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HEMATOPOIESIS
From the Laboratoire de Thérapie
Génique Hématopoïétique, Institut
Universitaire d'Hématologie, Hôpital Saint Louis,
Paris; and the Laboratoire de Chimie Bioorganique et de Biotechnologie
Moleculaire et Cellulaire, UMR7001 CNRS-ENSCP/Aventis Gencell,
Vitry Sur Seine, France.
High doses of recombinant human erythropoietin (rhEpo) are required
for the treatment of chronic anemia. Thus, it is clear that therapy for
chronic anemia would greatly benefit from an erythropoietin derivative
with increased erythropoietic activity rather than the native
endogenous hormone. In this report, the activity of a human Epo-Epo
dimer protein, obtained by recombinant technology, is described and
compared with its Epo monomer counterpart produced under identical
conditions. Although monomer Epo and dimer Epo-Epo had similar
pharmacokinetics in normal mice, the increase in hematocrit value was
greater with the dimer than with the monomer. Moreover, in clonogenic
assays using CD34+ human hematopoietic cells, the human
dimer induced a 3- to 4-fold-greater proliferation of erythroid cells
than the monomer. Controlled secretion of dimeric erythropoietin was
achieved in Erythropoietin (Epo) is a 34-kd glycoprotein
produced mainly by kidney paratubular cells in response to reduced
oxygen delivery.1,2 Epo stimulates erythroid progenitor
cell proliferation, differentiation, and maturation, and it inhibits
cell apoptosis, which results in increased erythrocyte
production.3-5 The recombinant human erythropoietin
(rhEpo) hormone is widely used to compensate for the reduced production
of endogenous Epo in renal failure and to correct the associated
anemia. Administration of rhEpo alleviates the necessity for blood
transfusion and greatly improves the quality of life for
patients.6-8 Clinical studies have shown that rhEpo can
also be effective in the treatment of other chronic
anemias,9 especially when endogenous Epo levels are
inappropriately low. When injected into Although high doses of Epo may be beneficial for patients with
Epo mimetic peptides18 are small and relatively easy
to produce in large quantity, but their activity is still very low compared with that of native Epo.19 It was found that the
dimerization of 2 Epo mimetic peptides strongly increased their
activity, though it remained inferior to that of native
Epo.20,21 The association of 2 Epo molecules obtained by
either chemical cross-linking or recombinant DNA-mediated fusion of
coding regions resulted in a more stable protein than the native
monomer with an increased in vivo life span.22,23
Based on evidence that the bridging of 2 adjacent Epo receptors
triggers a conformational change that initiates signal
transduction24,25 and that high- and low-affinity sites
for the Epo receptor are present on Epo,26-28 we
hypothesized that the fusing of 2 Epo molecules might confer an
increase in intrinsic activity by providing 2 closely associated
high-affinity domains.
In this study, we report the design and characterization of a
recombinant fusion protein made of 2 human Epo molecules linked by a
peptide linker of 9 amino acids. We show that this dimer has enhanced
erythropoietic activity, both in vitro on primary human erythroid
progenitors and in vivo in normal mouse compared with its monomer counterpart.
The following oligonucleotides were from Genset (Paris, France):
Epo1, 5'-CCTCTAGAGTCGAGCTCGACGG-3'; Epo2,
5'-CGGGATCCCCTGTCCCCTCTCCTGCAT-3'; Epo3,
5'-AACGGGCGCCGCTCCCCCACGCCTCATG-3'; Epo4, 5'-CGGAATTCAGCGCTTCGTACC-3'; Epo6, 5'-CGGGATCCTCTGTCCCCTGTCCTGCA-3'; Epo7,
5'-CGGAATTCTGGACACACCTGGTCATC-3'; Epo8,
5'-CGGAATTCGAGATGGGGGTGCACGAATG-3'; and Epo9,
5'-CATGCACGTGTCTGTCCCCTGTCCTGCAGG-3'.
Dimeric and monomeric mouse erythropoietin-expressing
plasmids
Dimeric and monomeric human erythropoietin-expressing
plasmids
Polymerase chain reactions were performed on 100 ng template with 300 nM primers, 200 µM each dNTP, 5% formamide, 1.5 mM
MgCl2, and 1 U Expand high-fidelity PCR enzyme mix (Roche
Diagnostics, Meylan, France) for 30 cycles of 30-second
denaturation at 94°C, 30-second annealing at 50°C, and 60-second
extension at 72°C.
