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Infection and Immunity, August 2001, p. 5131-5137, Vol. 69, No. 8
Microbiology Department, Chicago College of
Osteopathic Medicine, Midwestern University, Downers Grove,
Illinois 60515,1 and Department of
Medical Microbiology and Immunology, University of Wisconsin
Medical School, Madison, Wisconsin 537062
Received 15 February 2001/Accepted 10 April 2001
It was previously reported that female mice resolve a primary
Chlamydia trachomatis urogenital infection
independent of inducible nitric oxide synthase (iNOS). We now
report that although iNOS-deficient (NOS2 Mice resolve primary chlamydial
genital tract infections and become resistant to challenge infections,
as assessed by cessation of shedding of viable Chlamydia
trachomatis (1, 2). Recovery from chlamydial
urogenital infections in mice requires type 1 immune responses that are
at least partially dependent on gamma interferon (IFN- Persistent chlamydial infections develop in cell cultures in response
to low-level IFN- In the studies described here, we used NOS2 Mice.
Mice with a targeted disruption in the iNOS gene
(NOS2 Chlamydiae.
C. trachomatis MoPn (Weiss) was grown
in HeLa 229 cells and maintained by adaptation of previously described
methods (7, 11).
Infection and infection assessment.
For primary infection,
mice were pretreated with 2.5 mg of DepoProvera (P4; Upjohn, Kalamazoo,
Mich.) in 0.1 ml of saline administered subcutaneously 10 and 3 days
prior to infection (11). Mice were inoculated
intravaginally with 10 µl of MoPn suspension containing
104 inclusion-forming units, equivalent to
approximately 200 50% infective doses (10), in SPG
buffer (10 mM phosphate, 0.25 M sucrose, 5 mM L-glutamic acid).
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.8.5131-5137.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Chlamydia trachomatis Persistence in the Female
Mouse Genital Tract: Inducible Nitric Oxide Synthase and
Infection Outcome
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
/) mice
resolve culture-apparent infection in a fashion similar to that of
normal control (NOS2+/+) mice, they
sustain significantly increased rates of disease, as assessed by
hydrosalpinx formation. PCR amplification of ompA followed by Southern blot detection of amplicands revealed the presence
of chlamydial DNA in the lower genital tracts of both NOS2/ and NOS2+/+ mice at
120 days postinfection and in upper genital tract tissues at >120
days postinfection. However, only NOS2/ mice shed low
numbers of viable chlamydiae from the lower genital tract after
immunosuppressive treatment at 120 days postinfection. When cultured
primary murine lung fibroblasts were activated in the presence of gamma
interferon (IFN-
), inhibition of chlamydial growth occurred in both
NOS2+/+ and NOS2/ cells,
but the inhibition was reversible after removal of the cytokine in the
NOS2/ primary cell culture only. The iNOS-independent
inhibition was microbistatic but was independent of 2,3-indoleamine
dioxygenase activity. We conclude that chlamydial DNA and antigens
persist in mice subsequent to culture-apparent resolution. In addition, IFN- induces in vivo inhibition of chlamydial growth through microbistatic mechanisms in the absence of iNOS activity, but in the
presence of iNOS activity, IFN- is microbicidal and effects eradication.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
) (11,
28, 29) and major histocompatibility complex class II antigen
processing and presentation (20). A potentially important
mechanism of IFN--dependent chlamydial growth inhibition is the
production of reactive nitrogen intermediates (RNI) via
cytokine-inducible nitric oxide synthase (iNOS). RNI have been shown to
restrict chlamydial growth in vitro in murine epithelial cells
(16), fibroblasts (19, 27), and macrophages (8). However, RNI could not account for the sterile
eradication of C. trachomatis because in the presence
of the iNOS inhibitor NG-monomethyl-L-arginine
(L-NMMA), chlamydial growth was not completely restored to that of controls. This result indicates that other IFN--inducible mechanisms also might be important for controlling chlamydial infections in mice. It was previously found that in primary murine fibroblast cell cultures derived from iNOS
knockout (NOS2/) mice, IFN- can
restrict chlamydial growth via iNOS-independent mechanisms
(27). In vivo, NOS2/ or normal
control (NOS2+/+) mice treated with
L-NMMA are capable of resolving chlamydial genital tract infections (27). In human systems, the
induction of the tryptophan-decyclizing enzyme indoleamine
2,3-dioxygenase (2,3-IDO) has a central role in IFN--mediated growth
inhibition (37). However, induction of this enzyme in
response to chlamydial infections has yet to be observed in murine
systems (8, 27), although the 2,3-IDO gene is present
and active in mice in other situations (32, 35, 36).
