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Infection and Immunity, August 1999, p. 3757-3762, Vol. 67, No. 8
Department of Microbiology and Immunology,
Baylor College of Medicine, Houston, Texas 77030
Received 25 January 1999/Returned for modification 22 March
1999/Accepted 6 May 1999
To determine the importance of the O75 O antigen and the K5
capsular antigen in resistance to phagocytosis and phagocytic killing,
we used previously described O75 Escherichia coli strains
causing urinary tract infections, septicemia, and neonatal meningitis
typically belong to a restricted set of O and K antigen serogroups. The
O antigen is part of the complex carbohydrate lipopolysaccharide (LPS).
LPS also consists of lipid A (toxic portion of the molecule) and core
oligosaccharide. In addition to LPS, many pathogenic bacteria have an
outermost acidic polysaccharide or capsular antigen (K antigen). The
capsule is made of linear polymers of repeating carbohydrate subunits. Of the more than 160 different O antigens and 80 different K antigens, only a few are associated with disease (1, 18, 20). These major outer membrane antigens of pathogenic strains are thought to
provide resistance to the host defense system, promoting colonization and infection.
Complement-mediated lysis and phagocytosis are the first line of
defense against invading microorganisms. The bacteriolytic activity of
serum usually kills most gram-negative bacteria, but those that are
resistant may then be susceptible to ingestion and killing by
phagocytic cells (15). Polymorphonuclear leukocytes (PMNs)
and monocytes are the major phagocytic cells that phagocytize and lyse
bacteria. K antigens and in some cases O antigens are thought to
provide resistance to phagocytosis (9, 17, 25, 33). The
capsule may provide protection to the organism by masking the
underlying opsonized surface and serving as a physical barrier to the
phagocytic cell (5, 10, 17, 29).
Previously, we investigated the role of the O75 O antigen and the K5
antigen in resistance to complement-mediated lysis by using proven
isogenic mutants in serum resistance assays. The use of genetically
defined mutants is critical for the evaluation of the precise role of
putative virulence factors. We constructed an O75 The K1 antigen has been speculated to be crucial in resistance to
phagocytosis, a possibility supported by results of several studies
(2, 6, 11, 19, 34). However, comparison of K5+
and K1+ strains in phagocytosis assays suggested that the
K5 antigen did not have antiphagocytic properties (6).
Certain O antigens are also speculated to provide resistance to killing
(6, 16, 24). To determine whether the O75 O antigen and the
K5 capsular antigen have a significant role in resistance to
phagocytosis and to phagocytic killing, we analyzed the
O75 Bacterial strains, media, and reagents.
The O75:K5 E. coli strain GR-12 was originally isolated from a patient with
pyelonephritis (28). SMB20 (O
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Loss of Resistance to Ingestion and Phagocytic
Killing by O
and K Mutants of a
Uropathogenic Escherichia coli O75:K5 Strain
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
and K5
mutants from an O75+ K5+ wild-type
uropathogenic Escherichia coli strain in phagocytosis assays with polymorphonuclear leukocytes (PMNs) and monocytes. At a
10-to-1 ratio of bacteria to phagocytes and in the presence of 10%
serum, the parental strain GR-12 was resistant to both PMNs and
monocytes over a 2-h incubation period. The O75 and
K5 mutants were similar in sensitivity to killing by both
PMNs and monocytes, decreasing in viability by 80% in the first hour.
