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Infection and Immunity, June 1999, p. 3040-3046, Vol. 67, No. 6
Departments of Medical
Microbiology1 and Cell
Biology/Immunology,
Received 26 October 1998/Returned for modification 24 February
1999/Accepted 30 March 1999
The vacA and cagA geno- and phenotypes of
two mouse-adapted strains of Helicobacter pylori, SS1 and
SPM326, were determined. The SS1 strain, which had the
cagA+ and vacA s2-m2 genotype,
induced neither vacuole formation in HeLa cells nor interleukin-8
(IL-8) production in KATO III cells. In contrast, H. pylori
SPM326, with the cagA+ and vacA
s1b-m1 genotype, induced vacuoles as well as IL-8 production in vitro.
Furthermore, a spontaneous mutant of SPM326, which produced a
vacuolating cytotoxin but was not able to induce IL-8 production (SPM326/IL-8 Helicobacter pylori
infection nearly always leads to gastritis in humans. The infection is
not cleared by the gastric immune response, and after a prolonged
period of time more-severe clinical symptoms, such as peptic ulcer
disease, carcinoma, and lymphoma, may develop (4). Several
genes of H. pylori, such as vacA and cagA, are associated with this development (5).
vacA, which encodes a cytotoxin, shows mosaicism in the
signal (s) sequence and the middle (m) region of the gene. In addition
to the previously described s1a, s1b, and s2 alleles, recently a fourth
allele, s1c, was described. The m region is now expanded to three
alleles: m1, m2a, and m2b (2, 30). Several combinations of
these s and m alleles are present, but s2-m1 is not. The s1-m1 and
s1-m2 genotypes are associated with production of a vacuolating
cytotoxin, whereas the s2-m2 genotype is considered to be noncytotoxic
(2, 20, 23).
The CagA protein is about 128 kDa in size (9), and although
the function of CagA is unknown, its association with peptic ulcer
disease and carcinoma makes it a useful marker for virulent strains
(17, 32). cagA is part of the pathogenicity
island of H. pylori (1, 8). Some of the genes in
this island play a role in the induction of interleukin-8 (IL-8)
production by epithelial cells (1, 8, 28). IL-8 is a potent
chemoattractant and activator of neutrophils and T cells and is
considered to be an important contributor to inflammation
(22).
Several H. pylori strains are now used in animal models,
such as the mouse. However, detailed information on geno- and/or phenotype is often not available for these strains. In this study we
determined the vacA s and m genotypes of and the expression of an active cytotoxin by two mouse-adapted H. pylori
strains. Furthermore, the presence of cagA was determined,
as well as the ability of these H. pylori strains to induce
IL-8 production in a gastric cell line. C57Bl/6 mice and BALB/c mice
have previously been shown to have different inflammatory responses
after infection with Helicobacter felis (21, 25).
Using these two strains of mice we investigated the distribution of the
H. pylori strains in the stomach and the effects of their
colonization on the gastric immune response.
Bacterial strains and culture conditions.
The H. pylori strains used in this study were SS1 (18), SPM326
(19), ATCC 43504, and 93-1184 (isolated from a patient with
gastritis at the University Hospital of the Vrije Universiteit, Amsterdam, The Netherlands). Bacteria used for cytotoxin and IL-8 assays were grown in brucella broth supplemented with 5% (vol/vol) newborn calf serum (NCS) (Gibco BRL, Paisley, Scotland) at 37°C for
24 h in a microaerobic atmosphere with shaking. The bacteria that
were used in animal experiments were cultured on horse blood agar
plates with Dent supplement (Oxoid, Basingstoke, United Kingdom), as
described (31).
Genotyping by LiPA and AFLP.
DNA of H. pylori was
extracted by the CTAB method (3). vacA and
cagA genotypes of these strains were determined by PCR and a
single-step reverse hybridization line probe assay (LiPA), as described
previously (29, 30). In addition, SS1, SPM326, and a
spontaneous mutant of the latter strain lacking IL-8 induction capacity
(SPM326/IL-8 Assay for cytotoxin production by H. pylori
strains.
