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Clinical and Diagnostic Laboratory Immunology, May 2000, p. 463-467, Vol. 7, No. 3
1071-412X/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Antibody Response of Patients with
Helicobacter pylori-Related Gastric Adenocarcinoma:
Significance of Anti-CagA Antibodies
Christine
Vaucher,1
Blandine
Janvier,2,*
Jean-Baptiste
Nousbaum,3
Bernadette
Grignon,2
Leon
Pezennec,2
Michel
Robaszkiewicz,3
Herve
Gouerou,3
Bertrand
Picard,1 and
Jean-Louis
Fauchere2
Laboratoire de Bactériologie,
Faculté de Médecine de Brest-Université de Bretagne
occidentale,1 and Service de
Hépato-Gastro-Entérologie, CHU La Cavale
Blanche,3 29200 Brest, and Laboratoire
de Microbiologie A, CHU La Milétrie et Faculté de
Médecine et de Pharmacie, 86000 Poitiers,2
France
Received 12 May 1999/Returned for modification 27 July
1999/Accepted 22 February 2000
 |
ABSTRACT |
The aim of this study was to search for a specific antibody pattern
in sera from patients suffering from Helicobacter
pylori-related gastric adenocarcinoma (GAC). The serological
response of 22 patients suffering from GAC, 31 patients with
gastroduodenal ulcer, and 39 asymptomatic subjects was analyzed using
immunoblotting performed with three H. pylori strains:
strain ATCC 43579; strain B110, isolated from a patient with ulcers;
and strain B225, isolated from a patient with GAC. In addition, the
latex agglutination test Pyloriset Dry was used to analyze ambiguous
sera. H. pylori seropositivity was 75% in the GAC group,
61.3% in the ulcer group, and 56.4% in the asymptomatic group.
Anti-CagA antibodies were found more often in the GAC group (48.8%)
and in the ulcer group (47.3%) than in the asymptomatic group
(21.2%). These percentages depended on the strain used as an antigen:
in the GAC group, the anti-CagA frequencies were 93.3, 40, and 13.3%
with strains B225, B110, and ATCC 43579, respectively. Thus the
presence of anti-CagA antibodies was increased in patients suffering
from H. pylori-related GAC, in particular when the CagA
antigen was from a GAC strain. These data suggest the existence of a
CagA protein specifically expressed by H. pylori strains
isolated from GAC patients.
| |
INTRODUCTION |
Gastric adenocarcinoma (GAC) is the
second-most-common cancer in the world, with an estimated incidence of
700,000 new cases a year (32). Epidemiological studies
suggest that Helicobacter pylori-related gastritis, together
with environmental and genetic factors, plays a role in the initiation
of GAC (24). H. pylori is a gram-negative, spiral
shaped, flagellated microaerophilic bacterium that was identified in
the early 1980s (46). It colonizes the stomach of about 50%
of all humans and is responsible for the majority of chronic gastritis
and peptic ulcer cases (4, 7, 18). A history of H. pylori infection has been found in 50 to 90% of patients with GAC
(11, 50). Since 1991, H. pylori has been
classified as a group 1 carcinogen, and the relative risk of cancer has
been estimated to be 3.6 times higher in patients with H. pylori infection than in noninfected patients (16, 20, 33,
41). Nevertheless, H. pylori infection is more
widespread than gastric cancer in Japan, where the prevalence of
gastric cancer is high; 0.04% of Japanese subjects who are
seropositive for H. pylori suffer from gastric carcinoma
(1). Recently, the involvement of H. pylori
infection in gastric carcinogenesis has been confirmed in a Mongolian
gerbil experimental model (21, 47). Thus, Koch's postulates
for H. pylori as a cause of GAC seem to be fulfilled
(45).
These results led to research of the specific determinants of both host
and bacterium that predispose H. pylori-infected individuals to GAC. Two virulence factors have been found more frequently in
H. pylori strains isolated from patients with ulcers or
cancer than in strains isolated from patients with gastritis (5,
36, 44, 49). The first one is the vacuolating cytotoxin (VacA); the second one is the cytotoxin-associated antigen (CagA) that reflects
the presence of the CagA pathogenicity island, including about 30 genes
of unknown function (9).
