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Infection and Immunity, December 2001, p. 7703-7710, Vol. 69, No. 12
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.12.7703-7710.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Host Response to Infection: the Role of CpG DNA in
Induction of Cyclooxygenase 2 and Nitric Oxide Synthase 2 in
Murine Macrophages
Dipak K.
Ghosh,
Mary A.
Misukonis,
Charles
Reich,
David
S.
Pisetsky, and
J. Brice
Weinberg*
Department of Medicine, Veterans
Affairs and Duke University Medical Centers, Durham, North
Carolina
Received 16 May 2001/Returned for modification 24 August
2001/Accepted 13 September 2001
 |
ABSTRACT |
Depending on sequence, bacterial and synthetic DNAs can activate
the host immune system and influence the host response to infection.
The purpose of this study was to determine the abilities of various
phosphorothioate oligonucleotides with cytosine-guanosine-containing motifs (CpG DNA) to activate macrophages to produce nitric oxide (NO)
and prostaglandin E2 (PGE2) and to induce
expression of NO synthase 2 (NOS2) and cyclooxygenase 2 (COX2).
As little as 0.3 µg of CpG DNA/ml increased NO and PGE2
production in a dose- and time-dependent fashion in cells of the mouse
macrophage cell line J774. NO and PGE2 production was noted
by 4 to 8 h after initiation of cultures with the CpG DNA, with
the kinetics of NO production induced by CpG DNA being comparable to
that induced by a combination of lipopolysaccharide and gamma
interferon. CpG DNA-treated J774 cells showed enhanced expression of
NOS2 and COX2 proteins as determined by immunoblotting, with the
relative potencies of the CpG DNAs generally corresponding to those
noted for the induction of NO and PGE2 production as well
as to those noted for the induction of interleukin-6 (IL-6), IL-12, and
tumor necrosis factor. Extracts from CpG DNA-treated cells converted
L-arginine to L-citrulline, but the NOS
inhibitor
NG-monomethyl-L-arginine
(NMMA) inhibited this reaction. The COX2-specific inhibitor NS398
inhibited CpG DNA-induced PGE2 production and inhibited NO
production to various degrees. The NOS inhibitors NMMA, 1400W, and
N-iminoethyl-L-lysine effectively blocked NO production and increased the production of PGE2 in a
dose-dependent fashion. Thus, analogues of microbial DNA (i.e., CpG
DNA) activate mouse macrophage lineage cells for the expression of NOS2
and COX2, with the production of NO and that of PGE2
occurring in an interdependent manner.
| |
INTRODUCTION |
DNA is a complex
macromolecule whose immunological properties vary with base sequence
and backbone structure. While mammalian DNA is inactive, DNA from
bacteria can induce potent immunological effects (12, 18,
32). Bacterial DNA can activate several different cell types
(e.g., B lymphocytes, macrophages, and dendritic cells). As has been
shown through the use of synthetic oligodeoxynucleotides (ODNs),
the activity of bacterial DNA results from the presence of short,
6-base sequence motifs characterized by an unmethylated cytosine-guanosine dinucleotide within the context of flanking bases of
two 5' purines and two 3' pyrimidines. These motifs are called
immunostimulatory stimulatory sequences or CpG motifs, with DNA
containing these sequences being termed CpG DNA. Because of its immune
stimulatory properties, CpG DNA can activate innate immunity. In
addition to being important in the context of infection and
inflammation, these properties are relevant to the activities of DNA
vaccines and to the use of synthetic DNA as an adjuvant and
immunomodulator (13).
While CpG DNA has beneficial effects in stimulating host defense, it
can also induce inflammation and may promote tissue injury during
infection or local administration. For example, CpG DNA from bacteria
(or synthetic CpG DNA) causes severe arthritis when it is injected into
the joints of mice. This arthritis is characterized by synovial tumor
necrosis factor (TNF) mRNA expression and monocyte influx (3,
4). Similarly, the instillation of CpG DNA into the lungs of
mice elicits a marked exudate and inflammatory reaction (23). The proinflammatory effects of CpG DNA may result
from the production of cytokines (e.g., TNF-
), chemokines, and other mediators such as nitric oxide (NO). The contribution of these various
mediators in the response to CpG is not well understood and may reflect
both local and systemic effects.