Hemagglutinin tag-containing constructs
Polymerase chain reactions were performed on 100 ng template, with 300 nM primers, 200 µM each dNTP, 1.5 mM MgCl2, and 1 U Expand high-fidelity PCR enzyme mix (Roche Diagnostics) for 30 cycles of 30-second denaturation at 94°C, 30-second annealing at 55°C, and 90-second extension at 72°C. Erythropoietin-derived proteins Murine C2C12 myoblasts were derived from the skeletal leg muscle of an adult C3H mouse (CERDIC; Sophia Antipolis, France). Nontagged proteins were obtained from culture medium of C2C12 cells cotransfected with the transactivator-encoding plasmid, ptet-tTak (Gibco), and either ptet-hEpoM or ptet-hEpoD. Transfections were performed in 24-well plates with 2.5 µL polyethylenimine (Exgen 500; Euromedex, Souffelweyersheim, France), 100 ng ptet-tTak, and 400 ng pet-hEpo per well, in 200 µL Optimem (Gibco). Two hours after transfection, cells were cultured for 24 hours in Dulbecco modified Eagle medium containing 10% fetal calf serum and were grown in serum-free medium for another 24 hours. Secreted proteins were concentrated 100-fold with Centricon Plus 80 Centrifugal filter devices (Amicon, Beverly, MA). hEpoM-HA- and hEpoD-HA-tagged proteins were obtained as for the nontagged molecules after the transfection of C2C12 cells with the pMHhEpoM-HA and pMHhEpoD-HA plasmids, respectively.EPO assays Epo-derived molecules were assayed either biologically or by enzyme-linked immunosorbent assay (ELISA; R&D Systems, Oxon, United Kingdom; or Medac Diagnostika, Wedel, Germany). For the bio-assay, 2 types of cells were used the Epo-dependent mouse DAE7 cells, derived
from the nonerythroid hematopoietic DA-1(c1.14) cell
line,31 and spleen cells from phenylhydrazine-treated
mice.32 For the DAE7 assay, 3000 cells were seeded into
wells of a microtitration plate and incubated for 3 days with several
dilutions of samples or rhEpo (epoetin  ; Roche
Pharmaceuticals). Cell survival assays were performed 3 days
later using the WST-1 reagent as indicated by the manufacturer (Roche
Diagnostics). Concerning the spleen cells, Epo assays were based on
3H-thymidine incorporation, as described by
Krystal.32
Western blot analysis Samples were submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Hybond C extra membrane (Amersham Pharmacia, France) in methanol-containing buffer. Western blots were probed either with a goat anti-hEpo polyclonal antibody (N-19, Santa Cruz Biotechnology, Santa Cruz, CA, 1/1000) or with an anti-HA tag mouse monoclonal antibody (BAbCO, 1/1000). Horseradish peroxidase-conjugated anti-IgG antibodies were used as secondary antibody. The secondary antibody was either antigoat IgG-POD (1/2000; Sigma, Saint-Quentin Fallavier, France) to detect goat anti-Epo or antimouse IgG-POD (1/1000; Roche Diagnostics) to detect mouse anti-HA. Blotted antigens were detected by chemiluminescence using the Lumi-light Plus Western blotting substrate (Roche Diagnostics).Clonogenic assay Normal human bone marrow cells were subjected to Ficoll gradient (Seromed Biochrom KG, Berlin, Germany). Cells with densities lower than 1.077 g/cm3 were harvested and purified with the Dynal (Compiegne, France) CD34 progenitor cell selection system as specified by the manufacturer.For BFU-e assays, CD34+ hematopoietic cells were plated in methylcellulose-based medium (Methocult H4230; Stem Cell Technologies, Meylan, France) containing 1000 U/mL rhuIL-3 (TEBU; Le Perray en Yvelines, France) and 20 ng/mL rhuSCF (TEBU) at a density of 2.5 × 103 cells/mL per 35-mm Petri dish. Cells were incubated in a fully humidified atmosphere with 5% CO2 in air at 37°C for 14 days in the presence of different amounts of monomeric or dimeric erythropoietin. At day 14, colonies were counted and cells were harvested and cytocentrifuged. Percentages of erythroid cells were ascertained after May-Grünwald-Giemsa staining. Assays were performed in triplicate, and statistical analysis was performed using the 2-tailed Student t test. For late erythroid progenitor assays, 2.5 × 105 to 5 × 105 CD34+ cells were initially grown in 25-cm2 flasks in the presence of BIT 9500 (Stem Cell Technologies), IL-3, SCF, and IL-6 (TEBU) as described.33 Six days later, cells (late erythroid progenitors) were incubated with 1 µg monoclonal CD36 IgGI antibody (clone FA6-152; Immunotech, Marseilles, France) per 106 cells, and CD36+ cells were purified with Dynabeads M-450 goat antimouse IgG (Dynal) as specified by the