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Infection and Immunity, July 1999, p. 3566-3570, Vol. 67, No. 7
Division of Infectious Diseases,
Received 12 February 1999/Returned for modification 2 April
1999/Accepted 20 April 1999
The vast majority of cases of gram-negative meningitis in neonates
are caused by K1-encapsulated Escherichia coli. The role of
the K1 capsule in the pathogenesis of E. coli meningitis
was examined with an in vivo model of experimental hematogenous
E. coli K1 meningitis and an in vitro model of the
blood-brain barrier. Bacteremia was induced in neonatal rats with the
E. coli K1 strain C5 (O18:K1) or its K1 Meningitis remains a potentially
devastating disease. In the neonatal period Escherichia coli
is the most common gram-negative pathogen responsible for meningitis
(9, 31). It is associated with a mortality rate as high as
40%, and more than half of the survivors have neurologic sequelae
(9, 31). The poor outcome statistics, despite medical
advances, including bactericidal antibiotics and improved
intensive-care unit care, point to our incomplete knowledge of the
pathogenesis and pathophysiology of neonatal E. coli
meningitis. It is well documented that the majority of cases of
neonatal E. coli meningitis are caused by K1-encapsulated bacteria (26). The reasons for this association are myriad
and may include the neonatal immune system's incomplete ability to localize and fight infection and the propensity of certain strains of
E. coli to invade the central nervous system. In addition, studies of infants with meningitis and animal models of meningitis have
shown that a high level of bacteremia is required for the development
of E. coli meningitis (7, 15). Previous
investigations have determined that the K1 capsule contributes to this
high level of bacteremia by virtue of its serum resistance and
antiphagocytic properties (15).
In an effort to better understand how systemically circulating E. coli bacteria cross the blood-brain barrier, we have used an in
vivo model of neonatal rat meningitis (13, 15). This model
shares several characteristics with human neonatal meningitis, most
notably hematogenous infection of the meninges. In addition we have
used an in vitro model of the blood-brain barrier, with bovine brain
microvascular endothelial cell (BMEC) monolayers (11, 25,
29), to examine the process of invasion by K1+ and
K1 Bacterial strains.
The clinical isolate of K1-encapsulated
E. coli, strain C5 (O18:K1), and its unencapsulated mutant
C5ME have been characterized previously (15). Briefly,
strain C5 was isolated from the cerebrospinal fluid (CSF) of a newborn
infant with E. coli meningitis. Strain C5ME was obtained by
selection for resistance to the K1-specific bacteriophages. Strain C5ME
was examined for the loss of capsule production by the antiserum agar
technique, testing for agglutination with an anti-K1 monoclonal
antibody as well as lytic sensitivity to the K1-specific
bacteriophages, as described previously (15). Extensive
investigations have been undertaken to examine known virulence factors
in the K1 mutant in order to ensure that these phenotypic
characteristics remained intact. There were no phenotypic alterations
in virulence factors such as outer membrane protein, S fimbriae, O18
lipopolysaccharide (LPS), and the invasion protein Ibe10 (11, 15,
24). The parent K1+ strain and the K1 mutant strain
possess identical hemolysin, biochemical reactions, and patterns of
binding to homologous LPS monoclonal antibody (15). In
addition, these strains were found to have identical genotypes when
they were examined by multilocus enzyme electrophoresis
(15).
Animal model for E. coli bacteremia and meningitis.
E. coli bacteremia and meningitis, defined as a positive CSF
culture, were induced in 5-day-old rats by a method described previously (13, 15). Briefly, outbred,
specific-pathogen-free, pregnant Sprague-Dawley rats with timed
conception were purchased from Charles River Breeding Laboratories
(Wilmington, Mass.); the rats delivered in our vivarium 5 to 7 days
after arrival. Each adult rat and her pups (average litter size, 10;
range, 8 to 16) were housed in an opaque solid-polypropylene cage under a Small Animal Isolator (model 1894; Forma Scientific, Inc., Marietta, Ohio).