Yet, a significant difference in killing between the O75
and K5 mutants was observed in the first 15 min of
incubation. The K5 mutant decreased in numbers by almost
60%, while the O75 mutant increased in numbers similarly
to GR-12 in the first 15 min. The difference in killing was found not
to be due to the rate of opsonization. To further determine the
mechanism of resistance, a fluorescence assay was used to differentiate
attached and internalized bacteria. The K5 capsule hindered the
association of both the wild-type strain and the O75
mutant in the initial incubation time with PMNs. In conclusion, both
the K5 capsule and O75 O antigen play crucial roles in resistance to
phagocytosis over time.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
mutant
and a K5 mutant of the uropathogenic strain GR-12. The
O75 O antigen was demonstrated to be more important than the K5 antigen
in serum resistance. The K5 capsule antigen, in contrast, played only a minor role in resistance to serum (4). Although the K5
capsule was not crucial in serum resistance, it may play a role in
protection from phagocytic activity.
and K5 mutants along with GR-12 by
several different phagocytosis assays not only to determine the
importance of the antigens in providing resistance to phagocytosis but
also to differentiate the step at which resistance was provided by the
O and K antigens.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
mutant) and
SMB213 (K mutant) are defined chromosomal mutants derived
from GR-12 and were described previously (4).
Isolation of PMNs and monocytes. Human serum was collected from five healthy donors with no recent history of antibiotic therapy as previously described by Burns and Hull (4). Blood for separation of PMNs and monocytes was donated from an individual into heparin (143 USP units of sodium heparin) Vacutainer tubes (Becton Dickinson, Franklin Lakes, N.J.) in 20- to 40-ml volumes. A discontinuous density gradient was made by placing 1 ml of a 5:3 ratio of Mono-Poly resolving medium (M-PRM; ICN Biomedicals, Aurora, Ohio) and ddH2O onto 3 ml of M-PRM in a 15-ml polypropylene conical tube (Corning Inc., Corning, N.Y.). A 4-ml volume of heparin-treated blood was layered on top of the density gradient and separated by spinning in a tabletop centrifuge (Centra GP8R; International Equipment Company, Needham Heights, Mass.) at 750 × g for 50 min; the centrifuge was allowed to stop without the brake. For isolation of PMNs, 16 ml of blood was separated. When monocytes were isolated, 40 ml of blood was separated.
After separation, the plasma layer was discarded and the appropriate fraction either mononuclear or PMNs as described in the M-PRM manufacturer's directions, was removed and washed with DPBS. The washed cells were pelleted at 250 × g for 10 min. The buffer was discarded, and the cells were resuspended in the remaining buffer. The erythrocytes were lysed by adding cold ddH2O; after 20 s, the isotonicity was restored by adding 2× DPBS. The cells were pelleted at 130 × g for 8 min. The lysis step was repeated if necessary. Cells were resuspended in 1/10 the volume of blood originally collected. The viability and concentration of the cells were determined by trypan blue exclusion. In each experiment, at least 98% of the PMNs and 95% of the monocytes were viable. The purity of the preparation was determined by using 0.1 N HCl. The PMN fraction was an average of 96% PMNs. The mononuclear fraction contained on average of 26% monocytes, with only 1% contaminating PMNs.Phagocytosis killing assays. The procedure for the phagocytosis killing assay was adapted from that of Rest and Speert (22). Bacteria were grown to the exponential phase and counted in a hemocytometer. For certain experiments, bacteria were preopsonized by incubation in 10% human serum for 30 min at 37°C. Reactions consisted of a 10:1 ratio of bacteria to phagocytes, 10% serum or 10% heat-inactivated serum as a complement source, 1 mM MgCl2 and CaCl2, and DPBS as a diluent. A concentration of 4 × 106 PMNs/ml or 1.5 × 106 to 2.0 × 106 monocytes/ml was used in the assays as appropriate. The reactions were incubated in Falcon polypropylene snap cap tubes (Becton Dickinson) at 37°C, with rotation, for 2 h. Samples in duplicate were removed at 0, 15, 30, 60, and 120 min and diluted in ddH2O to lyse the phagocytes releasing internalized bacteria. To separate PMNs from unattached bacteria, additional samples were taken, placed on ice, and spun in a low-speed HF-120 capsule centrifuge (Tomy Seiko Co., Tokyo, Japan) for 10 s to pellet the PMNs. The supernatant was subsequently removed and diluted. Samples from dilutions were plated on LB plates and incubated at 37°C overnight, and viable counts were determined. Percentages for each time point were obtained by dividing the surviving viable count by the initial viable count. Statistical analysis was done by the two-sample t test (35).