treatment of host cells and in response to other
stressors (4). Persistent chlamydiae are characterized by
their ability to enter a metabolically inactive and noninfectious state
in response to stress and by the resumption of growth and the release
of infectious elementary bodies upon removal of the stress
(4). There is also indirect evidence suggesting that chlamydiae can persist during human ocular (5, 22),
genital (13), and joint (31) diseases and in
atherosclerotic lesions (18). In subhuman primates,
chlamydial DNA and RNA can be detected during a culture-negative state
(15). It was previously found that the culture-recoverable
mouse pneumonitis (MoPn) biovar of C. trachomatis remains in
the normal immunocompetent female mouse genital tract for only up to 5 weeks after culture-apparent clearance of the infection
(10). This finding could argue against long-term persistence contributing to potentially chronic genital tract disease in the immunocompetent murine system.
/
mice to determine if persistence can be extended in mice that are
deficient in the eradicating cytokine-induced activity. We observed
exacerbated disease subsequent to infection in the absence of
iNOS-derived RNI. Thus, we hypothesize that in the absence of iNOS
activity, sterile eradication of chlamydial infection does not occur
and that chlamydial antigen and viable chlamydial organisms persist subsequent to culture-apparent resolution of infection. This model may
be useful for studying the long-term fate of C. trachomatis in the murine genital tract subsequent to cessation of shedding of
viable organisms and for correlating persistent forms of C. trachomatis in the mouse with those previously studied using in vitro systems. Additionally, we seek to define the IFN--inducible mechanism or combination of mechanisms that leads to eradication of murine chlamydial infection in vitro and in vivo.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
/ mice) were obtained under a materials
transfer agreement with John Mudgett (Merck & Co., Rahway, N.J.), and a
colony was initiated at the Midwestern University Animal Resource
Facility. The knockout in these mice was confirmed with randomly
selected experimental animals by PCR of genomic DNA using primer pairs
for the targeting vector containing the neomycin resistance gene and
the NOS2 promoter as described elsewhere (27). B6129F1
control or C57BL/6 wild-type mice (NOS2+/+
mice) were purchased from Taconic Labs (Germantown, N.Y.). All mice
were at least 8 weeks of age at the start of each experiment. Knockout
mice were housed in microisolator cages under barrier conditions with a
10 h-14 h light-dark cycle and were fed sterile food and water ad
libitum. Control mice were housed in standard rodent cages with a
similar light-dark cycle.
70°C for later batch processing. Just prior to and
subsequent to immunosuppression, samples were placed on ice and
cultured on the day they were collected in an effort to increase the
sensitivity of the cell culture system. MoPn was isolated from thawed
swab samples using HeLa 229 cell cultures and enumerated by indirect
immunofluorescence as previously described (11).
80°C. After being thawed, tissues were
homogenized, sonicated, and cleared of large debris by low-speed
centrifugation (10 min at 500 × g at 4°C). Diluted supernatants of homogenates were plated on HeLa 229 cell monolayers in
24-well plates as described elsewhere (10), and culturing was performed as described above for swab samples.
Assessment of chlamydial DNA.
Swab samples for isolation of
viable MoPn and those for detection of DNA were never collected from
the same animal on the same day. Instead, either parallel groups of
mice were assessed or separate collection dates for the same mice were
used to measure these parameters. Vaginal swab samples for chlamydial
DNA detection were collected in phosphate-buffered saline (PBS)
containing 2.5 µg of amphotericin B (Sigma, St. Louis, Mo.) per ml
and were frozen at 20°C until processed. DNA was extracted from
either genital swab samples or excised UGT tissues using a commercially
prepared kit (QIAMP DNA Mini Kit; Qiagen Inc., Valencia, Calif.).