Cytotoxin production was determined as described
(12). Bacterial culture supernatants were filter sterilized
and stored at Assay for IL-8 induction in KATO III cells.
KATO III cells
(human gastric carcinoma cell line; kindly provided by A. van der Ende,
Academic Medical Center, Amsterdam, The Netherlands) were grown in RPMI
1640 supplemented with 10% (vol/vol) NCS, 10 U of penicillin per ml,
and 10 µg of streptomycin per ml. The IL-8 assay was performed as
described by Sharma et al. (26), with a few modifications.
Cells were seeded in 24-well tissue culture plates at 2 × 105 cells per well in a volume of 1 ml of tissue culture
medium and were cultured for 2 days. Twelve hours before stimulation,
the cells were washed with phosphate-buffered saline (PBS), and RPMI 1640 without NCS or antibiotics was added to the wells, to minimize the
IL-8 production induced by NCS. Six to eight single colonies of each
strain were expanded, and bacteria were harvested by centrifugation, washed with PBS, and resuspended at a concentration of 2 × 108 cells/ml. A bacterium:cell ratio of 100:1 was used, and
supernatant (50 µl) was collected after 2 and 4 h, centrifuged
at 15,000 × g, and stored at IL-8 ELISA.
IL-8 protein concentrations were determined with
an IL-8 sandwich ELISA kit (CLB, Amsterdam, The Netherlands) and
expressed as picograms per milliliter. The detection limit for IL-8 was 1 pg/ml. Concentrations of IL-8 were determined from a standard curve
(range, 1 to 240 pg/ml).
Inoculation of mice with H. pylori.
Female 6-week-old
specific-pathogen-free C57Bl/6 and BALB/c mice (Harlan CPB, Zeist, The
Netherlands) were housed under conventional conditions in our animal
facilities. The mice had free access to food and water. The Animal Care
Committee of the Vrije Universiteit of Amsterdam approved all animal
experimentation described. The inoculation with bacteria was done as
described previously (18, 19). The mice received bacteria
three times in a 5-day period. A volume of 0.15 ml of bacterial
suspension containing approximately 109 CFU/ml in PBS was
given orally with a feeding needle. Control mice received sterile PBS.
Animals were killed at various times postinoculation by cervical
dislocation, and their stomachs were removed.
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Helicobacter pylori-Associated Gastritis
in Mice is Host and Strain Specific
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
), was detected. C57Bl/6 and BALB/c mice were
infected with these three strains to investigate the colonization
pattern and the effect on the immune response in vivo. The SS1 strain
colonized the stomachs of all mice in large numbers which remained
constant over time. Colonization with the SPM326/IL-8+ and
SPM326/IL-8 strains was lesser, or even absent, and
decreased over time. At 5 weeks postinoculation all three H. pylori strains induced a mild increase of neutrophil count in the
gastric corpus of C57Bl/6 mice, which disappeared by 12 weeks. At both
5 and 12 weeks postinoculation C57Bl/6 mice colonized with
SPM326/IL-8+ showed an increased expression of major
histocompatibility complex (MHC) class II antigen in the cardia which
was accompanied by an increased number of T cells. C57Bl/6 mice that
were infected with SS1 and SPM326/IL-8 did not show
chronic inflammation. BALB/c mice colonized with SS1 and
SPM326/IL-8 also showed an increase in neutrophil count
at 5 weeks, which normalized again by 12 weeks postinoculation. At this
time point SS1-infected mice showed inflammation in the corpus and
antrum. At these sites an increased expression of MHC class II antigens and an increased number of T cells were observed. Although small lymphoid follicles were already observed 5 weeks after inoculation with
SS1, their incidence as well as their number was increased at 12 weeks.