manufacturer. Five thousand CD36+ cells were plated in semisolid medium (Methocult H4230; Stem Cell Technologies) containing rhuSCF (20 ng/mL) and incubated in a fully humidified atmosphere with 5% CO2 in air at 37°C for 7 days in the presence of different amounts of monomeric (hEpoM) or dimeric (hEpoD) erythropoietin. At day 7, colonies were counted, and cells were harvested and cytocentrifuged. Percentages of erythroid cells were ascertained after May-Grünwald-Giemsa staining. Assays were performed in triplicate, and statistical analysis was performed using the 2-tailed Student t test. In vivo bioactivity Three days before injection, groups of 6 C57/Bl6 mice (Iffa Credo, l'Arbresle, France) were ether-anesthetized for identification. Blood was withdrawn for hematocrit determination. Mice were injected intraperitoneally on days 1, 3, and 5 with human EpoM or EpoD. Resultant hematocrit was determined on day 8 by a standard micro-hematocrit method.In vivo pharmacokinetics Groups of 3 C57/Bl6 mice were injected subcutaneously, intraperitoneally, or intravenously with the same amounts of hEpoD and hEpoM. Plasma Epo levels were determined over a 24-hour period by ELISA (R&D Systems).Long-term and controlled secretion of dimeric erythropoietin in
-Thalassemic mice (Hbb-thal1) were kept in pathogen-free
animal facilities. A mixture of 2 µg ptet-Off (Clontech, Basel, Switzerland) and 20 µg ptet-mEpoD was injected into the leg tibial cranial muscle of 5 -thalassemic mice. Electric pulses were
delivered through external plate electrodes placed on each side of the
leg as described.34 Blood samples were obtained every 2 weeks by retro-orbital puncture under ether anesthesia. Plasma Epo
concentrations were measured by ELISA (R&D Systems). When specified,
tetracycline (Research Organics, Cleveland, OH) was added to the
drinking water at a final concentration of 1 mg/mL in 2.5% sucrose.
Statistical analysis Hematologic data are expressed as the mean ± SD. For each mouse group, discrete variables were compared by using a 2-tailed Student t test. Results were considered significant at P < .05.
Characterization and activities of Epo dimer and monomer C2C12 myoblasts were transfected with ptet-hEpoM (monomer) or ptet-hEpoD (dimer), together with ptet-tTAk. Two days after transfection, serum-free culture medium was harvested and concentrated. Human monomer and dimer were analyzed by SDS-PAGE and characterized with a polyclonal anti-Epo antibody raised against an N-terminal human Epo fragment. As expected, 2 distinct Epo immunoreactive species could be distinguished, with molecular weights of approximately 35 kd for the monomer and 70 kd for the dimer (not shown), which correspond to the glycosylated forms of erythropoietin. Epo bioactivity of the 2 molecules was initially assayed on the Epo-responding cell line DAE7 and on mouse spleen cells and was compared to rhEpo standards. No difference was found between the 2 in vitro bioassays. Based on these results, the Epo activities were measured by ELISA using tests from 2 different manufacturers (R&D Systems; Medac). Intrinsic activities of monomer and dimer were equivalent to those observed by proliferation assay with the Medac ELISA. With the R&D ELISA, the intrinsic activity of the human dimer was 5 times greater than that observed for its monomer counterpart. The affinity of the anti-Epo antibodies might have been different for the monomer and the dimer because of the presence of 1 or 2 epitopes; hence, ELISA values might simply not have reflected the relative number of Epo domains in the 2 molecules.To determine the concentration of human monomer and dimer in cell
culture supernatants more accurately, C-terminal HA-tagged human Epo
dimer and monomer were evaluated. Indeed, in these molecules, only one
immunoreactive epitope was detected. Epo values were determined by DAE7
proliferation assay and by ELISA calibrated with rhEpo. As for the
nontagged molecules, R&D ELISA overestimated the tagged dimer by a
factor of 5. Different concentrations of HA-tagged monomer and dimer
were subsequently run on SDS-PAGE, submitted to Western blot analysis,
and revealed by a monoclonal anti-HA antibody. As shown in Figure
2, similar intensities could be detected
when 5 times more dimer than monomer (as determined by R&D ELISA) were
transferred to the Western blot. These results confirmed that the R&D
ELISA overestimated the dimer by a factor of 5 compared to the monomer.