Detection of bacteria in CSF.
Two methods were used to
demonstrate morphologically the presence of bacteria in CSF. First,
selected CSF specimens were pooled and concentrated by cytospin
centrifugation, stained with acridine orange, and examined under a
fluorescence microscope (Olympus BH2, equipped with a wide-band BPu 95 filter set).
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
The Capsule Supports Survival but Not Traversal of
Escherichia coli K1 across the Blood-Brain Barrier
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
derivative, C5ME. Subsequently, blood and cerebrospinal fluid (CSF)
were obtained for culture. Viable bacteria were recovered from the CSF
of animals infected with E. coli K1 strains only; none of
the animals infected with K1 strains had positive CSF
cultures. However, despite the fact that their cultures were sterile,
the presence of O18 E. coli was demonstrated
immunocytochemically in the brains of animals infected with
K1 strains and was seen by staining of CSF samples. In
vitro, brain microvascular endothelial cells (BMEC) were incubated with
K1+ and K1 E. coli strains. The
recovery of viable intracellular organisms of the K1+
strain was significantly higher than that for the K1
strain (P = 0.0005). The recovery of viable
intracellular K1 E. coli bacteria was
increased by cycloheximide treatment of BMEC (P = 0.0059) but was not affected by nitric oxide synthase inhibitors or
oxygen radical scavengers. We conclude that the K1 capsule is not
necessary for the invasion of bacteria into brain endothelial cells but
is responsible for helping to maintain bacterial viability during
invasion of the blood-brain barrier.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
E. coli. While the use of two different
species of brain tissues may be questioned, in detailed experiments
performed in our laboratory, the interactions between E. coli and brain endothelial cells were found to be similar,
regardless of the cell's species of derivation (25, 29).
Given that bovine brain cells are more readily available, we opted to
use these cells for our in vitro experiments. The present research uses
these experimental models to examine, in part, the process by which
bacteria gain access to the central nervous system and remain viable.
We hypothesize that the K1 capsule is not necessary for the invasion of
brain endothelial cells. It is, however, an important virulence factor,
protecting E. coli from host defenses, and thus the
bacterium is able to cross the blood-brain barrier alive, ultimately
leading to meningitis.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
Immunocytochemical detection of bacteria in brains. Brains from infected animals were embedded in OCT compound (Tissue Tek; Sakura Finetek) and cut by using a B1-H1 cryostat. Sections were fixed in acetone, preincubated with 1% acetic acid to block endogenous alkaline phosphatase activity, and then blocked with 5% heat-inactivated serum to avoid nonspecific binding of immunoglobulin (Ig) to neuronal tissues. The sections were then incubated with the primary antibody to O18 LPS (murine IgG monoclonal anti-O18 antibody) (14), followed by incubation with the secondary antibody (biotinylated sheep anti-mouse IgG). The sections were further incubated with alkaline phosphatase-conjugated streptavidin. Visualization of the antigen-antibody complex (red color) was done with the Alkaline Phosphatase Substrate Kit I (Vector Laboratories), and sections were counterstained with hematoxylin. Controls used for tissue specimens as well as for antibodies included uninfected brains and omission of the primary and/or secondary antibody.