These results show that inflammation induced by H. pylori
depends both on the bacterial strain and the host.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
MATERIALS AND METHODS
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
) were analyzed by amplified fragment length
polymorphism (AFLP). The AFLP was performed as described
(16) with only minor modifications in the sequences of the
fluorescence-labeled Eco-O primer (5'-GACTGCGTACCAATTC-3') and the unlabeled Mse-O primer
(5'-ACGATGAGTCCTGAGTA-3').
20°C until use. HeLa cells (kindly provided by B. Kremer, Academic Center for Dentistry, Amsterdam, The Netherlands) were cultured in RPMI 1640 medium supplemented with 5% (vol/vol) NCS, 10 U
of penicillin per ml, and 10 µg of streptomycin (Gibco BRL) per ml.
Cells were harvested by trypsinization and seeded at a concentration of
3 × 102 cells/100 µl of tissue culture medium in a
microtiter plate. After 16 h of culture at 37°C in 95% air and
5% CO2 in a humidified incubator, twofold dilutions of
bacterial culture supernatants in RPMI 1640 were added. After overnight
incubation, the cells were washed with RPMI 1640 and a solution of
saline with 0.05% neutral red was added. After 5 min the cells were
washed with cold saline containing 0.2% NCS. A total of 100 cells were
inspected, and if more than 50 cells showed vacuoles the H. pylori strain was designated as cytotoxin positive
(12).
20°C until further
analysis of IL-8 protein by enzyme-linked immunosorbent assay (ELISA).
The type strain of H. pylori, ATCC 43504, that was
previously reported to induce IL-8 production in KATO III cells was
used as a positive control (10). Strain 93-1184, which is
cagA negative, and serum-free tissue culture medium were
used as negative controls.
(16 mice of
each strain for each H. pylori strain; see Table 2). The
control group consisted of eight C57Bl/6 mice and eight BALB/c mice.
Gastric colonization with H. pylori was assessed after 5 and
12 weeks. The stomachs of eight mice from each group inoculated with an
H. pylori strain and four control mice were resected at each
time point and divided longitudinally into two halves. One half of the
stomach was frozen immediately and was used for immunohistochemistry,
whereas the other half was used to assess colonization in a
semiquantitative way. The stomach tissue was gently rubbed over the
surface of freshly prepared blood agar plates (31)
containing Dent supplement (Oxoid) and 75 mg of bacitracin per liter.
After 3- to 5-day incubation at 37°C in a microaerobic atmosphere,
the numbers of H. pylori colonies present on the plates were
counted. H. pylori was identified by Gram's stain and
detection of positive reactions in urease, oxidase, and catalase tests.
The presence of H. pylori in Giemsa-stained sections was
also determined microscopically.
Immunohistochemistry. Longitudinal sections (thickness, 8 µm) of the stomach that contained mucosal tissue from the nonglandular part of the stomach, cardia, corpus, and antrum were picked up on gelatin-coated slides, air dried, and fixed in pure acetone for 10 min. A two-step immunoperoxidase method was used as described previously (31). Consecutive sections were stained with monoclonal antibodies RA3-6B2 (which recognizes B220 on B cells), 59-AD-22 (which recognizes Thy-1 on T cells), and M5.114 (which recognizes major histocompatibility complex [MHC] class II antigens) (31). These monoclonal antibodies were raised in our laboratory from hybridoma cell lines. At least three sections per mouse were investigated for the presence of inflammatory cells. Sections of the spleen were included on every slide as a positive control. Neutrophils were recognized by staining of endogenous peroxidase.
Statistical analysis. Differences between IL-8 concentrations and colonization levels were determined by unpaired t test and were considered significant when the P value was <0.05.
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RESULTS |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Genotypes and phenotypes of the H. pylori strains.
The genotypes of the strains used in this study were determined by LiPA
and are summarized in Table 1. The
strains 93-1184 and SS1, which showed the s2-m2 vacA
genotype, did not induce vacuolation in HeLa cells. The other strains
induced vacuolation in approximately 50% of the cells. The SS1 strain
was the only cagA-positive H. pylori strain that
did not produce a vacuolating cytotoxin.
|
. The vacA genotype and phenotype
of this strain were tested and were shown to be identical to those of
the wild-type strain, SPM326/IL-8+ (Table 1). To further
ensure that strain SPM326/IL-8 originated from the
wild-type strain, both strains were analyzed by AFLP. Identical
patterns were obtained for the two SPM326 strains, whereas the
SS1 strain showed a different pattern (results not shown).