To further characterize immune activities of CpG DNA in the present
study, we investigated its effects on macrophages for the production of
two important mediators (NO and prostaglandins [PGs]). NO is a
gaseous molecule with widespread biological activities, including
inflammation, while the PGs have a number of important immunomodulatory
activities. As a model for CpG DNA, we have used synthetic
phosphorothioate ODNs (Ps ODNs) containing CpG motifs and tested their
abilities to induce the production of NO and PG E2
(PGE2) by J774 mouse macrophage cell line cells. These ODNs contain the substitution of a sulfur for one of the nonbridging oxygens
in the phosphodiester backbone. This substitution leads to nucleic
resistance to degradation and increased immunological activities. Ps
ODNs are now being investigated as adjuvants as well as
immunomodulators to promote host defense and alter the Th1/Th2 balance.
We report here that certain Ps ODNs induce NO and PGE2
production as well as NO synthase 2 (NOS2) and cyclooxygenase 2 (COX2) expression by these cells in a time- and dose-dependent fashion. Furthermore, we show that inhibition of NO production leads to an
increase in ODN-stimulated PGE2 production. These results
indicate that pathways for the production of NO and PGE2
may be interdependent. Since NO and PGE2 can modulate
immune responses, the in vivo and in vitro activities of CpG DNA may
reflect the actions of these mediators, as well as those of induced
cytokines and chemokines.
| |
MATERIALS AND METHODS |
Cells and culture methods.
Cells of the mouse macrophage
cell lines J774 and RAW264 (American Type Culture Collection, Manassas,
Va.) were used as models of macrophages. They were cultured in
Dulbecco's modified Eagle medium with 10% heat-inactivated fetal
bovine serum. For these experiments, cells were generally seeded into
11-mm-diameter wells of 24-well plates at a concentration of
106 cells in 1 ml of medium with the various additives. For
selected experiments as noted in Results, some inhibitors were cultured with the cells before addition of CpG DNA.
Materials.
Endotoxin-free media were from GIBCO-BRL
(Gaithersburg, Md.). The Ps ODNs (Table
1) were purchased from Midland Certified Reagent Company (Midland, Tex.). All ODNs had undetectable endotoxin (lipopolysaccharide [LPS]) contents as determined by the
Limulus amebocyte lysate assay (<0.1 endotoxin unit
[EU]/ml). Escherichia coli LPS was from Sigma-Aldrich (St.
Louis, Mo.), and gamma interferon (IFN-
) was from R&D Systems
(Minneapolis, Minn.). The NOS-nonspecific inhibitor
NG-monomethyl-L-arginine (NMMA) and
the NOS2-specific inhibitors N-iminoethyl-L-lysine (L-NIL) and
1400W were from Alexis Biochemicals (San Diego, Calif.), and the
COX2-specific inhibitor NS398 was from Cayman Chemicals (Ann Arbor,
Mich.). All other chemicals were from Sigma-Aldrich.
NO, PG, and cytokine assays.
The NO oxidation
products nitrate and nitrite (NOx) were measured using nitrate
reductase and the Griess method as described before
(33). PGE2, interleukin-6 (IL-6), IL-12
p40/p70, and TNF were measured using enzyme-linked immunoassays
(R&D Systems).
NOS enzyme assay and immunoblots.
Cells were harvested by
scraping, washed, and suspended in a buffer containing 1 mM
phenylmethylsulfonyl fluoride, 5 µg of aprotinin/ml, 1 µg of
chymostatin/ml, and 5 µg of pepstatin A/ml. Cells were then lysed by
3 cycles of freezing and thawing. The lysate was centrifuged at
14,000 × g, and the supernatant was assayed
(24). Protein content was determined by the Bradford assay
(Bio-Rad, Hercules, Calif.). NOS activity was determined by an assay
converting L-[14C]arginine to
L-[14C]citrulline as noted previously
(34). In brief, the assay buffer contained 50 mM HEPES (pH
7.5), 200 µM NADPH, 1 mM dithiothreitol, 10 µM flavin adenine
dinucleotide, 100 µM tetrahydrobiopterin, and 10 µM
L-arginine with L-[14C]arginine
labeled in the guanido position (NEN, Wilmington, Del.). The
specificity of the reaction was determined by inhibition with NMMA.
