From the a Centre de Recherche en Infectiologie and Département de Biologie Médicale, i Unité de Recherche en Neuroscience and Département de Médecine, Centre Hospitalier Universitaire de Québec, Pavillon CHUL, Faculté de Médecine, Université Laval, Ste-Foy (Québec) Canada G1V 4G2, and h Department of Medicine, McGill University, Montréal,Canada H3A 1A1
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ABSTRACT |
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Phagocyte functions are markedly inhibited after
infection with the intracellular protozoan parasite
Leishmania. This situation strongly favors the installation
and propagation of this pathogen within its mammalian host. Previous
findings by us and others have established that alteration of several
signaling pathways (protein kinase C-, Ca2+- and
protein-tyrosine kinases-dependent signaling events) were directly responsible for Leishmania-induced macrophage
(MØ) dysfunctions. Here we report that modulation of
phosphotyrosine-dependent events with a protein tyrosine
phosphatases (PTP) inhibitor, the peroxovanadium (pV) compound
bpV(phen) (potassium
bisperoxo(1,10-phenanthroline)oxovanadate(Vi)), can control
host-pathogen interactions by different mechanisms. We observed that
the inhibition of parasite PTP resulted in an arrest of proliferation
and death of the latter in coincidence with
cyclin-dependent kinase (CDK1) tyrosine 15 phosphorylation. Moreover the treatment of MØ with bpV(phen) resulted in an increased sensitivity to interferon- stimulation, which was reflected by enhanced nitric oxide (NO) production. This enhanced IFN-
-induced NO
generation was accompanied by a marked increase of inducible nitric
oxide synthase (iNOS) mRNA gene and protein expression. Finally we have verified the in vivo potency of bpV(phen)
over a 6-week period of daily administration of a sub-toxic dose. The results revealed its effectiveness in controlling the progression of
visceral and cutaneous leishmaniasis. Therefore PTP inhibition of
Leishmania and MØ by the pV compound bpV(phen) can
differentially affect these eukaryotic cells. This strongly suggests
that PTP plays an important role in the progression of
Leishmania infection and pathogenesis. The apparent potency
of pV compounds along with their relatively simple and versatile
structure render them attractive pharmacological agents for the
management of parasitic infections.
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INTRODUCTION |
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Parasitic protozoa of the order Kinetoplastidae are the causative
agents of several subtropical and tropical diseases including leishmaniasis. This infection is estimated to affect more than 15 million people around the world with 400,000 new cases/year (1).
Leishmania donovani, the causative agent of visceral
leishmaniasis, is often fatal if left untreated, whereas other
Leishmania species are mainly responsible for cutaneous and
mucocutaneous afflictions. The incidence of leishmaniasis is rising
because of increased traveling, the lack of vaccines, difficulty in
controlling vectors, and an increase in resistance to chemotherapy (2).
In addition to Leishmania (3-7), numerous potentially
deadly intracellular pathogens, such as Yersinia (8), human
immunodeficiency virus (9, 10), and others can promote mononuclear
phagocyte dysfunctions that inhibit the ability of these cells to
elicit an effective immune response, which may favor persistent
infection. We previously reported that several of these
Leishmania-induced macrophage
(MØ)1 dysfunctions were
related in part to the alteration of Ca2+- and protein
kinase C-dependent signaling pathways (3, 4). More
recently, it has been demonstrated that dysregulation of protein-tyrosine kinase
(PTK)-dependent signaling
events in L. donovani-infected MØ
(11)2 could also account for
the inhibition of several PTK-regulated MØ functions (i.e..
IFN--inducible MØ major histocompatability complex class II
expression) (3, 4, 7, 12-18). It is necessary for cells that both the
protein tyrosine phosphatases (PTP) and PTK maintain their
physiological balance to sustain a normal regulation of their
Tyr(P)-dependent events. It was thus of interest to
determine if changes in the PTP/PTK homeostatic balance could lead to
protection against leishmaniasis.
Peroxide of vanadium (pV, a mixture of vanadate and H2O2) is an insulinomimetic agent and potent inhibitor of PTP (reviewed in Ref. 19). It was demonstrated that a number of chemically defined pV derivatives, each containing an oxo ligand, one or two peroxo anions in the inner coordination sphere of vanadium, and an ancillary ligand, were equally potent PTP inhibitors stable in aqueous solution (20) that can activate the insulin receptor kinase and mimic insulin biological action in vivo (21). Moreover, they have the capacity to inhibit the proliferation of nervous cell lines in vitro (22) and activate the response of immune cells (23).