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Colonization of C57Bl/6 and BALB/c mice with three H. pylori strains.
The total numbers of bacteria present in the
stomachs of C57Bl/6 and BALB/c mice were determined as part of a study
in which the immune response was investigated. The greatest number of
colonies was observed in mice inoculated with the SS1 strain, which
colonized all C57Bl/6 and BALB/c mice (Table 2). No difference in
bacterial density was observed between these two mouse strains during
the 12-week period of the experiment. The SPM326/IL-8+
strain colonized seven of eight C57Bl/6 mice at 5 weeks, and this
number decreased to two of eight mice at 12 weeks, accompanied by a
severely decreased number of bacteria. Surprisingly,
SPM326/IL-8+ was not able to colonize BALB/c mice. Even
after repeated in vivo passages in C57Bl/6 mice
SPM326/IL-8+ did not colonize BALB/c mice. The number of
C57Bl/6 mice colonized with SPM326/IL-8 decreased in a
way similar to the decrease observed for the parent strain, but the
bacterial density was significantly higher. In contrast with the parent
strain, SPM326/IL-8 was able to infect BALB/c mice. The
number of mice which were colonized with this strain was greater at 12 weeks than at 5 weeks, but the bacterial density decreased over time
(Table 2).
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Distribution of H. pylori in the stomachs of C57Bl/6
mice.
The distribution of H. pylori strains SS1,
SPM326/IL-8+ and SPM326/IL-8 in the stomachs
of C57Bl/6 mice was studied at 4 weeks postinoculation. The numbers of
colonies cultured from the cardia, corpus, and antrum are shown in
Table 3. The SS1 strain showed an equal
distribution of bacteria over the three parts of the stomach.
SPM326/IL-8+ and SPM326/IL-8 colonized the
stomach in fewer numbers than the SS1 strain. Surprisingly, both SPM326
strains showed the highest density of bacteria in the cardia, with
numbers decreasing from the cardia to the antrum (Table 3).
|
Immunohistological analysis of C57Bl/6 and BALB/c mice infected
with H. pylori.
Serial stomach sections of control and
infected mice were investigated for the presence of neutrophils and B
and T cells and for expression of MHC class II antigens. Mast cells
were detected in Giemsa-stained sections by the presence of large
Giemsa-stain-positive granules in the cytoplasm. At 5 weeks
postinoculation SS1-, SPM326/IL-8+-, and
SPM326/IL-8-colonized C57Bl/6 mice showed mild increases
of neutrophil counts in the submucosa and deep mucosa of the corpus. A
small lymphoid follicle, primarily consisting of B cells, was observed
in the cardia of one of eight SS1-infected mice. Besides increased
numbers of neutrophils in the corpus, in three of seven
SPM326/IL-8+-colonized C57Bl/6 mice an increase in the
number of T cells was also present in the mucosa of the cardia. In the
same area MHC class II antigen expression was increased. In control
mice MHC class II antigens were expressed only by small cells, which
were located between the gastric glands, whereas in
SPM326/IL-8+-infected mice MHC class II antigen expression
was extended to the cells of the glands. B cells or lymphoid follicles
were absent. After 12 weeks the number of neutrophils had normalized in
all infected C57Bl/6 mice. In C57Bl/6 mice infected with
SPM326/IL-8+ an increase of MHC class II antigen expression
and T-cell number similar to that observed at 5 weeks was observed. No
inflammation was observed in SS1- or SPM326/IL-8-infected
C57Bl/6 mice.
-infected BALB/c mice showed an
increase in neutrophil count (Fig. 2)
similar to that observed in C57Bl/6 mice at 5 weeks postinoculation.