We previously showed that a specific antibody response pattern is found
in the sera from patients suffering from H. pylori-associated peptic ulcer (3). The aim of this
study was to extend these data and to search for specific antibody
patterns in sera from H. pylori patients suffering from GAC;
subjects suffering from peptic ulcer and asymptomatic subjects served
as controls. To investigate antibody patterns, immunoblot assays were
carried out with three H. pylori strains: one strain
isolated from a patient with GAC, one strain isolated from a patient
with duodenal ulcers, and the reference strain, ATCC 43579.
| |
MATERIALS AND METHODS |
Patients.
Three groups of patients were included in this
study. The patients were hospitalized at the University Hospital Center
of Brest, France, between 1997 and 1998. The first group included 20 patients (9 men and 11 women) with GAC. The median age was 75.3 years
(range, 55 to 95 years) for the men and 73.1 years (range, 53 to 83 years) for the women. Twelve GACs were intestinal-type adenocarcinomas,
and eight cancers were diffuse-type adenocarcinomas, according to the
Lauren classification (27). The second group included 31 patients (26 men and 5 women) with gastroduodenal ulcers. The median
age was 61 years (range, 18 to 80 years) for the men and 72 years
(range, 51 to 84 years) for the women. The third group included 39 asymptomatic patients (17 men and 22 women). The median age in this
group was 70 years (range, 56 to 87 years) for the men and 76.3 years
(range, 63 to 91 years) for the women. No significant demographic
differences between the GAC group and the asymptomatic group were
present. Serum samples from the 90 patients were collected, aliquoted,
and stored frozen at 
70°C.
Serological assays.
The presence of antibodies to H. pylori in serum was determined using the rapid latex agglutination
test Pyloriset Dry (Orion Diagnostica, Fumouze, France) in accordance
with the manufacturer's instructions and a home-made immunoblot assay
with saline extracts from three strains: ATCC 43579, B110, and B225
(29). Strain B110 was isolated from a patient with a
duodenal ulcer, and strain B225 was isolated from a patient with GAC.
The three strains were cagA positive and had the s1
vacA signal sequence (2, 38). They were
consequently considered virulent. For the preparation of antigens,
H. pylori strains were cultivated on Pylori agar (bioMérieux, Marcy l'Etoile, France) and incubated at 37°C
under microaerobic conditions for 3 to 4 days. Saline extracts
corresponding to the water-soluble and surface-exposed antigens were
prepared according to a previously described method (3).
Briefly, bacterial cells were harvested in sterile 0.15 M NaCl,
vortexed for 5 min, and centrifuged (10,000 × g for 10 min at 4°C). The protein concentration of supernatants was
determined, and the saline extracts were stored frozen at 70°C.
Using a mini-gel apparatus (Bio-Rad, Richmond, Calif.), we carried out
sodium dodecyl sulfate-polyacrylamide gel electrophoresis with 50 µg
of saline extracts per gel (26). Molecular mass markers
ranging from 14 to 94 kDa (Pharmacia, Uppsala, Sweden) were included on
each gel. After migration, proteins were electrotransferred to a
nitrocellulose membrane. The membranes were cut into strips and
incubated for 1 h at room temperature with serum samples or with
monospecific serum (see below) at a 1:100 dilution. They were then
incubated for 1 h at room temperature with alkaline
phosphatase-conjugated anti-human, anti-rabbit, or anti-mouse
immunoglobulin G (Dakopatts, Copenhagen, Denmark). Color reactions were
developed with 5-bromo-4-chloro-3-indolylphosphate and nitroblue
tetrazolium (Sigma, St. Quentin Fallavier, France).
To aid identification of the immunoreactive bands detected by the
patient's serum, a set of eight monospecific polyclonal rabbit sera
raised to the 20-kDa ferritin-like protein (Felp), the 26- and 35-kDa
antigens, the 54-kDa catalase, the 60-kDa HspB, the 76-kDa fumarate
reductase, the 87-kDa VacA, and the 125 kDa CagA antigens and two
murine monoclonal antibodies raised to the 30-kDa UreA and 66-kDa UreB
antigens were used. These sera were generously supplied by AVENTIS
Pasteur (Marcy l'Etoile, France). Immunoreactive bands were also
identified with a calibration curve constructed by plotting the
migration distance of the various markers versus their respective
molecular masses. Immunoblots were considered to be positive for
antibodies to H. pylori when three or more immunoreactive
bands were present and when at least one of these three bands
corresponded to CagA (125 kDa), HspB (60 kDa), UreB (66 kDa), or UreA
(30 kDa) antigens (3). The Pyloriset Dry test was used to
analyze ambiguous sera: those sera that were positive by Western
blotting with only one H. pylori strain were considered to
be positive if positive with the Pyloriset Dry test also.