In vitro invasion assays. An in vitro model of the blood-brain barrier was developed with bovine BMECs. These cells were isolated and cultured as described previously (29) and were used in invasion experiments. Ten million bacteria in 500 µl of experimental media (Ham's F-12, medium 199, 1× Earle salts [1:1], 5% heat inactivated fetal bovine serum [FBS], 1% sodium pyruvate, and 0.5% glutamine) (Irvine Scientific) were added to confluent BMEC monolayers at a multiplicity of infection of 100. The cells and bacteria were incubated for 11/2 h at 37°C under 5% CO2 without shaking. The monolayers were then washed four times with M199 and reincubated with experimental medium containing gentamicin (100 µg/ml) for 1 h at 37°C to kill extracellular bacteria. In pilot experiments performed previously, there was no bacterial survival in the absence of BMECs if gentamicin was present. To determine the number of viable intracellular bacteria, the monolayers were washed five times with M199 and lysed with 100 µl of 0.1% Triton X-100 for 10 min. Four hundred microliters of M199 was then added to the wells for a final volume of 500 µl. Bacterial viability was not affected by this Triton X-100 treatment. CFUs were determined for each well by plating 50 µl undiluted and three serial 10-fold dilutions on blood agar plates. These experiments were conducted in triplicate and were repeated a minimum of three times.
Effects of eukaryotic inhibitors on E. coli
invasion.
Cycloheximide (20 µg/ml),
N
-nitro-L-arginine (NNLA) (1 mM),
N
-methyl-L-arginine (NMLA) (1 mM),
superoxide dismutase (SOD) (100 µg/ml), or catalase (5,000 U/ml) (all
from Sigma) was added to the wells in 400 µl of experimental medium
1 h before the bacteria. None of these inhibitors affected the
viability or morphology of the BMEC monolayers. Bacteria were then
added in 100-µl volumes to the wells, thereby decreasing the
concentrations of inhibitors by 20%. Following this, the procedure was
the same as that for the invasion assay described above. Experiments
with cycloheximide were run in multiples of 3, 6, or 9 and repeated
five times, while those done with other inhibitors were performed in
replicates of 3 or 6 and repeated three times.
Statistics. Nonparametric (Mann-Whitney), unpaired, two-tailed tests were used to examine the differences between experimental study groups.
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RESULTS |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Demonstration of live bacteria in the CSF from the animal
model.
To examine whether the entry of E. coli K1 into
the central nervous system requires the capsule, E. coli
bacteremia and meningitis were induced in 5-day-old rats with a
K1-encapsulated and a K1 E. coli strain. The
isolation of E. coli from CSF was, as expected, observed in
animals infected with the K1+ strain, who developed a high
degree of bacteremia (e.g., >105 CFU/ml of blood).
Overall, 14 of the 24 (58%) animals who were infected with strain C5
and had levels of bacteremia greater than 105 CFU/ml of
blood were found to have positive CSF cultures. In contrast, none of
the 20 animals infected with the K1 strain (C5ME) were
found to have positive CSF cultures, despite the fact that all animals
developed similarly high levels of bacteremia. However, we were not
able to exclude the possibility that these CSF specimens might contain
viable bacterial counts below the lower limit of detection, e.g., <10
CFU/total CSF, assuming that the total CSF of a 5-day-old rat is
approximately 50 to 100 µl.
Detection of bacteria in CSF.
Due to the observation that
animals infected with K1 bacteria did not develop
meningitis, despite reaching high levels of bacteremia, we sought to
determine if the K1 capsule was necessary for blood-brain barrier
traversal or if it might function to facilitate bacterial survival
instead. If the latter hypothesis is true, one would expect to find
evidence of nonviable K1 E. coli in the CSF or
brains of animals infected with this strain. The presence of bacteria
was demonstrated in the CSF of these animals by two methods as
described above. Figure 1 demonstrates the presence of bacteria with a morphology consistent with that of
E. coli in acridine orange-stained cytospin specimens from animals infected with K1 E. coli whose CSF
cultures were sterile. Similarly, methylene blue staining of semithin
sections of pelleted and fixed sterile CSF specimens from animals
infected with C5ME (K1) revealed the presence of bacteria
with a bacillus morphology consistent with that of E. coli
(data not shown). These data suggest that the K1 strain
is able to cross the blood-brain barrier and thus that sterile CSF
cultures may not represent failure of the organism to invade the
central nervous system. Instead, these organisms may be unable to
survive the invasion process.