Attached versus internalized bacteria. Bacteria grown to the exponential phase were pelleted and washed twice with saline. Approximately 3 × 109 CFU of bacteria were resuspended in 1 ml of FITC (0.15 mg/ml) in DPBS. The suspension was protected from light and rotated at 37°C for 30 min to label the bacteria. Viability was determined before and after labeling. Bacteria were viable after labeling with 0.15 mg of FITC per ml in DPBS. The reactions were set up as described above; 50-µl samples were taken at 0, 5, 15, and 30 min and immediately placed on ice. The PMNs were separated from unattached bacteria as described above, resuspended in an equal volume of DPBS, and placed on ice. To quench the extracellular fluorescence of attached bacteria, 2.5 µl of a 0.1% ethidium bromide solution was added to the samples prior to visualization as described by Arduino et al. (3). The mixture was placed on a glass slide and overlaid with a coverslip, and the number of attached and/or internalized bacteria was determined by examination with a Leitz fluorescence microscope (Wetzlar, Germany) with an FITC filter under oil immersion (magnification of ×1,000). Bacteria attached to the PMNs appeared yellow or yellow-green with yellow-orange centers, whereas the internalized bacteria were an intense green. Twenty-five individual PMNs were examined per sample, and attached and ingested bacteria per PMN were counted. The results were averaged for four individual experiments.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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To investigate whether the O75 O antigen and the K5 capsular
antigen provide resistance to phagocytosis or phagocytic killing, phagocytosis assays were performed with an O75 mutant
(SMB20), a K5 mutant (SMB213), and the parental strain
(GR-12). Previously, an O75 mutant and a K5
mutant were constructed in the uropathogenic E. coli strain
GR-12 by mutating the rfbD gene and the kfiC
gene, respectively (4).
PMN phagocytosis killing assays.
The wild-type strain and
mutants at the exponential phase of growth were mixed with PMNs at a
10:1 ratio of bacteria to PMNs with 10% serum or 10% heat-inactivated
serum added. The reaction mixtures were incubated with rotation at
37°C for 2 h, and viable counts were determined in duplicate at
0, 15, 30, 60, and 120 min. To control for sensitivity of the bacterial
strains to 10% serum, the same reactions were performed without PMNs
added. The cumulative results of seven different assays using blood
from seven different donors are shown in Fig.
1 as percent viability. A 15-min time
point was performed for only five of the seven assays. The wild-type
strain (GR-12) maintained its number for the first hour and then
increased in number in the second hour of incubation. Both SMB20
(O75 mutant) and SMB213 (K5 mutant)
decreased approximately 80% in viability in the first and second hours
of incubation. These results were significantly different from the
sensitivity exhibited by GR-12 (Table 1). The K5 mutant was more sensitive to killing by PMNs in
the first 15 min of incubation compared to GR-12 and the
O75 mutant. An average of only 42.5% ± 11.8% of the
K5 mutant was still viable in the first 15 min, compared
to 148.8% ± 61.3% and 130.0% ± 34.1% for the O75
mutant and GR-12, respectively (n = 5; P < 0.02
for SMB213 compared to SMB20 and P < 0.001 for SMB213
compared to GR-12). Yet, at 30 min there was no statistical difference
in the survival of the O75 mutant compared to the
K5 mutant. GR-12 and the mutants were not sensitive to
10% serum alone (Fig. 1). When 10% heat-inactivated serum was used as
the complement source, GR-12 and the mutant strains were not sensitive to killing by PMNs (data not shown).
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Preopsonization phagocytosis killing assays.
To determine
whether the difference in killing of SMB20 (O75 mutant)
and SMB213 (K5 mutant) was due to a difference in the
kinetics of opsonization of the bacteria, GR-12 and the mutants were
preopsonized in 10% serum for 30 min to equalize the amount of time of
opsonization prior to the addition to PMNs. The K5 mutant
was still killed more rapidly than the O75 mutant despite
preopsonization (Fig. 2). We noted an
approximately 60% decrease in viability of SMB213 and an increase in
viability of SMB20 at the 15-minute time point.