For immunoblots, cells were lysed in 50 µl of 40 mM EPPS
(
N-hydroxyethyl]piperazine-
N'-[3-propanesulfonic
acid) buffer containing
10% glycerol, 150 mM NaCl, 50% Beeper II
detergent (Pierce Chemicals,
Rockford, Ill.), 1 mM phenylmethylsulfonyl
fluoride, and leupeptin
and aprotinin (5 µg/ml each) by incubating on
ice with occasional
shaking for 30 min. The lysate was centrifuged at
14,000 ×
g,
and the supernatant was analyzed by
immunoblotting as noted above
by using the ECL technique
(Amersham, Piscataway, N.J.). Anti-mouse
NOS2 and COX2 antibodies were
from Transduction Laboratories (Lexington,
Ky.).
| |
RESULTS |
NO and PGE2 production and NOS2 and COX2
expression.
To assess the effects of CpG DNA on the production of
NO and PGE2, we treated J774 cells with a panel of Ps ODNs
and assessed mediator production. As shown in Fig.
1, certain of the ODNs tested increased
the production of both NO and PGE2. Increased production was noted with as little as 0.3 µg of CpG DNA/ml and occurred without
preactivation of the cells with either LPS or IFN-. Activation of
the cells for NO and PGE2 production was sequence specific, with the 74, 75, 115, and SAK2 ODNs showing the highest activity. SAK2
was the most potent inducer of NO and PGE2 production.
SAK1, an ODN that does not contain a CpG motif, was generally the least effective of the agents. CpG DNA enhanced NO and PGE2
production by cells of the mouse macrophage line RAW 264 (data not
shown) as well as by J774 cells.
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FIG. 1.
NOx (A) and PGE2 (B) production by J774
cells after stimulation with CpG DNAs. Each symbol represents the mean
of results for triplicate samples. Cells were cultured with the
indicated additives, and supernatants were analyzed after 48 h of
culture. These results are from one experiment, which is representative
of four that were done. Variation was small, and error bars fall within
the areas of the symbols.
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|
We tested the kinetics of the response to analyze further the
production of these mediators and the relationship of this
response
to responses induced by other stimulants (Fig.
2A and
B). As these
data indicate, production of
NO and PGE
2 was noted by 4 to 8 h
after initiation of
cultures with SAK2, with the kinetics of NO
production induced by CpG
DNA being comparable to that induced
by LPS-IFN-
. Under these
conditions, however, LPS, IFN-
, and
LPS-IFN-
generally induced
very little PGE
2 production or none
at all.
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FIG. 2.
Time course for production of NOx (A) and
PGE2 (B) by J774 cells after stimulation with CpG DNAs.
Each symbol represents the mean of results from triplicate samples.
Cells were cultured with 10 µg of the indicated ODN/ml and with 100 ng of LPS/ml plus 100 U of IFN-/ml. Supernatant media were assayed
at the indicated times. These results are from one experiment, which is
representative of three that were done. Error bars show the SEMs. Some
SEMs fall within the areas of the symbols and are not seen.
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|
Production of NO was paralleled by induction of NOS enzyme activity.
Lysates of SAK2- or LPS-IFN-
-simulated J774 cells had
an increased
ability to convert
L-arginine to
L-citrulline,
while
SAK1-stimulated cells showed little or no increase in their
ability.