Protein tyrosine phosphorylation events are also playing an important role in the regulation of Kinetoplastidae growth (24, 25), and PTP activities were previously detected in L. donovani promastigote extracts (26). Several findings also support the pivotal role of PTK-dependent signaling in agonist-induced MØ functions including cytokine-induced nitric oxide (NO) generation (12-16). PTP could also play a pivotal role in Leishmania pathogenesis since several important immune functions necessary for the development of a protection against leishmaniasis are PTK-regulated, and their Leishmania-induced inhibitions are correlated with host signaling alterations (3-7, 11). Thus, we have evaluated the effectiveness of bpV(phen) in vivo in controlling the development of the cutaneous lesions and inflammation of the hind footpad in BALB/c mice induced by Leishmania major infection. The effect of similar in vivo PTP inhibition on the development of visceral leishmaniasis has also been evaluated. The present study firmly establishes that the pV compound bpV(phen) can modulate both Leishmania and MØ cellular physiology to effect protection against leishmaniasis.
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EXPERIMENTAL PROCEDURES |
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Materials--
Isotopes were obtained from ICN Pharmaceuticals
Canada Ltd. (Montréal, QC, Canada). Recombinant murine IFN-
(2 × 105 units/ml) was purchased from Life
Technologies, Inc. Phosphospecific CDK1 antibody was purchased from New
England Biolabs (Beverly, MA). The antiphosphotyrosine antibody (clone
4G10) was purchased from Upstate Biotechnology Inc. (UBI, Lake Placid,
NY), and the inducible nitric oxide synthase (iNOS) antibody was from
Cedarlane (Hornby, ON, Canada). The iNOS inhibitor
L-NG-monomethylarginine (L-NMMA)
was purchased from BioMol (Plymouth Meeting, PA). The peroxovanadium
complexes (PTP inhibitors) used in this study are
K(VO(O2)2phen)·3H2O, bpV(phen);
K(VO(O2)2bipy)·5H2O, bpV(bipy);
K2(VO(O2)2pic)·H2O,
bpV(pic);
K2(VO(O2)23-OHpic)·H2O, bpV(OHpic) were synthesized as we previously described (20). Sodium
orthovanadate (Vi) was purchased from Sigma. BALB/c and C57BL/6 (6-8-weeks-old female, 20-30 body weight) were purchased from
Charles River (St-Constant, QC, Canada).
Cell Culture-- Leishmania promastigotes were grown at room temperature and maintained in the laboratory by weekly transfers in SDM-79 culture medium as described previously (17, 27). For specific experiments, parasites were transferred (5 × 106 log phase promastigotes in 100 µl) into 10 ml of fresh SDM-79 culture medium in the presence or absence of PTP inhibitors. The growth of the parasites was followed over 6 days by measuring the absorbance at 610 nm using an automated microplate reader (Organon Teknika). The murine macrophage cell line J774 was maintained in Dubelcco's modified Eagle's medium (Life Technologies, Inc.) supplemented with 10% fetal calf serum + streptomycin (100 mg/ml) and 2 mM L-glutamine at 37 °C and 5% CO2. All cells mentioned above and used in this study were obtained from the American Type Culture Collection (Manassas, VA).
Flow Cytometric Cell Cycle Analysis-- 107 pre-stationary parasites were transferred into 10 ml of fresh SDM-79 culture medium in the presence or absence of Vi (10 µM) and bpV(phen) (10 µM). After 48-72 h, 106 promastigotes were collected, washed with phosphate-buffered saline (PBS) (pH 7.4), and fixed for 1 h in 1 ml of 70% methanol, PBS. The cells were resuspended in PBS containing 10 mg/ml of RNase A (20 min at 37 °C), labeled with propidium iodide (PI, 50 µg/ml; Sigma), and analyzed using a Coulter EPICS 753 pulse cytometer (Hialeah, FL) to estimate the DNA content of each cell.