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Separation of nonassociated bacteria from PMNs.
The assay
described above (without preopsonization) was repeated except that PMNs
were separated from nonassociated bacteria and the viable number of
bacteria in the supernatant was determined. The bar graph in Fig.
3 compares GR-12 and the mutants by the number of viable bacteria in the supernatant compared to the initial number of viable bacteria in the reaction. At time zero, all bacteria are nonassociated, as approximately 100% are in the supernatant. The
numbers of viable SMB213 (K5 mutant) decreased by 80% in
the supernatant at 15 min, while the viable numbers of GR-12 and SMB20
(O75 mutant) exhibited no decrease in the supernatant
fraction. By 30 min, the number of viable bacteria in the supernatant
for both mutants had declined to 20%, while 65% of the parent
remained in the supernatant. These results indicate that the
K5 mutant associated with PMNs faster than GR-12 and the
O75 mutant in the first 15 min, but by 30 min equal
numbers of the K5 and O75 mutants were
associated with the PMNs.
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Uptake versus attachment assays.
Since preopsonization of the
mutants did not predispose SMB20 (O75 mutant) to faster
killing by PMNs, similar to findings for SMB213 (K5
mutant), we investigated whether the difference in killing was due to
the kinetics of association. We performed a phagocytosis assay
developed by Arduino et al. in which FITC-labeled bacteria are used
along with ethidium bromide to quench the extracellular fluorescence of
the attached bacteria, allowing distinction between attached and
ingested bacteria (3). Bacteria were labeled with slight
modification, using a lower concentration of FITC (0.15 mg/ml) in DPBS
to retain bacterial viability as described in Materials and Methods.
Average viabilities after 30 min of labeling for GR-12, SMB213, and
SMB20 were 96.5, 94.5, and 87.0%, respectively. Labeled bacteria were
added as before; samples were taken at 0, 5, 15, and 30 min and
immediately placed on ice. After separation of PMNs from nonassociated
bacteria, ethidium bromide was added to quench extracellular
fluorescence of bacteria immediately before viewing under the microscope.
mutant). For GR-12 and SMB20 (O75
mutant), however, less than one bacterium was attached and/or ingested
per PMN. At 15 min, the K5 mutant increased its
association with PMNs to 5.05 ± 0.77 ingested and 1.51 ± 0.13 attached, compared with 0.66 ± 0.37 ingested and 0.70 ± 0.18 attached for the O75 mutant. GR-12 was similar to
the O75 mutant at 15 min, with approximately one
bacterium attached and one ingested per PMN. Although at 30 min GR-12
and the O75 mutant were similar in the number of
associated bacteria per PMN, the O75 mutant was ingested
by PMNs in greater numbers than was GR-12. For GR-12, 2.79 ± 1.00 bacteria were attached per PMN, compared with only 0.51 ± 0.22 attached for the O75 mutant. This difference was
reversed, however, when the numbers of ingested bacteria were compared:
for the O75 mutant, 2.78 ± 0.80 bacteria were ingested per PMN, while for GR-12 the number
was only 1.44 ± 0.80. For the K5 mutant, the
numbers of bacteria associated with PMNs were similar for the 15- and
30-min time points. Shown in Fig. 4B are the results of using
heat-inactivated serum as a complement source. After 60 min, the
K5 mutant was both associated with and internalized by
PMNs, while the O75 mutant and GR-12 were relatively
resistant to both attachment and ingestion.
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Monocyte phagocytosis killing assays.
Although PMNs are a
major defense mechanism against invading bacteria, monocytes are also
thought to play a role in clearance of bacteria. The phagocytosis
killing assay was repeated with monocytes at the same ratio, 10:1. The
results of these experiments are presented in Fig.