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Immunocytochemical detection of bacteria in the brain.
Corroborating evidence that K1 organisms could be found
in the brains of animals infected with the K1 mutant, despite their having sterile CSF cultures, comes from immunocytochemical studies of
brain sections from K1-infected animals. The presence of
O18 E. coli was demonstrated in the brain sections of two
animals infected with E. coli K1 and two animals infected
with its K1 derivative. Figure
2 shows representative brain cortex
slices from three different experimental conditions: infection with
E. coli K1 C5 (panel A), omission of primary antibody (panel
B), and infection with C5ME (K1) (panel C). The brain of
an animal with a positive CSF culture infected with strain C5 revealed
red precipitates, indicating E. coli stained by an O18 LPS
antibody in the brain cortex (Fig. 2A). Controls (uninfected brains or
brain sections for which the primary anti-O18 antibody was omitted) did
not show any red precipitates (Fig. 2B), supporting the specificity of
O18 E. coli interaction with the anti-O18 antibody.
Similarly, E. coli bacteria, stained by O18 LPS antibody,
were demonstrated in the brain of an animal infected with strain C5ME
(O18+ K1) despite the fact that its CSF
cultures were sterile (Fig. 2C). This methodology does not permit the
identification of the cellular location of these bacteria.
Nevertheless, these results suggest that animals infected with
K1 E. coli had bacteria present in their
brains, despite having sterile CSF cultures.
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Invasion assay and effects of eukaryotic inhibitors. Our hypothesis, as supported by the previous data, is that the K1 capsule is not required for the invasion of E. coli into BMECs but does serve to protect the bacteria from being killed during the invasion process. To better understand the role of the K1 capsule in the invasion of BMECs by E. coli, we performed the following in vitro tissue culture invasion assays.
An in vitro model of the blood-brain barrier was developed with BMECs as described above. Experiments with the K1+ and K1 E. coli strains revealed a fourfold
increase in the intracellular recovery of viable C5 (K1+)
organisms over that of viable C5ME (K1) organisms
(P = 0.0005) after invasion of BMEC monolayers (Fig. 3). This correlates with our in vivo
finding that viable bacteria were recovered only from CSF specimens of
animals infected with E. coli K1.
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E. coli (C5ME) organisms but
not in that of K1+ E. coli (C5) organisms (Fig.
3). These findings suggest that a newly synthesized endothelial-cell
product might be responsible for the decreased recovery of the
K1 strain. Nitric oxide (NO) and oxygen intermediates
have been shown to have antimicrobial properties (2, 8, 20).
NO and/or nitric oxide synthase (NOS) has been isolated from a variety of different types of endothelial cells (12, 23), including cerebrovascular endothelium (5, 21). Superoxide and hydrogen peroxide have also been detected in cultured endothelial cells (4,
27). To see if NO and/or oxygen radicals produced by BMECs might
be involved in the killing of K1 organisms, one of the
NOS inhibitors NNLA (1 mM), NMLA (1 mM), SOD (100 µg/ml), and
catalase (5,000 U/ml) (all from Sigma) was added to wells 1 h
before the bacteria. None of these inhibitors affected the viability or
morphology of the BMEC monolayers. Bacteria were then added in 100-µl
volumes to the wells. As shown in Fig. 4,
the addition of either of two NOS inhibitors (NNLA and NMLA), SOD-catalase, or a combination of SOD-catalase with NNLA or NMLA did
not affect the intracellular recovery of K1 E. coli; the level of bacterial killing was similar to that in the
standard invasion assay. Therefore, these agents could not duplicate
the survival benefit seen with cycloheximide.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In the study of the pathogenesis of E. coli meningitis,
we employed both in vitro and in vivo models of the blood-brain
barrier, using BMEC monolayers and experimental hematogenous meningitis in neonatal rats. Our present findings revealed that both
K1+ and K1 E. coli bacteria can
invade the central nervous system and BMECs; however, significantly
fewer K1 bacteria remain alive after invasion. The CSF
clinical isolate of wild-type O18:K1+ E. coli,
strain C5, and its K1 derivative, C5ME, have been shown
to be identical in terms of several phenotypic properties examined
(e.g., outer membrane protein, O18 LPS, S fimbriae, and invasion
proteins) except for the presence of the capsule (11, 15,
24). Therefore, the differences between the recovery of live
K1+ and K1 E. coli bacteria can
likely be ascribed to the capsule. Because the genetic basis of the
loss of the K1 capsule for the K1 phage-derived C5ME is undetermined,
work to construct a genetically defined K1 mutant strain is in
progress. This strain will contribute to further understanding of the
role of the K1 capsule in central nervous system invasion by E. coli K1. Despite this limitation, we believe that the present
study supports our hypothesis that the capsule is a crucial component
of the bacterium's armamentarium, allowing it to cross the blood-brain
barrier and remain viable.