This means that the dimer is as effective as the monomer in inducing
DAE7 and spleen cell proliferation. Because of these results,
subsequent experiments (in vitro and in vivo) were performed using
equal amounts of monomer and dimer, as determined by proliferation
bioactivity.
In vivo erythropoietic activity of monomeric and dimeric hEpo Two groups of mice were injected intraperitoneally with 300 U/kg (as determined by proliferation assays) of human Epo-derived molecules on days 1, 3, and 5. Hematocrit values measured 8 days after the first injection were compared with values obtained 3 days before injection (Figure 3A-B). Mean hematocrit values were slightly raised, from 49.6% ± 1.1% and 49.4% ± 2.9% to 51.6% ± 1.5% and 54.4% ±2.4% for the monomer and the dimer, respectively. The mean increase was significantly higher (P < .05) for the dimer (5.0% ± 2.2%) than for the monomer (2.0% ± 1.9%).
To compare the relative in vivo efficacy of the 2 molecules, 5 groups
of mice were injected with 300, 900, and 1800 U/kg monomer or 300 and
600 U/kg dimer (Figure 4). The mean
hematocrit increase was significantly higher with 300 U/kg dimer
(3.6% ± 1.4%) than with 300 U/kg monomer (1.1% ± 1.4%;
P = .01) and 900 U/kg monomer (1.6% ± 1.5%;
P = .04) but was not significantly different between 1800 U/kg monomer and 300 U/kg dimer. The mean hematocrit increase was
significantly higher with 600 U/kg dimer (5.5% ± 1.1%) than with
all other groups (P = .000004 for NaCl;
P = .00001 for 300 U/kg monomer; P = .0005
for 900 U/kg monomer; P = .01 for 1800 U/kg monomer;
P = .03 for 300 U/kg dimer). Based on these results, it
appeared that the dimeric human Epo is approximately 6 times more
active than the monomer on a per mole basis and approximately 3 times
more active on a per weight basis.
Pharmacokinetics of monomeric and dimeric hEpo Six groups of mice were injected intraperitoneally, intravenously, or subcutaneously with monomeric and dimeric human Epo (200 U/kg as determined by proliferation assay). Blood samples (100 µL) were obtained either 15 minutes or 1, 2, 4, or 8 hours after intraperitoneal or intravenous injection or, alternatively, after 2, 8, and 24 hours after subcutaneous injection. Because the R&D ELISA is not sensitive to mouse erythropoietin, human erythropoietin level was determined by this assay. Noninjected control mice were included in the study to determine whether endogenous mouse erythropoietin induced by the bleedings might have interfered with the ELISA measurement. At 8 hours, endogenous mouse erythropoietin was indeed detectable in mice bled at 15 minutes and at 1, 2, 4, and 8 hours after injection. Therefore, results are given only for the first 4 hours. When mice were bled only 3 times (after 2, 8, and 24 hours), no endogenous erythropoietin was detected. Results are given in Figure 5 as the percentage of the maximal values, observed 15 minutes, 1 hour, or 2 hours after intravenous (Figure 5A), intraperitoneal (Figure 5B), or subcutaneous (Figure 5C) injection, respectively. As shown, the erythropoietin kinetics did not differ between the 2 groups and decreased to half the maximum value in approximately 50 minutes, 3 hours, and 7.5 hours after intravenous, intraperitoneal, or subcutaneous injection, respectively.