For example, NOS activity in lysates of control cells was
659
± 29 pmol of
L-citrulline/mg (mean ± standard error of the mean
[SEM]); in cells treated with
LPS-IFN-
, it was 1,438 ± 58; in
cells treated with SAK2, it
was 1,609 ± 90; and in cells treated
with SAK1, it was 714 ± 38. Increases in NO production induced
by the ODNs 74, 75, 115, 139, and SAK2 were inhibited by more
than 90% after inclusion of the
NOS2-specific inhibitor 1400W
(0.5 mM) during the culture period. 1400W
was not toxic for the
cells (data not shown). Similarly, inclusion of
0.5 mM 1400W or
2 mM NMMA in the NOS activity assay using cell extracts
inhibited
activity by more than 95%.
NOS2 and COX2 protein expression.
We studied the effects of
CpG DNA on enzyme protein expression to investigate the mechanisms for
the increase in NO and PGE2 production. J774 cells treated
with CpG DNA showed enhanced expression of NOS2 and COX2 protein as
determined by immunoblotting (Fig. 3A and
B). In general, the relative potencies of
the ODNs corresponded to those noted for the induction of NO and
PGE2 production. Levels of NOS2 and COX2 protein expression
did not correspond directly with the levels of NO and PGE2
produced, respectively. Such discrepancies have been noted by others
and may relate to numerous factors, including mRNA and protein
stability and cofactor abundance (17, 30). They may also
relate to differences in the times of the various measurements. The
intensity of NOS2 expression was greatest in IFN-- and
LPS-IFN--treated cells. The ODNs 74 and SAK2 were the most potent
inducers of COX2 protein. As noted in Fig.
4, COX2 and NOS2 proteins induced by SAK2
or LPS-IFN- were noted as early as after 4 to 8 h of culture,
with expression peaking at about 16 h. Cultures with no addition
showed no increase in NOS2 protein over time (Fig. 4A and B) but showed
a small increase in COX2 protein over time. SAK1 induced no increase in
NOS2, but it did cause an increase in COX2. Overall, LPS-IFN- and
SAK2 were the most potent inducers of NOS2 and COX2 protein.
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FIG. 3.
NOS2 and COX2 expression in J774 cells after treatment
with CpG DNAs or LPS-IFN-. Immunoblots for NOS2 (A) and COX2 (B)
proteins are shown for extracts of cells cultured for 48 h with 10 µg of the indicated ODN/ml and with 100 ng of LPS/ml plus 100 U of
IFN-/ml. The blot and a bar graph showing the relative density of
each band are shown.
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FIG. 4.
Immunoblot analysis of the time course for NOS2 and COX2
induction after treatment of J774 cells with CpG DNAs or LPS-IFN-.
(A) Shown is the time course for the appearance of NOS2 (133 kDa) and
COX2 (70 kDa) in cells after treatment with 10 µg of SAK1 (S1) or
SAK2 (S2)/ml or 100 ng of LPS/ml plus 100 U of IFN-/ml (L/I). Lanes
O, no addition. (B and C) Relative densities of the bands for NOS2 (B)
and COX2 (C).
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|
IL-6, IL-12, and TNF expression.
CpG DNA has the capacity to
induce cytokine expression by macrophages (14, 19). To
determine the relationship between the inductions of NO,
PGE2, and cytokines, we tested the CpG DNAs for their
abilities to induce the production of IL-12, IL-6, and TNF by J774
cells. As these data indicate, SAK2 was a potent inducer of these
cytokines while SAK1 had low activity; the other ODNs showed
intermediate levels of activity (Fig. 5).
The relative abilities of ODNs to induce the cytokines were thus
comparable to those to induce NO and PGE2 production, while
SAK2 was much more potent than LPS-IFN- in inducing cytokine
production.
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FIG. 5.
Time course for production of IL-12 (A), IL-6 (B), and
TNF- (C) by J774 cells after treatment with CpG DNAs or
LPS-IFN-. Cells were cultured with 10 µg of the indicated ODN/ml
and with 100 ng of LPS/ml plus 100 U of IFN-/ml. Supernatant media
were assayed at the indicated times. These results are from one
experiment, which is representative of two that were done. Results are
displayed as means + 1 SEM. No add, no addition.
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|
Influence of NOS and COX inhibitors on NO and PGE2
production.