Prior in vivo studies of E. coli K1 meningitis have shown
that the K1 capsule is a critical factor in the development of
meningitis by virtue of its serum resistance and antiphagocytic
properties (15). This is again demonstrated in this study by
the differences in inoculum size required for the K1+ and
K1 strains to reach high levels of bacteremia in neonatal
rats. An inoculum approximately 104- to
105-fold greater was required for the K1
strain to achieve a high degree of bacteremia (e.g., >105
CFU/ml of blood) compared to the parent K1+ strain. The
sterile CSF cultures from animals infected with K1
strains were previously interpreted to mean that the K1 capsule was
necessary for the bacterial crossing of the blood-brain barrier (15). Several lines of evidence presented in this paper now suggest that the capsule is not necessary for invading BMECs but is
responsible for maintaining the viability of the bacteria inside the
BMECs. This is supported by the in vivo observation that while both
K1+ and K1 E. coli bacteria are
found in brains by immunocytochemical assays and in CSF by acridine
orange and methylene blue staining, CSF cultures did not reveal any
viable K1 E. coli organisms. These bacteria
were therefore able to enter the central nervous system but were
presumably killed in the process. We cannot completely exclude the
possibility that sterile CSF cultures from the animals infected with
K1 E. coli might represent viable counts below
the limit of detection, e.g., <102 CFU/ml of CSF. We also
recognize that the use of pooled specimens may not be the optimal
experimental design; however, the volume of CSF that can be removed
from a rat pup is small (15 to 20 µl) and necessitates the pooling of
specimens. Therefore, to support these data, several different
techniques (e.g., cytospins and fixed sections of pelleted CSF, and O18
LPS monoclonal antibody staining of brain sections) were used to
demonstrate the presence of O18 bacteria in the central nervous systems
of these animals, despite their having sterile CSF cultures. The
concept that bacteria can be demonstrated in the central nervous
systems of K1 E. coli-infected animals,
without the evidence of viable bacteria, is a novel observation and
potentially important for our understanding of the pathogenesis of
E. coli meningitis.
Corroborating the in vivo finding are our tissue culture invasion data,
which show that fourfold-fewer K1 bacteria can be
recovered from BMECs in invasion assays. In addition, our in vitro data
showed that by inhibition of BMEC protein synthesis with cycloheximide,
the K1 strains were protected from killing. Our
interpretation of these data is that brain endothelial cells may
produce a substance that is bactericidal to E. coli strains
without a capsule. The nature of this eukaryotic substance is unknown.