However, in some SS1-infected mice the neutrophils not only were
present in the submucosa but also extended to the upper part of the
corpus mucosa. In SPM326/IL-8-infected BALB/c mice the
neutrophils present in increased numbers were located in the antrum.
The neutrophil response decreased over time in a way similar to
the decrease observed in C57Bl/6 mice. One of eight SS1-infected BALB/c
mice showed a follicle in the cardia at 5 weeks. At 12 weeks
postinoculation, areas with increased MHC class II antigen
expression were present in both the corpus and the antrum of
SS1-colonized BALB/c mice (Fig. 3A). At
these locations increased numbers of T cells were present (Fig. 3B).
The number of SS1-infected mice with lymphoid follicles increased from
one mouse (of eight mice) at 5 weeks to five mice (of 8 mice) at 12 weeks postinoculation. These small lymphoid follicles were observed in
the deep mucosa of the cardia and occasionally also in the corpus (Fig.
3C), whereas these structures were absent in control mice. Most
follicles consisted primarily of B cells. Chronic inflammation was not
observed in SPM326/IL-8-infected BALB/c mice.
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DISCUSSION |
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In this study the vacA and cagA genotypes of two mouse-adapted H. pylori strains as well as the phenotypic expression of these genes were determined. C57Bl/6 and BALB/c mice were infected with these strains, which differed in cytotoxin expression and ability to induce IL-8 production, and the patterns of colonization and gastric immune response were compared.
The induction of vacuoles in HeLa cells by H. pylori SPM326, which has vacA genotype s1b-m1, and the lack of vacuolization by the vacA s2-m2-possessing strain SS1 are consistent with the data of other studies (2, 20). However, recently it has become clear that the ability of the m1 or m2 cytotoxin to induce vacuoles strongly depends on the cell line that is used in the assay (20, 22). Whether H. pylori strains with the s2-m2 genotype are able to induce vacuoles in other cell lines remains to be established.
The presence of cagA is associated with IL-8 production by
epithelial cells (10). Although both mouse-adapted strains
were cagA positive, only SPM326 induced a significantly
higher level of production of IL-8 in KATO III cells compared with a
cagA-negative H. pylori strain. In addition, a
spontaneous mutant of SPM326 which induced vacuolization but did not
induce IL-8 production (SPM326/IL-8) was isolated. It has
been shown that mutations in several genes of the cag
pathogenicity island affect IL-8 production by epithelial cells
(1, 8, 28). Whether one of these genes is mutated in the SS1
and SPM326/IL-8 strains is currently under investigation.
Whether these three H. pylori strains with different
cytotoxin and IL-8 induction phenotypes also showed different
properties in vivo was investigated in C57Bl/6 and BALB/c mice.
Culture of H. pylori from gastric biopsy specimens was
found to be more sensitive to assess colonization than analysis of
Giemsa-stained sections by microscopy; this result is in
agreement with data reported by Shomer et al. (27). The SS1
strain showed the greatest number of colonies in culture
(104 to 105 CFU/stomach) and was easily
detected in Giemsa-stained sections, whereas both SPM326 strains showed
fewer colony numbers and were hardly detectable in sections. The
detection limit for H. pylori in sections was approximately
103 bacteria/mouse stomach. The SS1 strain colonized both
mouse strains in large numbers, whereas SPM326/IL-8+ and
SPM326/IL-8 colonized in smaller numbers and
SPM326/IL-8+ was not able to colonize BALB/c mice at all.
Although SPM326 has been reported to show a constant colonization rate
in CD1 mice for up to 52 weeks (14), we observed a decrease
in the numbers of SPM326/IL-8+-and
SPM326/IL-8-colonized C57Bl/6 mice as well as in
bacterial density over time. These results suggest that the SPM326
strain is more susceptible to host factors than the SS1 strain.