We next assessed the effects of inhibitors of NO and
PGE2 production. Inclusion of the nonspecific NOS inhibitor
NMMA or of the NOS2-specific inhibitor 1400W or L-NIL (data
not shown) in cultures inhibited CpG DNA-induced NO production. The
COX2-specific inhibitor NS398 also inhibited CpG DNA- and
LPS-IFN--induced NO production to various degrees (Fig.
6A). NS398 completely blocked PGE2 production induced by any of the stimuli (Fig. 6B).
CpG DNA-induced PGE2 production was enhanced in cells
incubated with either NMMA (Fig. 6B) or 1400W (data not shown). Figure
7 displays the effect of a wide range of
concentrations of NMMA on CpG DNA-induced PGE2 production.
As the amount of NMMA included in the cultures increased, there was a
reduction in SAK2-induced NO production and a reciprocal increase in
PGE2 production, with doses as low 92 µM having an enhancing effect.
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FIG. 6.
Influence of treatment with a NOS inhibitor (NMMA) or a
COX2 inhibitor (NS398) on production of NO (A) or PGE2 (B)
by J774 cells after treatment with CpG DNAs or LPS-IFN-. Cells were
cultured with 10 µg of the indicated ODN/ml and with 100 ng of LPS/ml
plus 100 U of IFN-/ml for 48 h. The NOS inhibitor NMMA (2 mM)
or the COX2 inhibitor NS398 (100 µM) was present in the cultures as
indicated. Cells were cultured with the indicated additives, and
supernatants were analyzed after 48 h of culture. These results
are from one experiment, which is representative of three that were
done. Results are displayed as the means of results from triplicate
samples.
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FIG. 7.
Effect of various doses of NMMA on PGE2 and
NO production by J774 cells after treatment with SAK2 CpG DNA. Cells
were cultured with no treatment or with 10 µg of SAK2/ml. "No Rx"
signifies cultures to which no SAK2 was added. Various concentrations
of NMMA were present throughout the culture period as indicated.
Supernatant media were assayed after 48 h of culture. These
results are from one experiment, which is representative of two that
were done. NOx values are expressed as NOx divided by 2 to allow easy
comparison to PGE2 values. Results are displayed as the
means ± SEMs of results from triplicate samples. For some data
points, the error bars are obscured by the symbol.
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| |
DISCUSSION |
Our results provide new insights into the immunological properties
of CpG DNA and show that this stimulator can induce both NO and
PGE2 production by mouse macrophage lineage cells.
Bacterial DNA as well as synthetic ODNs with certain sequences can
potently stimulate B and T lymphocytes, NK cells, mononuclear
phagocytes, and dendritic cells (see references 11 and 18
for reviews). These activities may be beneficial in inducing innate
immunity and providing adjuvant activity. They may also promote
inflammation, however, and may induce or exacerbate conditions such as
arthritis or pneumonitis. Although cytokines such as TNF have been
implicated as mediators of CpG DNA-induced inflammation, our findings
suggest that PGE2 and NO may also contribute to this
action. Furthermore, PGE2 and NO, by virtue of their
effects on various lymphoid and myeloid populations, may influence the
overall host response to CpG DNA.
We demonstrate here that CpG DNA can, in the absence of prior treatment
with IFN- or LPS, directly activate mouse J774 macrophage cells for
not only NOS2 expression and NO production but also COX2 expression and
PGE2 production. These results contrast with those of
studies by other investigators who showed that CpG DNA-induced NO
production in murine macrophages requires priming with IFN- or LPS
(6, 25, 27). The causes of the differences among these
studies are not known, but they could relate to either the sequences of
the Ps ODNs used, the cell population studied, or the conditions for
cell culture.
Among Ps compounds tested, SAK2 (an ODN with two CpG sequences) was the
most potent inducer of NO, PGE2, and cytokines. Other CpG
compounds had less activity, and a control compound without a CpG was
generally inactive. This result is consistent with those of other
studies demonstrating that the stimulation by an ODN is determined by a
CpG motif and its flanking sequences. Furthermore (although we did not
study phosphodiester ODNs), Ps ODNs are in general more active than
other ODNs. The activities of these compounds most likely relate to the
stability conferred by the Ps backbone. Thus, both base sequence and
backbone structure contribute to the overall activity of CpG DNA.