Western Blotting--
Cells were collected
(106-107), lysed in buffer containing 20 mM Tris-HCl (pH 8.0), 0.14 M NaCl, 10%
glycerol (v/v), 1% Nonidet P-40 (v/v), 25 µM nitrophenyl
guanidinobenzoate, 10 µM NaF, 1 mM
Vi, 25 µg/ml leupeptin and aprotinin. The lysates (20 µg/lane) were submitted to SDS-polyacrylamide gel electrophoresis,
the separated proteins were transferred on a polyvinylidene difluoride membrane (Millipore). Membranes blocked overnight in Tris-buffered saline/Tween containing 1% gelatin were washed and incubated with an
anti-phosphotyrosine antibody (clone 4G10; UBI), washed with Tris-buffered saline/Tween, incubated with anti-mouse horseradish peroxidase-conjugated antibody (Life Technologies, Inc.), and developed
using ECL Western blotting detection system (Amersham Pharmacia
Biotech). In addition, iNOS antibody has been used to reveal the level
of expression of MØ iNOS protein in cells treated or not with PTP
inhibitors and IFN-. Leishmania CDK1 hyperphosphorylation was assessed using a phosphospecific CDK1 antibody recognizing the
yeast and human CDK1 (P34) Tyr-15 phosphorylated residue. The doublet
signal was revealed according to the manufacturer's protocol (New
England Biolabs).
Nitric Oxide (NO) Production--
Macrophages were seeded in
24-well dishes (5 × 105cell/well) and cultured in the
presence or absence of Vi or bpV(phen) for 1 h,
IFN- (100 units/ml) was then added, and the cells were further incubated for 24 h. The iNOS inhibitor L-NMMA (BioMol)
has been used in some experiments at a concentration of 5 µM. The NO production was evaluated by measuring the
accumulation of nitrite in the culture medium as described previously
(28).
PTP Activity Determination--
Cells were grown in SDM-79
culture medium in the presence or absence of Vi or
bpV(phen) (10 µM). After 6 h, 107 cells
were collected, rinsed 3 times in serum-free medium, resuspended, and
disrupted in a buffer containing 50 mM Tris-HCl (pH 7.0 at 25 °C), 0.1 mM EDTA, 0.1 mM EGTA, 0.1%
-mercaptoethanol (v/v), 25 µg/ml aprotinin, and 25 µg/ml
leupeptin. The PTP activity was determined in total cell preparation by
measuring the dephosphorylation of 32P-labeled
poly(Glu-Tyr) (Glu/Tyr ratio, 4:1). In some experiments, PTP activity
was also monitored over a 24-h period (data not shown). Poly(Glu-Tyr)
was phosphorylated by partially purified insulin receptor kinase from
rat hepatic endosomes as described previously (20).
PTK Activity Measurement--
J774 cells (5 × 105) incubated in 24-well dishes in the presence of
Vi or bpV(phen) (10 µM) over a 1-h period at
37 °C were lysed in buffer containing 1 M Tris-HCl (pH
8.0), 3 M NaCl, 100% glycerol, 10% Nonidet P-40, 0.5 M NaF, 50 µM nitrophenyl guanidinobenzoate, and protease inhibitors (5 µM aprotinin and leupeptin).
After 1 h on ice with gentle mixing, the lysate was spun at
15,000 × g for 30 min at 4 °C in a microfuge. The
phosphorylation reaction was initiated by the addition of a reaction
mixture (25 µM ATP in 50 mM Hepes (pH 7.4),
40 mM MgCl2, 2.5 mg/ml synthetic substrate, poly(Glu-Tyr) (4:1) (Sigma), and 5 µM
([-32P])ATP (New England Biolabs) to a total volume of
100 µl]. After incubation (10 min at 22 °C), the reaction was
terminated by spotting 50 µl of reaction solution onto Whatman No.
3MM square paper (2.5 × 2.5 cm). The paper was extensively washed
with 10% trichloroacetic acid containing 10 µM sodium
pyrophosphate with anhydrous ethanol for 10 min, air-dried, and counted
(LKB rackbeta) using universol (ICN).
Northern Blot Analysis--
Expression of gene iNOS
in Vi- (10 µM)- and bpV(phen)-treated (10 µM, 1 h) and -untreated J774 cells in response to
IFN--stimulation (100 units/ml, 8 h) was evaluated by a
Northern blot of total mRNA, as we described previously with some
modifications (29). Briefly, after incubation under appropriate
conditions, cells were washed twice with Hepes-buffered saline
solution, and total RNA was extracted using TRIzol reagent (Life
Technologies, Inc.). Ten to 20 micrograms of RNA were loaded onto 1%
agarose gels, and equal loading and RNA integrity were confirmed by
ethidium bromide staining. RNA was then transferred onto Hybond-N
filter paper and hybridized with random primer-labeled cDNA probes.