The distribution of H. pylori in the mouse stomach was assessed to investigate whether colonization correlated with the gastric immune response. In both C57Bl/6 and BALB/c mice the number of neutrophils was increased at 5 weeks but not at 12 weeks postinoculation. The presence of neutrophils in the corpus or antrum was associated neither with the highest density of bacteria nor with the IL-8 induction phenotype of the colonizing H. pylori strain. This may not be very surprising, because mice do not express a homologue of human IL-8 (7). However, these results show that activation of neutrophils in mice occurs via mechanisms other than those observed in humans.
In contrast with the active inflammation, the presence of chronic inflammation was correlated with the highest bacterial density. In SS1-infected BALB/c mice chronic inflammation was present in the cardia, corpus, and antrum, whereas in SPM326/IL-8+-infected C57Bl/6 mice chronic inflammation was restricted to the cardia. Although SS1-infected BALB/c mice showed a density of bacteria similar to that for SS1-infected C57Bl/6 mice, chronic inflammation was absent in the last group. These observations are not in agreement with those reported by Lee et al. (18), who observed the opposite phenomenon: chronic inflammation was present at an earlier time point in SS1-infected C57Bl/6 mice than in infected BALB/c mice. This discrepancy could be due to the higher level of colonization with SS1 in C57Bl/6 mice reported by Lee et al. (18).
The chronic inflammation in the cardia of SPM326/IL-8+-infected C57Bl/6 mice was characterized by increased numbers of T cells in an area in which MHC class II antigen expression was extended to epithelial gland cells. In SS1-infected BALB/c mice a similar colocalization of T cells and MHC class II antigen expression by gland cells was observed, but this was found in the corpus and antrum. In addition, lymphoid follicles were present in the deep mucosa of the cardia and corpus and their presence was not correlated with MHC class II antigen expression by gland cells. The early signs of chronic gastritis in SS1-infected BALB/c mice clearly show that differences exist between H. felis infection and H. pylori infection, as in H. felis-infected BALB/c mice chronic inflammation occurs only after 22 months (11). Although the chronic inflammation of SS1-infected BALB/c mice does not mimic the active and chronic inflammation observed in humans, it could be used as a model to study the interaction between T cells and the gastric epithelium.
The absence of chronic inflammation in
SPM326/IL-8-infected C57Bl/6 and BALB/c mice makes this
mutant an interesting strain that could be used to unravel the
mechanism of induction of inflammation. This strain might be mutated in
its adherence to epithelial cells. The attachment of the bacterium to
epithelial cells is very important in the process of cytokine
production (24). The importance of attachment of H. pylori cells to epithelial cells has also been studied in a mouse
model, i.e., transgenic mice that express Lewis b, which has
been reported to be an adhesin for H. pylori (6), in the gastric glands. These transgenic mice show a more severe inflammatory response than nontransgenic mice and produce
autoantibodies after infection with H. pylori
(15).
The cytotoxin of H. pylori is able to induce vacuolation of cells in vitro (2, 20, 23), and the oral administration of sonic extracts of cytotoxin-positive H. pylori strains or purified cytotoxin induces gastric lesions in mice (13). However, under these experimental conditions the concentration of cytotoxin is probably much higher than under the physiological conditions of colonization with living bacteria, as no epithelial damage or vacuolation was observed in any of the H. pylori-infected mice.
In conclusion, this study showed that the mouse-adapted H. pylori strains with the vacA s1 genotype induced vacuolation in HeLa cells. The IL-8-inducing ability of these strains could not be predicted from the cagA-positive genotype. The infection of two strains of mice with H. pylori strains with different vacA and cagA phenotypes showed that the inflammatory response depended on both the host and the H. pylori strain.
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FOOTNOTES |
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* Corresponding author. Mailing address: Vrije Universiteit, School of Medicine, Department of Medical Microbiology, Van der Boechorststraat 7, 1081 BT Amsterdam, The Netherlands. Phone: 31-20-4448296. Fax: 31-20-4448318. E-mail: F.Namavar.MM{at}med.vu.nl.
Editor: J. R. McGhee
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Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Infection and Immunity, June 1999, p. 3040-3046, Vol. 67, No. 6
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