Immunosuppression. Immunosuppression was used to reveal the presence of viable chlamydiae following culture-apparent recovery from the primary infection (10). To enhance the reactivation of chlamydial shedding subsequent to immunosuppression, mice were treated with P4 at 110 and 117 days postinfection. At 120 days postinfection, mice were treated with 200 mg of cyclophosphamide (Cy)/kg (5 mg/mouse) administered intraperitoneally in approximately 0.2 ml of sterile PBS. On each of the succeeding 9 days, mice were treated with 40 mg of Cy/kg (1 mg/mouse) in 0.2 ml of PBS. Control mice were treated with equivalent volumes of PBS only. To assess the reactivation of chlamydial infection, swab samples were collected at 120, 124, 127, and 130 days postinfection for the isolation of MoPn in HeLa 229 cells as described above. Swab samples for the detection of chlamydial DNA were collected 117, 123, 126, and 129 days postinfection. Also at each time point after immunosuppression, some of the animals were sacrificed by cervical dislocation. UGT tissues (uterine horns and oviduct) were aseptically removed and divided at the bifurcation of the uterine horns. They were then prepared for chlamydial isolation in cultures or for extraction of DNA as described above.
Assessment of pathological outcome. Hydrosalpinx formation has been used as a means to assess gross UGT pathology in mice subsequent to chlamydial infection, and some researchers have reported its use as a surrogate marker for infertility (24, 34). At the time of necropsy, hydrosalpinx formation was assessed by gross macroscopic or microscopic observation using a dissecting microscope with ×10 magnification. In most cases, hydrosalpinx formation was readily observable without the aid of a microscope. Also noted at necropsy were any organ changes suggestive of disseminated infection or disease, including iliac lymph node adenopathy, splenomegaly, visceral adhesions, and morphological changes in the lungs and uterus.
The development of infertility subsequent to MoPn infection has been used to delineate susceptible and resistant strains of mice and as a further measure of pathological outcome that may be related to hydrosalpinx formation (14, 26). Fifty-six days subsequent to infection, all mice were assessed for fertility with an adaptation of the method of De La Maza et al. (14). Briefly, experimental or control female mice were placed three or four per cage with a proven breeder male. Baseline weights were recorded initially and subsequently 7, 10, 14, and 18 days later. In addition, at the end of each time interval, abdomens were visually checked and palpated for pregnancy. Obviously pregnant mice were sacrificed at 18 days, and embryos in the left and right uterine horns were counted. If a mouse was not obviously pregnant at the end of 21 days, she was monitored for an additional 1 week in the absence of a male and then introduced to a different male who had successfully mated in the first round of breeding. If at the end of the second round the mouse remained not pregnant, she was considered infertile, sacrificed, and necropsied. Fertility rates were compared between groups of infected mice and uninfected age-matched progesterone-treated control mice.Primary cell cultures.
Lung cells were collected for primary
culture experiments for convenience because processed lung tissue
yielded consistent and reproducible cultures (27).
Single-cell suspensions of lungs from uninfected
NOS2/ and NOS2+/+
mice were derived as described elsewhere and consisted of 90% fibroblasts (27). After the cells reached confluence,
culture medium was aspirated and the cells were treated with either (i) IFN- (50 ng/ml; Pharmingen, San Diego, Calif.) plus
lipopolysaccharide (LPS) (100 ng/ml; Sigma), (ii) IFN- plus LPS plus
1 mM L-NMMA (Calbiochem, La Jolla, Calif.), (iii)
fresh medium plus L-NMMA, or (iv) fresh medium
only for 48 h prior to infection. To confirm iNOS activation,
nitrite accumulation was assessed by the Greiss reaction
(33). Some samples also were tested for the presence of
2,3-IDO as described previously (6). Treatment of cell
cultures with IFN- plus LPS routinely resulted in marked nitrite
accumulation, which could be blocked by the addition of 1 mM
L-NMMA (27). Cultures were infected
with viable MoPn at a multiplicity of infection of 1.0. Infection was
established for 40 h, after which the medium was changed and
replenished with medium without IFN- and LPS (reactivation). If
present during the initial treatment, L-NMMA was
also used when the medium was changed. Parallel triplicate cultures
were fixed in methanol at the following time points after replenishment
of the medium as described above: 0, 24, 48, 60, 72, and 96 h.