Equal loading of RNA was also confirmed by hybridization with
glyceraldehyde-3-phosphate dehydrogenase cDNA probe. All washes
were performed under stringent conditions. The mRNA hybridizing
with the cDNA probe was visualized by autoradiography. Probes have
been kindly provided by Dr. Danuta Radzioch from the Montreal General
Hospital Research Center (McGill University, Montréal,
Québec, Canada).
Determination of Total MØ Phosphotyrosyl Phosphorylation by
Intracellular Flow Cytometry--
Evaluation of IFN--induced MØ
intracellular phosphotyrosine contents was performed by flow cytometry
as we previously described (23). Briefly, J774 cells (5 × 105) were treated with PTP inhibitors (10 µM,
1-2 h) and further stimulated or not with IFN-
(100 units/ml, 10 min). Next, MØ were washed in PBS (pH 7.4), pelleted, and fixed with
25 µl of reagent A (Fix & Perm cell permeabilization kit from CALTAG
Laboratories, South San Francisco, CA) for 15 min at room temperature.
After further PBS washing, cells were resuspended in reagent B (25 µl) in the presence of the 4G10 anti-phosphotyrosine monoclonal
antibody (UBI) and incubated at room temperature for 15 min. Cells were subsequently washed with PBS and 1% NaN3 and resuspended
with 100 µl of PBS containing a fluorescein isothiocyanate-labeled goat anti-mouse (1 µg total) and further incubated for 15 min at room
temperature. Finally, cells were centrifuged and resuspended in 1%
paraformaldehyde in PBS before being analyzed by flow cytometry (EPICS XL, Coulter Corporation, Miami, FL).
In Vivo Protozoan Infections and pV Treatments-- The mice were injected daily with Vi, bpV(phen), or phenanthroline (2.5 µmol/30 g (500 nM) of body weight, intraperitoneal injection) 2 days before their infection with parasites and further injected daily for a period of 2-6 weeks post-infection. In different experimental groups, animals were inoculated with L. major (106-5 × 106 stationary phase promastigotes, subcutaneous injection in hind footpad) and L. donovani (107 stationary phase promastigotes, injected intravenous in tail vein). For the in vitro infection, MØ cultures were incubated for 6 h with L. major and L. donovani stationary phase promastigotes at a 20:1 parasite to cell ratio as described previously (17). After several washes, infected cells were further cultured for 24 h in the presence or not of Vi or bpV(phen) at a concentration of 5-10 µM. Pretreatment of cells before infection did not further increase the reduction of parasitic load at time point 24 h (data not shown).
Statistical Analyses-- Statistically significant differences between groups were performed with the analysis of variance (ANOVA) module of SAS software (version 6.07, SAS Institute, Cary, NC) using the Fisher least significant difference test. P values <0.05 were considered to be statistically significant (P values are given in the figure legends). All data are presented as mean ± S.E.
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RESULTS AND DISCUSSION |
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Attenuation of MØ and other host immune functions by Leishmania infection has been correlated with alterations of several signaling pathways including PTK activation (3-7, 11-18). Involvement of PTK-dependent events in the regulation of cell proliferation, including that of protozoan parasites, and in the modulation of immune cell functions has been increasingly recognized (14, 24, 25, 30). Modulation of these cellular processes using PTP inhibitors has been widely studied in a range of in vitro systems including activation of T lymphocytes (23). With the exception of experiments in animal diabetes, few studies have tested the in vivo effects of PTP inhibitors, including the recently characterized pV compounds (20), on the modulation of PTK-dependent cellular events. In this study, we have thus evaluated the role of PTP in the development of murine leishmaniasis using the pV compound bpV(phen) to inhibit both the parasite and host cell PTP activities.