5 and Table 1. There was donor-to-donor variability in killing, as depicted by the large error bars in the
graph. Nonetheless, the difference in the means between GR-12 and the
mutants were significant at all time points, as shown in Table 1. The
difference in the killing of the O75 and K5
mutants at the 15-min time point was also significant. The viability of
the K5 mutant at 15 min was 46 ± 20, compared to
105 ± 45 and 162 ± 32 for the O75 mutant and
GR-12, respectively. There was no significant difference in killing of
the mutants at subsequent time points.
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and K5 mutants. Monocytes are able to
kill the mutants just as efficiently as PMNs. There was a significant
difference in killing by PMNs and the monocytes at the 2-h time point,
at which monocytes were more effectively able to kill GR-12 than were
PMNs (P < 0.05).
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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The use of genetically defined mutants of a pathogenic strain
allows for more conclusive determination of the importance of a
specific virulence factor. We previously constructed proven isogenic
mutants in a wild-type pathogenic strain, creating an O75
mutant and a K5 mutant. The mutants were analyzed
phenotypically and tested in serum resistance assays. The O75 O antigen
was shown to be more important than the K5 antigen in serum resistance
(4). Although the K5 antigen was not critical in serum
resistance, we sought to determine if it played a role in resistance to phagocytosis.
This is the first study in which isogenic mutants were used in
phagocytosis assays. Previous studies to determine the role of O and K
antigens in resistance to phagocytosis are for the most part
inferential. Genetically defined mutants are superior for determining
the importance of a virulence factor because it is possible to
attribute the cause for loss or gain of a specific attribute to the
mutation. In our study, we can directly compare an O75+
K5+ strain with an O75 strain and a
K5 strain to reliably determine the role of the O75 and
K5 antigens in resistance to phagocytic killing. The wild-type strain
and the O75 and K5 mutants were tested in
phagocytosis killing assays with PMNs. The wild-type strain was
resistant to killing, whereas the mutants were sensitive. The
O75 and K5 mutants were equally sensitive
to killing at 30-min and subsequent time points. These results support
the evidence that certain O and K antigens provide resistance to
killing by phagocytes. In this study, the K5 capsule imparts resistance
to phagocytic killing, as shown by comparison of the viability of the
wild-type strain (bearing the K5 capsule) to that of the
K5 mutant in the phagocytosis killing assay. The O75 O
antigen also imparts resistance to phagocytic killing similarly to the
K5 antigen.
Despite similar killing of the mutants at 30-min and later time points,
there was a significant difference in killing of the mutants at the
15-min time point, with the K5 mutant exhibiting a more
rapid decrease in viability than the O75 mutant. Thus,
the K5 capsule was more important in the survival of the organism
during the initial incubation with PMNs. We demonstrated that the
difference was not due to a difference in opsonization because despite
preopsonization, there was still a significant difference in killing of
the two mutants.
The kinetics of phagocytic killing may be divided into several stages:
rate of opsonization, rate of attachment, rate of internalization, and
rate of killing (7, 10). Since the difference in the killing
of the mutants is not due to the rate of opsonization, we further
analyzed the association of the mutants with PMNs and demonstrated that
the K5 mutant associated more rapidly with PMNs. To
confirm the difference in rate of association and to differentiate the
type of association (attached versus internalized bacteria), we used a
fluorescence assay which distinguishes between attached and
internalized bacteria (3). Our results indicate that the K5
capsule prevented initial association with the PMNs, and bacteria
lacking the K5 capsule quickly attached and were internalized by PMNs.
After the O75 mutant eventually associated with PMNs, it
was internalized faster than the wild-type strain. This indicated that
in the presence of the K5 capsule, the O75 O antigen hinders
internalization. Thus, we conclude that the K5 antigen prevents
attachment to phagocytic cells and that both the O75 and K5 antigens
confer protection from internalization.