After fixation in methanol, wells were stained for chlamydial
inclusions by indirect fluorescent-antibody staining (11).
Control cultures treated and replenished with medium only were used as
the basis for percentage-of-control-data calculations.
Statistics. Rates of hydrosalpinx formation and infertility were compared by Fisher's exact test. Reactivation of infection in vitro was analyzed by a repeated-measures analysis of variance (group and time point postreactivation), and posthoc analysis was completed with a Tukey-Kramer test.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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NOS2/ mice sustain greater pathological outcome
subsequent to infection than NOS2+/+ controls.
While
it has been reported that iNOS-derived RNI are not essential for the
resolution of chlamydial urogenital infection of mice, the role they
play in sterile eradication of the pathogen and disease pathogenesis is
unknown (27). To address the possible role of RNI in
disease pathogenesis, NOS2/, C57BL/6
NOS2+/+, and B6129F1
NOS2+/+ mice were infected intravaginally
with MoPn. Infection in all mice was confirmed by collection of
cervicovaginal swab samples and subsequent culturing. The infection was
then monitored with a subset of 10 mice in each group by further
collection of swab samples at 4, 7, 10, and 14 days postinfection and
every 7 days thereafter until resolution of the infection was
confirmed. We found that NOS2/ mice were
capable of resolving infection in a manner not significantly different
from that of NOS2+/+ mice (data not shown),
confirming previous observations (27).
/ and
NOS2+/+ mice subsequent to primary
infection in two separate experiments. The results are day 56 or later
necropsy and fertility results and necropsy results at day 120. These
time points represent approximately 3 weeks and 12 weeks,
respectively, postinfection resolution in most mice.
NOS2/ and B6129F1
NOS2+/+ mice had similar rates of
hydrosalpinx formation at 56 days postinfection that was significantly
greater than that of C57BL/6 NOS2+/+ mice.
However, when monitored to 120 days postinfection, the rate of
hydrosalpinx formation declined in the infected
NOS2+/+ mice compared to the
NOS2/ mice. Infertility rates at day 56 or
later also correlated well with hydrosalpinx formation. Infertility
rates were not assessed in mice at 120 days postinfection due to the
possibility that age-related fertility decline would complicate the
interpretation of results.
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/ mice sacrificed and necropsied at day
120, an exacerbated hydrosalpinx and grossly distended and dilated
uteri were observed either unilaterally or bilaterally (Fig.
1A). Hematoxylin-eosin-stained sections
revealed that uterine distension and dilation were likely due to the
formation of severe endometrial cysts and cystic hyperplasia (Fig. 1B). While uterine cysts and distension occasionally develop
postinfection in C57BL/6 NOS2+/+ mice
(10), the extent of uterine dilation and distension has not been previously observed by us but is reminiscent of that described by Darville et al. for IFN--deficient mice
(12). We did not observe similar results for any B6129F1
NOS2+/+ or C57BL/6
NOS2+/+ mice in these experiments.
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Detection of chlamydial nucleic acids subsequent to primary
urogenital tract infection.
Considering that exacerbated disease
occurred in NOS2/ mice, we hypothesized that
long-term persistent infections occurred due to microbistatic
suppression of chlamydial growth. The presence of persistent chlamydiae
could potentially provide an impetus for heightened or protracted
inflammatory responses and thus greater pathology. To investigate the
nature and extent of persistent chlamydial infections, we infected
NOS2/ and NOS2+/+
animals as before. Infection in all mice was confirmed on day 4 postinfection by culturing viable MoPn from cervicovaginal swab samples. Subgroups of NOS2/and
NOS2+/+ mice were then monitored for 120 days. Additional swab samples were collected, and total DNA was
extracted and subjected to PCR amplification targeting a portion of the
ompA gene. All PCRs were screened for the presence of bands
of the appropriate size on agarose gel electrophoresis and subsequently
assessed by Sb hybridization to confirm the specificity of the
amplification and to enhance sensitivity when no band was visible on
the gels. Table 2 shows the summarized
results, and Fig. 2 shows individual
results from representative NOS2/ and
NOS2+/+ mice.