Statistical analysis.
The chi-square test was used to
compare the frequencies of the immunoreactive bands. Factorial analysis
of correspondence was done for every immunoblot series, one series for
each of the three H. pylori strains (19, 28, 42).
This analysis was used to compare entire immunoblot patterns. The data
of the immunoblots were transferred into three two-way tables, one
table for each strain. Each table had 90 rows, with 1 row for each
serum, and 10 columns, with 1 column for each immunoreactive band. The
presence or the absence of each band was encoded as follows:
present = 2, absent = 1. Factorial analysis of correspondence
(SPAD.N software; Cisia, Saint-Mandé, France) was performed from
each table, first considering all sera and then only the seropositive sera.
| |
RESULTS |
Protein profiles of the saline extracts used as antigens in
immunoblots.
Saline extracts prepared from the three H. pylori strains included in the study were resolved by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis in a 12% acrylamide
gel. They showed qualitative and quantitative differences for numerous
proteins (Fig. 1). In particular, the
CagA protein was present in a larger amount in strain ATCC 43579 than
in strains B225 and B110.
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FIG. 1.
Electrophoretic profiles of the saline extracts prepared
from the three H. pylori strains: B110 (lane 1), B225 (lane
2), and ATCC 43759 (lane 3). Proteins were resolved in a 12%
acrylamide gel and silver stained. MW, molecular weight markers in
thousands.
|
|
H. pylori serological status of the 90 patients.
The sera of 31 subjects (34.4%) were negative by both serological
tests, the agglutination assay and immunoblotting. The agglutination assay was positive in the sera of 44 (48.9%) of the 90 subjects. Depending on the H. pylori strain used as an antigen in the
immunoblot, the number of positive sera ranged from 49 (54.5%) with
strain B225, 51 (56.7%) with strain ATCC 43579, to 52 (57.8%) with
strain B110. The sera of 42 (46.7%) of the 90 subjects were positive with all three strains; the sera of 11 of 90 subjects (12.2%) were
positive with two strains. Among these 53 subjects, the sera of 41 were
positive by the agglutination assay. These 53 subjects were considered
seropositive. The sera of six subjects (6.7%) were positive with only
one strain; of these six subjects, three had sera positive by the
agglutination test and were therefore considered seropositive; the
three others were considered seronegative.
Thus, 56 of 90 (62.2%) subjects were considered
H. pylori
seropositive: 15 of the 20 patients belonging to the GAC group
(75.0%),
19 of the 31 patients belonging to the ulcer group (61.3%),
and
22 of the 39 subjects belonging to the control group (56.4%).
The
prevalence of seropositive subjects in the three groups was
not
significantly different. There was no influence of sex on
seropositivity: 60.5% of the women and 63.5% of the men were
seropositive.
H. pylori seropositivity increased with age:
25% of those under
50 and 64% of those over 50 were seropositive. In
the GAC group,
there was no significant difference in seropositivity
between
patients with diffuse-type tumors and those with
intestinal-type
tumors.
Antibody patterns of the 90 patients.
For each serum sample,
the number and nature of the immunoreactive bands observed on the blots
varied substantially according to the H. pylori strain used
to prepare the antigen. The average number of bands was 5.5, 10.5 and
11.75 with B225, B110, and ATCC 43579, respectively. Figure
2 shows the reactivity patterns of one
negative and one positive serum sample tested by immunoblotting with
the three H. pylori strains.
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FIG. 2.