Nitric oxide has been found to possess antimicrobial properties
(8, 20) and is produced by endothelial cells via NOS
(12, 23). NO has been shown, in vitro and in animal models,
to be active against a wide variety of pathogens, including, but not
limited to, the following organisms: bacteria (Mycobacterium
spp., E. coli, Salmonella typhimurium [19, 22, 30]), viruses (herpes simplex virus type 1 and Japanese encephalitis virus [6, 18]), fungi
(Cryptococcus neoformans [1]), and
parasites (Leishmaniae major [17, 28]). We
examined the possibility that NO or oxygen intermediates might be the
agent of E. coli killing in our in vitro BMEC invasion assays. Using two different NOS inhibitors, which are analogues of the
NOS substrate L-arginine, at concentrations found to
inhibit NO production in endothelial cells (10), we were
unable to reproduce the survival advantage to K1 E. coli seen with cycloheximide. This suggests that neither NO nor
peroxynitrites, which are formed when NO reacts with oxygen radicals
(3, 32), are responsible for the bacterial killing in this
system. We also examined the possible effects of superoxides and other
oxygen radicals, which are known antimicrobial products of professional
phagocytes (2) and are produced by endothelial cells
(4, 27), in the invasion process of meningitic E. coli K1 and its capsule-negative derivative. Experiments using SOD and catalase, which are scavengers of oxygen radicals, were performed. In contrast to the results of experiments with cycloheximide-treated BMECs, there was no increase in levels of viable intracellular K1 E. coli bacteria in these experiments.
Because SOD and catalase are large proteins, it is possible that they
do not freely enter endothelial cells, the putative site of the
bacterial killing. There is some evidence that certain cells, such as
hepatocytes, do actively take up SOD (16), but this has not
been investigated in BMECs. Further experiments are needed to correlate
BMEC oxygen radical production with bacterial killing and its
inhibition with an increase in the survival of unencapsulated bacteria.
In summary, using an in vivo neonatal rat model of hematogenous
meningitis and an in vitro model of the blood-brain barrier, we showed
that both K1+ and K1 E. coli
strains were able to penetrate BMECs and enter the central nervous
system. However, only infections caused by K1+ strains
resulted in positive CSF cultures in our animal model, and in vitro
experiments yielded a significantly higher recovery of viable
K1+ intracellular organisms compared to that of
K1 strains. This strongly suggests that the K1 capsule
has, in addition to its well-recognized serum resistance and
antiphagocytic properties, a novel role in the transversal of E. coli K1 across the blood-brain barrier. It serves to protect
K1-encapsulated E. coli strains from killing during invasion
of the central nervous system, thus helping to explain the predominance
of K1-encapsulated bacteria in neonatal E. coli meningitis.
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ACKNOWLEDGMENTS |
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This work is supported in part by United States Public Health Service grant R01-NS-26310 and by a Pediatric Infectious Disease Society Fellowship Award sponsored by Lilly Research Laboratories.
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FOOTNOTES |
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* Corresponding author. Mailing address: Division of Infectious Diseases, Children's Hospital Los Angeles, 4650 Sunset Blvd., Box 51, Los Angeles, CA 90027. Phone: (323) 669-2509. Fax: (323) 660-2661. E-mail: KSKim{at}chla.usc.edu.
Editor: E. I. Tuomanen
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REFERENCES |
|---|
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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|---|
| 1. |
Alspaugh, J. A., and D. L. Granger.
1991.
Inhibition of Cryptococcus neoformans replication by nitrogen oxides supports the role of these molecules as effectors of macrophage-mediated cytostasis.
Infect. Immun.
59:2291-2296 |
| 2. | Babior, B. M. 1978. Oxygen-dependent microbial killing by phagocytes. N. Engl. J. Med. 298:659-668[Medline]. |
| 3. |
Beckman, J. S.,
T. W. Beckman,
J. Chen,
P. A. Marshall, and B. A. Freeman.
1990.
Apparent hydroxyl radical production by peroxynitrite: implications of endothelial injury from nitric oxide and superoxide.