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/ and
NOS2+/+ mice for 120 days postinfection. In
several cases, Sb hybridization yielded positive reactions when no
band was present on agarose gels. Uninfected mice yielded negative
results. It thus appears that in this model, chlamydial nucleic acids
persist well beyond the ability to culture the organism regardless of
whether animals are capable of generating RNI.
Evidence for persistent viable C. trachomatis in
NOS2/ mice subsequent to primary infection.
We
also further investigated the nature of chlamydial persistence in this
model by seeking to determine if the origin of chlamydial nucleic acids
was residual nonviable components of the organisms or viable organisms.
It was previously found that infection can be reactivated by the
administration of Cy to various strains of
NOS2+/+ mice for up to 5 weeks past
culture-proven resolution (10). We therefore postulated
that the time frame for reactivation would be protracted in
NOS2/ mice compared to that of controls.
Animals were again treated at days 110 and 117 with P4 and at day 120 with either Cy or PBS. Genital swab samples were collected for
culturing or PCR-Sb hybridization on days 117, 123, 126, and 129 or 130 for all mice. Three to five mice were sacrificed in each group at days
124, 127, and 130, and UGT tissues were excised for culturing,
histological assessment, or PCR-Sb hybridization of DNA extracts. Table
2 also shows the summarized results of this experiment. We observed
renewed shedding of viable chlamydial elementary bodies in 6 of 14 NOS2/ mice treated with Cy but in none of the
PBS-treated NOS2/ or similarly treated
NOS2+/+ animals. It should be noted that
only small numbers of inclusions were observed in these cultures
(<102), whereas during the peak of the primary
infection, between 105 and
107 inclusion-forming units are normally isolated
per swab sample (10).
/ mice from which viable organisms had
been isolated. This finding was likely due to the effect of P4
pretreatment, which often results in a great deal of mucus, a known
inhibitor of PCR (9). Although the presence of large
amounts of mucus was also observed following P4 treatment in the
primary infection, the effect on PCR was not observed, likely due to
the large numbers of organisms present during that time frame.
Reactivation of MoPn infection in primary cell cultures derived
from NOS2/ and NOS2+/+ mice.
It was
previously shown that primary cell cultures derived from
NOS2/ mice restrict chlamydial growth upon
stimulation with IFN- and LPS (27). The results shown
in Fig. 3 support and extend these findings. Primary cell cultures derived from
NOS2/ or NOS2+/+
mice were treated for 48 h with IFN- and LPS.
NOS2+/+ cells showed iNOS activity, as
demonstrated by the Greiss assay (Table
3). NOS2/ cells
showed no evidence of iNOS activity, and activity was substantially reduced in NOS2+/+ cells when incubated in
the presence of 1 mM L-NMMA. Forty-eight hours
posttreatment, cultures were infected with MoPn. At 40 h postinfection (0 h postreactivation), medium was removed and replaced with medium containing neither IFN- nor LPS. Beginning at 24 h
postreactivation, chlamydial replication was restored in all NOS2/ cell cultures and in
NOS2+/+ cells in which iNOS was chemically
blocked by the presence of 1 mM L-NMMA. These
results indicate that IFN- induces as-yet-undefined mechanisms that,
in conjunction with RNI, are microbicidal for MoPn but that, in the
absence of RNI, are microbistatic. Cells also were tested for 2,3-IDO,
a known IFN--induced microbistatic enzyme, but neither
NOS2/ nor NOS2+/+
cells were observed to catabolize tryptophan after cytokine activation (Table 3).
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The present findings, taken together with previous observations (27), indicate that while iNOS activity is not essential to resolution of the infection, it may provide a degree of protection against chronic but not acute disease. We also conclude that disease susceptibility varies in the wild-type mice used as controls in most gene knockout mouse studies (C57BL/6 and B6129F1) and should be a planning factor in study designs that assess disease outcome. Hydrosalpinx formation may not necessarily represent a permanent state in mice in that significantly lower rates of disease were noted for wild-type B6129F1 NOS2+/+ mice at day 120 than at day 56 postinfection, thus indicating a tendency toward resolution of this particular outcome in immunocompetent mice.