Variability of the immunoblots obtained with one
seronegative patient (A) and one seropositive patient (B) according to
the three H. pylori stains used as antigens: B110 (lanes 1),
B225 (lanes 2), and ATCC 43759 (lanes 3).
|
|
We focused our study on the 10 bands which were most often encountered
on the blots. These bands, identified by their molecular
mass and by
reactivity with monospecific antisera, corresponded
to eight antigens
identified as CagA, VacA, fumarate reductase,
UreB, HspB, catalase,
UreA, and ferritin-like protein and to two
unidentified antigens of 35 and 26 kDa, respectively. For each
patient serum sample, the
frequencies of each band obtained with
each of the three antigenic
preparations were established, and
the mean frequency of each band was
calculated (Fig.
3). Significant
differences of mean frequencies between seropositive and seronegative
subjects were found for all antibodies. Catalase, HspB, and UreB
were
the antigens that were most often recognized by sera from
seropositive
subjects. However, antibody to catalase had a low
level of specificity,
since it was detected in 83.3% of the seropositive
subjects but also
in 55% of the seronegative subjects. Antibody
to HspB had the best
ratio of sensitivity to specificity.
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FIG. 3.
Mean frequencies (as percentages) of the 10 immunoreactive bands detected by immunoblot assay in the sera of 56 seropositive subjects (columns in black) and the 34 seronegative
subjects (columns in grey). Antibodies were directed to CagA (columns
1), VacA (columns 2), fumarate reductase (columns 3), UreB (columns 4),
HspB (columns 5), catalase (columns 6), p35 (columns 7), UreA (columns
8), p26 (columns 9), and ferritin like protein (columns 10).
|
|
Factorial analysis of correspondence performed with the data of the
immunoblots obtained with the three strains confirmed
the distinction
between the immunoblots of seropositive and seronegative
subjects. Data
points for the latter were clearly focused on the
factorial plane by
the negative values of the F1 axis (Fig.
4).
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FIG. 4.
Factorial analysis of correspondence of the 90 immunoblots performed with strain B110.  , projection of the 34 seronegative subjects;  , projection of the 56 seropositive
subjects.
|
|
Antibody patterns of seropositive subjects.
We next compared
the antibody patterns of the seropositive subjects, including 15 GAC
patients, 19 ulcer subjects, and 22 asymptomatic control subjects, to
search for a specific pattern of GAC. Of the 10 antibodies studied, the
mean frequencies of only 3 antibodies depend on the clinical origin of
the serum. Anti-CagA antibodies were found more often in sera from the
GAC patients (48.8%) and the ulcer subjects (47.3%) than in sera from asymptomatic subjects (21.2%) (P < 0.0001). Anti-VacA
antibodies were more frequent in the asymptomatic control subjects
(37.8%) than in the ulcer group (14%) and in the GAC group (20%)
(P < 0.0001). Anti-ferritin-like protein antibodies
were more frequent in the ulcer group (68.8%) than in the GAC group
(31%) and in the asymptomatic control group (33.3%) (P < 0.00001).
There was no statistical association between the frequencies of the
antibodies and the strain used to prepare the antigen,
except for
anti-CagA antibodies, which were statistically more
frequently detected
in the three groups of patients when GAC strain
B225 was used as an
antigen (Fig.
5). In particular in the
GAC
group, anti-cagA antibodies were present in 93.3, 40, and 13%
of
the sera when strains B225, B110, and ATCC 43579 were used
as antigens,
respectively. Thus, anti-CagA antibodies of subjects
suffering from GAC
seemed to be more easily detected when immunoblotting
was performed
with a strain isolated from a patient suffering
from the same disease.
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FIG. 5.
Frequency (as percentages) of anti-CagA antibody in the
three groups according to the H. pylori strain used as an
antigen to perform the immunoblots.
|
|
Finally, factorial analysis of correspondence restricted to
seropositive subjects was realized to analyze entire immunoblot
patterns. Data points for seropositive patients were widely scattered
on the plan, and the data did not focus according to the clinical
origin of the patients, whatever the strain used as an antigen
(data
not
shown).
| |
DISCUSSION |
In this study, we analyzed the serological responses of patients
suffering from H. pylori-related GAC. The age and sex
distributions of GAC patients studied were representative of our area.
The risk that asymptomatic subjects would be affected by GAC was very
low; hence, our control group can be considered free from cancer,
despite the fact that no upper gastroduodenal endoscopy was performed.