We measured the effects of vanadate and different pV compounds on the growth of L. donovani promastigotes (Fig. 1A). Only bpV(phen) was found to inhibit L. donovani growth in a dose- and time-dependent manner. No inhibition by vanadate or other pV compounds tested (bpV(pic), bpV(OHpic), bpV(bipy)) was observed, showing that the nature of the ancillary ligand is important. Similar effects have been observed on the growth of several Leishmania species including L. major (data not shown). The inhibitory effect of bpV(phen) on L. donovani was observed at a dose of 10 µM and was characterized by an increase in the number of cells at the SG2/M phase of the cell cycle (Fig. 1B) when compared with vanadate and untreated parasites. In parallel, PTP activity was measured (20) in whole Leishmania extracts using 32P-labeled poly Glu-Tyr as a substrate. As shown in Fig. 1C, incubation of L. donovani for 6 h with 10 µM bpV(phen) reduced parasite PTP activity by more than 90%. Similar levels of inhibition were measurable from 1 to 24 h after treatments (data not shown). One direct effect of this inhibition was further documented by the Tyr-15 hyperphosphorylation of CDK1 observed upon incubation with bpV(phen) (Fig. 1D). This showed that CDK1 is an endogenous target. Vanadate, albeit less efficient, was also able to inhibit PTP activity in these conditions and induced a slight increase in CDK1 phosphorylation. This observation may in part explain the absence of Leishmania growth inhibition in response to vanadate treatment in vitro. These results are in accordance with the report of Morla et al. (31) concerning the hyperphosphorylation of CDK1 by vanadate (50 µM) in 3T3 cells and the observation of Faure et al. (22) on the effect of bpV(phen) to promote the inhibition of mitosis by blocking progression at the SG2/M interphase in coincidence with CDK1 hyperphosphorylation and loss of catalytic activity. These observations suggest that, as in mammalian cells (22, 31), the protein tyrosine phosphatase Cdc25 is an important endogenous target in protozoan cells.
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Leishmania can inhibit several MØ functions to improve its
survival and propagation. It is also well established that phagocytes play a key role in controlling Leishmania infection in
vivo by secreting molecules such as NO (32), which are regulated
by Tyr(P)-dependent events (14). We thus evaluated the
effect of bpV(phen) on MØ functions since an inhibition of MØ PTP
activities may contribute to increased responsiveness toward cytokine
stimulation. The effects of bpV(phen) treatment on murine MØ
responsiveness to IFN- are shown in Fig.
2A. NO production was
significantly increased in bpV(phen)-treated cells over untreated or
vanadate-treated MØs at various doses and inhibitable by the iNOS
inhibitor L-NMMA. Similarly, IFN-
-stimulated
bpV(pic)-treated MØ has generated NO levels comparable to that of
bpV(phen)-treated cells (data not shown). In addition, the inhibition
of MØ PTK by genistein led to an almost complete abrogation of
inducible NO production as previously reported by others (14). These
results established that PTP inhibition by bpV(phen) can modulate
events in cells as different as the pathogen Leishmania and
its host cell, the macrophage. We then assessed whether this bpV(phen)
dual effect may attenuate parasite persistence within its host cells.
As shown in Fig. 2B, bpV(phen) at a dose of 10 µM can effectively reduce the parasitic load of
Leishmania-infected MØ by more than 75% in comparison to
control cells. In addition, we observed that the NO inhibitor
L-NMMA almost completely reversed this bpV(phen)-mediated protection against Leishmania infection, suggesting that
NO is a key player in pV-mediated leishmanicidal activity. MØ PTP
activities, as for Leishmania, were substantially inhibited
by bpV(phen) (data not shown). This was paralleled by augmented levels
of Tyr(P) proteins in MØ (Fig. 2C), which may result from
enhanced MØ PTK activity (Fig. 2D). Total MØ Tyr(P)
protein levels were further induced by IFN-
stimulation as revealed
by intracellular flow cytometry determinations (Fig. 2E). In
addition, bpV(phen) treatment enhanced iNOS gene mRNA
and iNOS protein expressions both in the basal state and in response to
IFN-
stimulation in comparison to their respective controls (Fig.
2F). Altogether, these observations document the capacity of
bpV(phen) to favor PTK-dependent signaling and to prime MØ
for enhanced responsiveness toward stimulants. Regulation of
iNOS has been previously reported to involve the participation of the transcription factor NF-
B (33). Our recent observations that several pV compounds can strongly induce NF-
B nuclear translocation in lymphoid and monocytoid cells (23) support the
hypothesis that nuclear translocation of NF-
B is involved in the
effect of bpV(phen) to increase iNOS mRNA expression. Furthermore, the activity of iNOS may be directly influenced by tyrosine phosphorylation (34).