The reactivation data indicate that although iNOS-derived RNI
undoubtedly contribute to the eradication of chlamydial infection, other, unidentified cytokine-mediated mechanisms also exist. In the
presence of RNI, the cumulative effects are microbicidal for MoPn, but
in the absence of iNOS activity, they are microbistatic. This
conclusion was indicated by reactivation of infection subsequent to
removal of IFN- only in primary cell cultures derived from NOS2/ mice or in those derived from
NOS2+/+ mice but treated with
L-NMMA. This conclusion was further confirmed by
the ability to reactivate the infection in vivo for prolonged periods
of time and in a larger proportion of NOS2/
mice than of NOS2+/+ mice.
While the exact contributions of additional IFN--inducible
mechanisms remain undefined, known mechanisms may include restriction of iron or some other key nutrients (17, 21, 30) or
production of defensins, protegrins, or other antimicrobial peptides
(38). Others have reported low 2,3-IDO activity in the
RAW264.7 murine macrophage line upon IFN- treatment, but added
tryptophan failed to reverse chlamydial growth inhibition
(8). Given previous results (27) and the data
presented here, we conclude that 2,3-IDO induction and subsequent
tryptophan catabolism are not important factors in this model. In light
of the recent findings by Perry et al. (25) that MoPn is
less sensitive to the effects of IFN- than human strains of C. trachomatis, delineation of the exact IFN--inducible mechanisms
that possess microbicidal and microbistatic activities for various
chlamydiae is of utmost importance. This is true not only for the
interpretation of results in the murine model but also for defining key
protective immune mechanisms evoked during human chlamydial infections
and other animal models of chlamydial infections.
Our finding of persistent chlamydial DNA subsequent to the resolution
of primary infection supports and extends the findings of Beale using
the murine model (3) and Holland et al. using the primate
model of chlamydial ocular infection (15). It also lends
credence to the use of this model for the study of chlamydial pathogenesis because chlamydial DNA or other chlamydial components are
often isolated from culture-negative patients (5, 13, 22,
31). In addition, although Cy administration is known to
reactivate chlamydial infection for a few weeks after primary infection
(3, 10), reactivation out to 120 days postinfection, as
documented here, provides evidence that true long-term persistence is
possible using NOS2/ mice. We recognize that
treatment with Cy reflects a harsh, global form of suppression, and we
understand the need to develop reactivation methods that are less
severe and more informative. For example, specific in vivo depletion
with monoclonal antibodies may be more useful in defining cell types or
cytokines that are responsible for suppressing chlamydial infection
subsequent to culture-proven resolution of infection in either
NOS2/ or NOS2+/+ mice.
It is interesting that NOS2/ mice sustained
more severe pathological outcome subsequent to infection than
NOS2+/+ controls. Viewed through the
paradigm that persistent chlamydial antigen induces immunopathological
host responses, it is tempting to link enhanced disease outcome in
NOS2/ mice to the persistence of potentially
viable but nonculturable chlamydiae. However, we have not yet
conclusively associated these two observations, and they may ultimately
represent independent outcomes in this model. Certainly, establishing a
clear cause-and-effect relationship between chlamydial persistence and
exacerbated disease may prove difficult. Irrespective of whether
this is an attainable goal or not, our model of chlamydial
persistence in NOS2/ mice may have greater
utility in characterizing the biological and antigenic nature of
persistent chlamydial infections in vivo. For example, in vivo host and
pathogen gene profiling studies may be possible since genomic
information has become available to do these types of studies.
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ACKNOWLEDGMENTS |
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This work was supported by Public Health Service grants AI37807 (to K.H.R.) and AI19782 (to G.I.B.).
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FOOTNOTES |
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* Corresponding author. Mailing address: Microbiology Department, Chicago College of Osteopathic Medicine, Midwestern University, 555 31st St., Downers Grove, IL 60515. Phone: (630) 515-6165. Fax (630) 515-7245. E-mail: kramse{at}midwestern.edu.
Editor: R. N. Moore
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REFERENCES |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Infection and Immunity, August 2001, p. 5131-5137, Vol. 69, No. 8
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.8.5131-5137.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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