The seroprevalence of H. pylori and its epidemiological
characteristics were similar to the data reported by others (13, 14). We found no significant difference in H. pylori
seroprevalence between GAC patients with diffuse-type tumors and those
with intestinal-type tumors, in agreement with some studies but not
with others (8, 10, 22, 34). The seroprevalences in the
three clinical groups were also similar to the results of other studies
(10, 13, 40).
Serology is recognized as one of the most reliable methods for
diagnosis of H. pylori infection. Therefore, the
seropositive patients may be considered H. pylori infected,
and the seronegative may be considered H. pylori free
(23, 35, 43). Among the serological methods used to study
the antibody response to H. pylori, immunoblotting is
certainly one of the most powerful (3, 43). This method
allows the detection of antibodies and a determination of the
specificity of these antibodies. The interpretation of the antibody
patterns in H. pylori-infected patients has not been well
established so far, even if some attempts have already been made
(6, 15). Nevertheless, because the antibody response reflects the features of both the infecting strain and the host response, it may be a helpful tool to predict the risk of an H. pylori-infected patient developing severe disease. For many years, several studies had sought correlations between antibodies against H. pylori and diseases related to this bacterium, using
enzyme-linked immunosorbent assay or immunoblot assay, but only one
considered all antibodies on immunoblot patterns of GAC patients
(25). In our study, we analyzed all antibody patterns by
immunoblotting using the factorial analysis of correspondence method.
We did not find an antibody pattern specific for GAC. Nevertheless, we found an association between antibodies to ferritin-like protein and
ulcer disease (12, 17). To our knowledge, this association has never been reported. In contrast to the majority of the studies, we
found that anti-VacA antibodies were more often present in sera from
asymptomatic subjects than in sera from the two other patient groups
(3, 37, 39, 50). At present, we have no explanation for this
finding. As with the majority of the studies, we found an association
between anti-CagA antibodies and GAC or gastroduodenal ulcer (11,
25, 37, 39, 48). There was no correlation between the expression
of the CagA proteins and their recognition by human serum. Strain ATCC
43579 expresses high CagA protein levels, and strain B225 expresses
lower levels. The CagA protein was not observed with strain B110 under
our analysis conditions (Fig. 1). However, CagA was more frequently
recognized when strain B225 was used as an antigen, a finding which
indicates qualitative differences in the CagA proteins.
The most adequate immunoblotting assay would probably be realized with
an antigenic extract prepared from the patient strain. However, the
infecting strains are not always available and, practically speaking,
such an assay would not be feasible. Thus, it may be helpful to define
what kind of strain is more relevant in obtaining an antigenic
preparation designed for immunoblot assays. Our results suggest that in
searching for anti-CagA antibodies in the sera of patients suffering
from GAC, the best choice would be a strain isolated from a patient
suffering from the same disease. Moreover, the specific recognition of
anti-CagA antibodies present in sera from GAC subjects and the CagA
antigen of strain B225 could suggest that GAC-associated H. pylori strains express a specific type of CagA protein. To confirm
these findings, it would be interesting to test more strains isolated
from patients in the three clinical groups included in the study and
also to analyze and compare the sequence of the cagA genes
in the three strains. The structure of the cagA gene of
H. pylori strain B225 is currently under investigation.
In conclusion, we confirmed the high diversity of the antibody response
to H. pylori and the strong association between anti-CagA and the
presence of GAC. This association is stronger if the CagA protein used
as an antigen comes from a strain isolated from a patient with GAC,
suggesting the existence of a type of CagA protein more implicated in
gastric carcinogenesis.
| |
ACKNOWLEDGMENTS |
We thank the Ligue contre le Cancer and the Université de
Poitiers for their financial support.
We are very grateful to B. J. Appelmek for his help in the
improvement of the manuscript.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratoire de
Microbiologie A, CHU La Milétrie, 86021 Poitiers Cedex, France.
Phone: 33 (5) 49 44 43 53. Fax: 33 (5) 49 44 38 88. E-mail:
blandinejanvier{at}hotmail.com.
| |
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Clinical and Diagnostic Laboratory Immunology, May 2000, p. 463-467, Vol. 7, No. 3
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Copyright © 2000, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