Erythroid activity of monomeric and dimeric hEpo Erythropoietic activities of monomer and dimer were evaluated on human CD34+ hematopoietic progenitors. This was performed by counting the total number of erythroid cells after 2 weeks of culture in the presence of monomeric and dimeric human Epo molecules, at various concentrations. An example of such an experiment performed in triplicate is shown in Figure 6. Whereas the number of recruited BFU-e was not significantly different between monomer and dimer (Figure 6A), 0.4 to 0.6 U/mL human dimer induced a 3- to 4-fold increase (P < .02) of the total number of erythroid cells (Figure 6B). At higher Epo concentrations (greater than 1 U/mL), the maximum number of erythroid cells was reached by using both monomer and dimer (not shown).
In semisolid medium, a 5-fold increase in the number of colonies from
late erythroid progenitors (CFU-e) was observed in the presence of 0.1 U/mL dimer compared to the value observed in the presence of the same
concentration of monomer (Figure 7A). At a higher erythropoietin concentration (0.4 U/mL), no difference in the
number of colonies could be observed in the presence of either monomer
or dimer, suggesting that the Epo receptors were saturated by
erythropoietin. In this assay, the increase in the total erythroid cell
number (Figure 7B) was similar to the increase in the number of
colonies, indicating that the increased effect of the dimer occurred
mostly at the CFU-e level.
Epo dimer and hematocrit increase in -thalassemic mice and would induce
antibodies, dimeric mouse erythropoietin was produced in vivo. The
mouse erythropoietin dimer encoding plasmid ptet-mEpoD and the
tetracycline-controlled transactivator encoding plasmid
pCMV-tTA35 were co-electrotransferred in the tibial
cranial skeletal muscle of -thalassemic mice. Erythropoietin was
measured during 26 weeks by R&D ELISA, and hematocrit changes were
observed for a period of 38 weeks. Tetracycline was added to the
drinking water from weeks 4 to 8, 16 to 20, and 26 to 29.5. Circulating
Epo immunoreactivity was high during the first 2 weeks after
electrotransfer and then slightly decreased (Figure
8B). This peak and then slight decrease
in erythropoietin production has also been observed with a pCMV-mEpo
plasmid constitutively expressing erythropoietin in -thalassemic
mice.36 As expected, tetracycline blocked erythropoietin
secretion (Figure 8B) and reduced hematocrit to values similar to those
for untreated -thalassemic mice (Figure 8A). Thirty-eight weeks (9 months) after electrotransfer, tetracycline withdrawal still led to an
increase in hematocrit levels. These results suggest that the partial
decline of erythropoietin expression observed 1 to 3 weeks after
injection was not due to the presence of antibodies raised against the
mouse erythropoietin dimer but to a slight variation of expression from
the injected plasmid. The long-lasting hematocrit increase observed as
late as 9 months in the absence of tetracycline further confirmed the absence of an antibody response against dimeric Epo.
We have designed and characterized a dimeric Epo-Epo protein obtained by recombinant DNA-mediated fusion of Epo coding regions linked by the Gly-Ser-Gly4-Ser-Gly-Ala peptide. Based on the working hypothesis that Epo-Epo fusion may trigger the conformational change of the Epo receptor to its active state, this study has investigated the intrinsic activity of the Epo-Epo dimer in vitro and in vivo after gene transfer into mice. Dimeric and monomeric forms of human and mouse recombinant Epo and their HA-tagged homologs were produced by C2C12 mouse myoblast cells, known to be efficient for transgenic Epo production both in vitro and in vivo.37,38 The activity of the monomeric and dimeric forms of Epo was compared in vitro on a mouse cell line (DAE7), on phenylhydrazine-induced mouse spleen cells, on primary human erythroid cells, and in vivo in normal mice. Equivalent numbers of Epo molecules, as determined by Western blot analysis using the antitag antibodies, had the same proliferative activity in vitro when added to DAE7 or spleen cells. In contrast, ELISA yielded different results. Whereas Medac ELISA findings were in agreement with the activity determined by proliferation assay, R&D ELISA findings overestimated the dimeric Epo by 5-fold. Dimeric Epo most likely had a higher binding affinity for the antierythropoietin antibody than monomeric Epo. Similar differences in biologic and immunologic activities of human monomeric and dimeric Epo have already been described.39 DAE7 proliferation assay calibrated with human recombinant Epo
determined that the Epo dimer induced a 6-fold-higher increase in
hematocrit compared with the monomeric form when injected on days 1, 3, and 5 in normal mice at concentrations of 300 U/kg. A similar effect on
hematocrit has been observed with chemically linked Epo dimer injected
in rabbits22 and with an Epo-Epo fusion protein injected
in mice.23 It has been postulated that this effect is
mainly the result of an enhanced blood lifetime of the dimeric form.