When heated serum was used in the fluorescence assay, greater numbers
of the K5 mutant than of the O75 mutant or
parent strain GR-12 associated with PMNs. This finding indicates that
complement is not necessary for the attachment and internalization of
the unencapsulated mutant, but complement is necessary for attachment
of the encapsulated strains. It is also interesting that although the
K5 mutant was internalized by PMNs in the presence of
heat-inactivated serum, it was not killed. Thus, despite ingestion of
the K5 mutant, heat-labile factors were not present to
completely stimulate the mechanisms necessary for killing the mutant.
In addition to PMNs, monocytes are an important defense to invading microorganisms. The rate and pattern of killing by monocytes paralleled those of the PMNs. These results support the findings of other groups who have found similar killing activity by monocytes (21, 23) but are in conflict with studies by Verbrugh (32), who found that monocytes had a lower bactericidal activity than PMNs. We also report that monocytes were significantly better at killing GR-12 at the 2-h time point. Grunwald et al. proposed that a CD14-defined phagocytosis pathway by monocytes may be able to destroy gram-negative pathogens which are resistant to both complement-mediated lysis and phagocytosis by PMNs (8). Our finding that GR-12 is more sensitive to killing by monocytes than PMNs supports this hypothesis.
Capsules are thought to impair phagocytosis by several mechanisms: by their hydrophilic nature, by serving as camouflage, and by masking the underlying opsonized surface (5, 10, 17, 29). Most investigations have focused on the K1 antigen, and more studies on the role of other K antigens and O antigens in phagocytosis are needed. The K5 capsule may prevent initial association with PMNs because the phagocytic cell does not recognize the opsonized bacteria. O antigens activate complement components that are recognized by complement receptors present on phagocytic cells. Antibodies to LPS which opsonize the bacterium are also formed. If the opsonized surface is covered by a capsule, the phagocytic cell will not recognize the opsonized bacteria. Kim et al. showed that the K5 capsule prevented anti-LPS monoclonal antibody from interacting with its target because it conferred protection to an unencapsulated but not an encapsulated, bacterial strain in a neonatal rat model (14).
Furthermore, the K5 capsule is poorly immunogenic due to its identity with an intermediate in the synthesis of heparin (30). The K5 capsule avoids immune recognition, and thus no antibodies against the K5 capsule are produced (13). The K5 capsule is also thought to not activate the alternative pathway (26). Thus, the K5 capsular surface is not opsonized by antibodies or complement.
The capsule may prevent association with the PMN simply due to its
hydrophilicity and negative charge (17, 24, 31), characteristics that result in repulsion from the phagocytic cell (12). The K5 mutant associates with PMNs in
the absence or presence of complement, although faster in the presence
of complement. Little is known about opsonization in the urinary tract
other than the work of Suzuki et al. showing that there is a small
amount of opsonic activity in urine (27). In the bladder,
bacteria lacking a capsule may be phagocytized more efficiently than
encapsulated strains and thus cleared from the bladder either by
phagocyte killing or by voiding of the urine. Thus, if there is not
enough complement present to effectively opsonize the bacteria, the K5
capsule plays a crucial role in preventing attachment and internalization.
This is the first study in which defined O75 and
K5 mutants were used in phagocytosis killing assays to
determine the role of the O75 and K5 antigens in resistance to
phagocytic killing. We have shown that both the K5 capsular antigen and
the O75 O antigen are important in resistance to phagocytic killing.
However, the K5 antigen plays a more critical role in the initial
encounter between bacteria and phagocytic cells.
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ACKNOWLEDGMENTS |
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This research was supported by Public Health Service grant NIAID21009 to S.I.H.
We greatly appreciate Robert Rakita and Holly Birdsall for their insightful and helpful discussions and Phillip Wyde for assistance with fluorescence microscopy.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Microbiology and Immunology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Phone: (713) 798-4021. Fax: (713) 798-7375. E-mail: sb691010{at}bcm.tmc.edu.
Editor: T. R. Kozel
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Infection and Immunity, August 1999, p. 3757-3762, Vol. 67, No. 8
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