921B1 (fr) | 2008-07-18 |
FR2840921A1 (fr) | 2003-12-19 |
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