Proc. Natl. Acad. Sci. USA
87:1620-1624 |
| 4. | Carter, W. O., P. K. Narayanan, and J. P. Robinson. 1994. Intracellular hydrogen peroxide and superoxide anion detection in endothelial cells. J. Leukoc. Biol. 55:253-258[Abstract]. |
| 5. | Catalan, R. E., A. M. Martinez, M. D. Aragones, and F. Hernandez. 1996. Identification of nitric oxide synthases in isolated bovine brain vessels. Neurosci. Res. 25:195-199[Medline]. |
| 6. | Croen, K. D. 1993. Evidence for antiviral effect of nitric oxide. Inhibition of herpes simplex virus type 1 replication. J. Clin. Investig. 91:2446-2452. |
| 7. |
Dietzman, D. E.,
G. W. Fischer, and F. D. Schoenknecht.
1974.
Neonatal Escherichia coli septicemia bacterial counts in blood.
J. Pediatr.
85:128-130[Medline].
|
| 8. | Fang, C. F. 1997. Mechanisms of nitric oxide-related antimicrobial activity. J. Clin. Investig. 99:2818-2825[Medline]. |
| 9. | Feigin, R. D. 1977. Bacterial meningitis in the newborn infant. Clin. Perinatol. 4:103-116[Medline]. |
| 10. | Gross, S. S., E. A. Jaffe, R. Levi, and R. G. Kilbourn. 1991. Cytokine-activated endothelial cells express an isotype of nitric oxide-dependent synthase which is tetrahydrobiopterin-dependent, calmodulin-independent and inhibited by arginine analogs with a rank order of potency characteristic of activated macrophages. Biochem. Biophys. Res. Commun. 178:823-829[Medline]. |
| 11. | Huang, S. H., C. A. Wass, Q. Fu, N. V. Prasadaroa, M. Stins, and K. S. Kim. 1995. Escherichia coli invasion of brain microvascular endothelial cells in vitro and in vivo: molecular cloning and characterization of invasion gene ibe10. Infect. Immun. 63:4470-4475[Abstract]. |
| 12. |
Janssens, S. P.,
A. Shimouchi,
T. Quertermous,
D. B. Bloch, and K. D. Bloch.
1992.
Cloning and expression of a cDNA encoding human endothelium derived relaxing factor/nitric oxide synthase.
J. Biol. Chem.
267:14519-14522 |
| 13. |
Kim, K. S.
1985.
Comparison of cefotaxime, imipenem-cilastin, ampicillin-gentamicin, and ampicillin-chloramphenicol in the treatment of experimental Escherichia coli bacteremia and meningitis.
Antimicrob. Agents Chemother.
28:433-436 |
| 14. | Kim, K. S., J. H. Kang, A. S. Cross, B. Kaufman, W. Zollinger, and J. Sadoff. 1988. Functional activities of monoclonal antibodies to the O side chain of Escherichia coli lipopolysaccharides in vitro and in vivo. J. Infect. Dis. 157:47-53[Medline]. |
| 15. | Kim, K. S., H. Itabashi, P. Gemski, J. Sadoff, R. L. Warren, and A. S. Cross. 1992. The K1 capsule is the critical determinant in the development of E. coli meningitis in the rat. J. Clin. Investig. 90:897-905. |
| 16. |
Kyle, M. E.,
D. Nakae,
I. Sakaida,
S. Miccadei, and J. L. Farber.
1988.
Endocytosis of superoxide dismutase is required in order for the enzyme to protect hepatocytes from the cytotoxicity of hydrogen peroxide.
J. Biol. Chem.
263:3784-3789 |
| 17. | Liew, F. Y., S. Millott, C. Parkinson, R. M. J. Palmer, and S. Moncada. 1990. Macrophage killing of Leishmania parasite in vivo is mediated by nitric oxide from L-arginine. J. Immunol. 144:4794-4797[Abstract]. |
| 18. | Lin, Y. L., Y. L. Huang, S. H. Ma, C. T. Yeh, S. Y. Chiou, L. K. Chen, and C. L. Liao. 1997. Inhibition of Japanese encephalitis virus infection by nitric oxide: antiviral effect of nitric oxide on RNA virus replication. J. Virol. 71:5227-5235[Abstract]. |
| 19. |
MacMicking, J. D.,
R. J. North,
R. LaCourse,
J. S. Mudgett,
S. K. Shah, and C. F. Nathan.
1997.