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In view of this, experiments were done to determine whether bpV(phen) (21) could modulate the course of the infection in a murine leishmaniasis model (32). Infection by L. major, the causative agent of cutaneous leishmaniasis, was inhibited by 60% (p < 0.05) in bpV(phen)-treated BALB/c mice (Fig. 3, A and B). As shown in Fig. 3A, mice treated daily with bpV(phen) (2.5 µmol/30 g (500 nM) of body weigh, intraperitoneal injection) over a 6 week period showed significantly reduced footpad inflammation compared with control, vanadate, and phenanthroline-treated groups. The bpV(phen) treatment not only reduced the inflammation of the footpads but also completely blocked the development of the cutaneous lesion (Fig. 3B). The remarkable reduction of these typical features of cutaneous leishmaniasis was further evidenced by the almost complete disappearance of parasite from the popliteal lymph node and complete absence of hepatic involvement (Table I). The effect of bpV(phen) on the course of murine visceral leishmaniasis was also tested and found to be even more striking. As noted in Fig. 3C, bpV(phen) was capable of completely reducing the liver parasitic load at two weeks post-infection, whereas hepatic infestation of control C57BL/6 was still at peak levels. These data strongly suggest that in vivo modulation of PTK/PTP balance by pV compounds (35) can restrict and favor regression of leishmaniasis in mammals.
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Various pathogens, including viruses (36) and bacteria (8), exploit signaling systems modulated by PTP for their growth and pathogenesis. Furthermore, cellular defenses utilize killing strategies modulated by PTP (14, 32). It is thus plausible that the inhibition of PTP could influence the course of various infections (e.g. Yersinia). Presently, we cannot say with certainty the extent to which the in vivo protective and curative effects of bpV(phen) are due to direct actions on the growth of the parasite or on the potentiation of MØ functions. Nevertheless our in vitro experiments strongly suggest the pivotal role played by NO in the control of Leishmania infection (Fig. 2, A and B). Indeed, we have evidence that NO is effectively the key molecule that restrains the progression of infection in pV-treated animals, since bpV(pic) was similarly capable as bpV-(phen) to significantly abolish L. major-induced footpad inflammation and lesion development.3
In conclusion, the results of the present study emphasize the important role that modulation of PTP plays in the development of Leishmania infection. Our findings highlight the fact that the apparent potency of pV compounds, along with their relatively simple and versatile structure, may represent a new avenue for the development of novel therapeutic agents against parasitic infections.
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ACKNOWLEDGEMENTS |
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We thank André Marette for its critical review of this paper and Danuta Radzioch for iNOS and glyceraldehyde-3-phosphate dehydrogenase cDNA probes.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
b Contributed equally to this work.
c Supported by funds from the Medical Research Council of Canada. To whom correspondence should be addressed: Centre de Recherche en Infectiologie, RC-709, Centre Hospitalier Universitaire de Québec, Pavillon CHUL, 2705 boul. Laurier, Ste-Foy (Québec), Canada G1V 4G2. Tel.: 418-654-2705; Fax: 418-654-2715; E-mail: martin.olivier{at}crchul.ulaval.ca.
d Members of a Medical Research Council group grant in infectious diseases.
e Hold Junior 2 scholarship award from the Fonds de la Recherche en Santé du Québec.
f Recipient of a studentship from the Ministère de la Science et de l'Éducation du Québec.
g Supported by Medical Research Council grants.
j Holds a grant from the Natural Sciences and Engineering Research Council of Canada.
1 The abbreviations used are: MØ, macrophage; iNOS, inducible nitric oxide synthase; pV, peroxovanadium; phen, phenanthroline; bpV(phen), potassium bisperoxo(1,10-phenanthroline)oxovanadate; PTK, protein-tyrosine kinase; PTP, protein tyrosine phosphatase; Vi, sodium-orthovanadate; L-NMMA, L-NG-monomethylarginine; IFN, interferon; PBS, phosphate-buffered saline; PI, propidium iodide; Tyr(P), phosphotyrosine.
2 M. Olivier, J. Blanchette, R. Faure, N. Racette, and K. Siminovitch, unpublished data.
3 M. Olivier, C. Matte, J. F. Marquis, P. L. Janvier, and P. Gros, manuscript in preparation.
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REFERENCES |
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