A variety of stimuli can activate cells for both NOS2 and COX2
expression, but in experiments reported here, we observed differences between the effects of CpG DNA and the effects of LPS and IFN-. CpG
DNA induced high levels of COX2 and NOS2, but IFN-, LPS, and
LPS-IFN- increased expression of COX2 in the J774 cells less well.
These results suggest that, while the activation pathways induced by
CpG DNA and LPS are similar, they are likely not identical. This
conclusion is supported by previous studies of the kinetics of cytokine
induction by LPS and CpG DNA and the differences in the responses of
C3H/HeJ mice. These mice, with a mutation in TLR4, can respond to CpG
DNA but not to LPS (9, 20).
Although NOS2 and COX2 differ in their levels of
activation by CpG DNA, there is nevertheless significant cross talk
between these systems. Eicosanoids can reduce NOS2 expression and NO
production (5, 10, 15, 16, 29), and NO modulates
PGE2 formation (22, 26, 28, 31). Stimuli that
enhance NOS2 expression and NO formation may also induce COX2
expression, but the time courses for induction may differ. Furthermore,
arginine analogues such as NMMA may lead to inhibition of both COX2 and
NOS (21), while aspirin (in high doses) inhibits both COX
and NOS2 (2). Studies to define these relationships
further have produced somewhat divergent results that may in part be
related to the use of different cells and experimental conditions.
An important finding in our study concerns the effects of NO inhibition
on the production of PGE2 induced by CpG DNA. We found that
while NMMA, 1400W, and L-NIL inhibited NO production, these agents increased PGE2 production in a dose-dependent
fashion. These results suggest that blocking NO production with a NOS
inhibitor reduces the inhibitory effect of NO on COX2 expression and
activity. A variety of mechanisms could account for this response. The
modulating effects of NO could result from altered transcription of COX
genes, modulation of COX activity, or modification of PG-metabolizing enzymes. In this regard, peroxynitrite, derived from NO and
O2
, activates the COX activities of COX1 and
COX2 by serving as a substrate for the enzymes' peroxidase activities
(7). Furthermore, NO may inhibit the enzymatic activity of
COX2 by reacting with the iron in the COX2 heme group in a fashion
comparable to that noted for NOS2 (8).
While the mechanism by which NOS inhibitors increase PGE2
production is unknown, this effect has implications for the therapy of
inflammatory disease. To the extent that NO modulates PGE2 production, treatment of inflammation with a NOS2 inhibitor alone might
result in increased PGE2 production and increased
inflammation. An interrelationship between the NO and PG systems has
also been observed in a study of cartilage (1). These
findings suggest that the cross talk we have observed with CpG DNA
stimulation may be a general phenomenon and not confined to this mode
of stimulation.
In summary, we have found that CpG DNA coordinately induces high levels
of expression of COX2 and NOS2 and high levels of production of
PGE2 and NO. These mediators may contribute to the immune
activity of CpG DNA and modulate the effects of the chemokines and
cytokines induced by CpG DNA. Furthermore, we have shown that the
inhibition of NO formation enhances PGE2 production. While the interplay of the PG and NO systems may influence the effects of CpG
DNA, our findings suggest a broader relevance for the treatment of
inflammatory disease with agents that modify the production or
activities of these mediators.
| |
ACKNOWLEDGMENTS |
This work was supported in part by the Veterans Affairs Research
Service, the James Swiger Hematology Research Fund, and NIH grant
AR-39162.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: VA and Duke
University Medical Centers, 508 Fulton St., Durham, NC 27705. Phone:
(919) 286-6833. Fax: (919) 296-6891. E-mail:
brice{at}acpub.duke.edu.
Editor:
E. I. Tuomanen
| |
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Infection and Immunity, December 2001, p. 7703-7710, Vol. 69, No. 12
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.12.7703-7710.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