Identification of nitric oxide synthase as a protective locus against tuberculosis.
Proc. Natl. Acad. Sci. USA
94:5243-5248 |
| 20. | Moncada, S., R. M. J. Palmer, and E. A. Higgs. 1991. Nitric oxide: physiology, pathophysiology, and pharmacology. Pharm. Rev. 43:109-142[Medline]. |
| 21. | Morin, A. M., and A. Stanboli. 1994. Nitric oxide synthase localization in cultured cerebrovascular endothelium during mitosis. Exp. Cell Res. 211:183-188[Medline]. |
| 22. |
Pacelli, R.,
D. A. Wink,
J. A. Cook,
M. C. Krishna,
W. Degraff,
N. Friedman,
M. Tsokos,
A. Samuni, and J. B. Mitchell.
1995.
Nitric oxide potentiates hydrogen peroxide-induced killing of Escherichia coli.
J. Exp. Med.
182:1469-1479 |
| 23. | Palmer, R. M. J., A. G. Ferrige, and S. Moncada. 1987. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 327:524-526[Medline]. |
| 24. |
Prasadarao, N. V.,
C. A. Wass,
J. Hacker,
K. Jann, and K. S. Kim.
1993.
Adhesion of S fimbriated Escherichia coli to brain glycolipids mediated by sfaA gene-encoded protein of S fimbriae.
J. Biol. Chem.
268:10356-10363 |
| 25. |
Prasadarao, N. V.,
C. A. Wass, and K. S. Kim.
1996.
Endothelial cell GlcNAc 1-4GlcNAc epitopes for outer membrane protein A enhance traversal of Escherichia coli across the blood-brain barrier.
Infect. Immun.
64:154-160[Abstract].
|
| 26. | Robbins, J. B., G. H. McCracken, E. C. Gotschlich, F. Orskov, I. Orskov, and L. A. Hanson. 1974. Escherichia coli K1 capsular polysaccharide associated with neonatal meningitis. N. Engl. J. Med. 290:1216-1220. |
| 27. |
Rosen, G. M., and B. A. Freeman.
1984.
Detection of superoxide generated by endothelial cells.
Proc. Natl. Acad. Sci. USA
81:7269-7273 |
| 28. |
Stenger, S.,
H. Thuring,
M. Rollinghoff, and C. Bogdan.
1994.
Tissue expression of inducible nitric oxide synthase is closely associated with resistance to Leishmania major.
J. Exp. Med.
180:783-793 |
| 29. | Stins, M. F., N. V. Prasadarao, L. Ibric, C. A. Wass, P. Luckett, and K. S. Kim. 1994. Binding characteristics of S fimbriated Escherichia coli to isolated brain microvessel endothelial cells. Am. J. Pathol. 145:1228-1236[Abstract]. |
| 30. | Umezawa, K., T. Akaike, S. Fujii, M. Suga, K. Setoguchi, A. Ozawa, and H. Maeda. 1997. Induction of nitric oxide synthase and xanthine oxidase and their roles in the antimicrobial mechanism against Salmonella typhimurium infection in mice. Infect. Immun. 65:2932-2940[Abstract]. |
| 31. | Unhanand, M., M. M. Mustafa, and G. H. McCracken, Jr. 1993. Gram-negative enteric bacillary meningitis: a 21-year experience. J. Pediatr. 122:15-21[Medline]. |
| 32. | Zhu, L., C. Gunn, and J. S. Beckman. 1992. Bactericidal activity of peroxynitrite. Arch. Biochem. Biophys. 298:452-457[Medline]. |
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