However, identical pharmacokinetics of monomeric and dimeric Epo
injected through different routes To understand the hematocrit increase induced by the dimeric Epo when compared to its monomeric counterpart, human CD34+ cells were studied with various concentrations of dimeric and monomeric Epo forms. Although a small relative increase in the number of BFU-e induced by the Epo dimer was observed, this trend was not significant, and this contrasted with the severalfold increase in the total erythroid cell number above that was induced by monomeric Epo (Figure 6). This difference in the increase in erythroid cell number was parallel to the increase in the CFU-e (Figure 7), indicating that dimeric Epo stimulated the late progenitors or prevented their apoptosis. Stem cell factor added to the culture medium, which has a major erythropoietic-stimulating activity on the early stages of erythroid differentiation,40,41 might have masked the effect of dimeric Epo, if any, on the recruitment of late BFU-e. One important point is the parallel between the increase in the erythropoietic activity induced by the Epo dimer added in vitro and the increased hematocrit induced by the Epo dimer in normal mice. Erythropoietin binds to the Epo receptor through 2 binding domains, one with high affinity for the receptor and the other with low affinity; the latter is required for the activation of the receptor.27,28 When the low-affinity binding moiety of Epo is mutated, the Epo receptor is no longer activated. However, when mutated Epo monomers are linked together by a peptide containing 7-glycine residues, the presence of a second high-affinity binding motif in the dimeric Epo molecule restores the erythropoietic transduction activity.39 These results suggest that the presence of 2 molecules of Epo might bring together 2 high-affinity binding sites and facilitate binding to the Epo receptor. A 5-fold increase in the number of erythroid cells was detected when erythroid progenitors were induced by the dimeric Epo form. This contrasted with the similar proliferation activity of the dimeric and monomeric Epo forms in DAE7 cells and phenylhydrazine-induced spleen cells (present results) or UT7 cell lines.39 These differences suggest that the environment of the Epo receptor at the cell surface or that the various components of the culture medium (present in fetal calf serum or added factors) might have modified the binding or the transduction activity of the dimeric Epo in comparison with the monomeric form. The Epo dimer did indeed induce a hematocrit increase in normal mouse, which was 6-fold higher than with the Epo monomer, even though there was no difference in erythropoietin pharmacokinetics. This provides further evidence that the dimer had a specific erythroid activity that was higher (approximately 6 times on a per mole basis and 3 times higher on a per weight basis) than the monomer. Long-term expression of mouse Epo dimer in In conclusion, we have observed an increase in the biologic specific activity of an Epo dimer in comparison with the activity of the Epo monomer. This increase in activity was shown in vitro on primary erythroid cells and in vivo in mice. Thus, the presently proposed Epo dimer could reduce the amount of therapeutic Epo required for the treatment of various chronic anemias. In addition, the availability of a more active Epo molecule could be useful for the development of Epo-based gene therapy approaches.
We thank E. Turpin for the sequencing reactions, L. Michel for spleen cell proliferation measurement, P. Leboulch for helpful discussion and critical reading of the manuscript, and Dr Ferrero for careful reading and correction of the manuscript.
Submitted December 28, 2000; accepted February 28, 2001.
Supported by the Institut National de la Santé et de la Recherche Médicale (INSERM). E.P. was supported by the Fondation de France and INSERM. M.B. was supported by the Ligue Nationale Contre le Cancer.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Emmanuel Payen, Laboratoire de Thérapie Génique Hématopoïétique, Institut Universitaire d'Hématologie, Hôpital Saint Louis, 1, avenue Claude Vellefaux, 75475 Paris Cedex 10; e-mail: letg{at}chu-stlouis.fr.
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Abstract
Introduction
3) and 8 days after (D8) the first injection. Values are
given for each mouse.
