Download Antibodies to Chlamydia trachomatis heat shock proteins in women

Human Reproduction vol.14 no.8 pp.1969–1973, 1999
Antibodies to Chlamydia trachomatis heat shock proteins
in women with tubal factor infertility are associated
with prior infection by C. trachomatis but not by
C. pneumoniae
K.Persson1,5, S.Osser2, S.Birkelund3, G.Christiansen3
and H.Brade4
1Department
of Clinical Microbiology and 2Gynecology and
Obstetrics, Malmö University Hospital, S-205 02 Malmö, Sweden,
3Department of Medical Microbiology and Immunology, University
of Aarhus, Denmark and 4Division of Medical and Biochemical
Microbiology, Forschungszentrum, Borstel, Germany
5To
whom correspondence should be addressed
The antibody response to heat shock proteins 60 and 10
were studied in 163 patients with tubal factor infertility
and in 163 age-matched pregnant women. The associations
of these antibodies with specific antibodies to Chlamydia
trachomatis and to Chlamydia pneumoniae as well as with
antibodies to the common chlamydial lipopolysaccharide
antigen were studied. Patients with tubal factor infertility
had significantly higher frequencies and titres of all antibodies except to C. pneumoniae. In a logistic regression
model an association was found between the prevalence of
antibodies to the heat shock proteins and to C. trachomatis
but no independent influence of antibodies to C. pneumoniae. No interaction between C. trachomatis and C. pneumoniae suggesting a synergistic effect was found although
the heat shock proteins from these two organisms are
immunologically similar. Antibodies to the chlamydial lipopolysaccharide also seemed to be related to C. trachomatis
and not to C. pneumoniae in these women.
Key words: Chlamydia trachomatis/Chlamydia pneumoniae/
hsp60/hsp10/infertility
Introduction
Tubal factor infertility has been linked to Chlamydia trachomatis infection in several sero-epidemiological studies
(Punnonen et al., 1979; Jones et al., 1982; Moore et al., 1982;
Cevenini et al., 1982; Gump et al., 1983; Kane et al., 1984).
The risk of infertility increases after repeated episodes of
salpingitis (Weström, 1980). Infertility after salpingitis is due
to occlusion of the Fallopian tubes by fibrous scarring. Such
scarring may occur as the result of an immunopathological
response after chronic or recurrent infection by C. trachomatis.
However, the severity of inflammation observed at laparoscopy
during the acute phase of salpingitis also seems directly to
influence the long-term fertility prospects (Weström, 1980).
Tubal occlusion after pelvic inflammatory disease is reminiscent of the situation in trachoma where relapsing or chronic
chlamydial infection results in tarsal scarring. Animal studies
have been helpful to uncover the pathogenic processes which
© European Society of Human Reproduction and Embryology
are likely to be involved. An extract of Chlamydia after
treatment by the detergent, Triton X-100, produced a follicular
response in monkeys previously immunized by a serovar B of
C. trachomatis but had no effect in immunologically naive
animals (Taylor et al., 1987). Triton extracts from either
C. trachomatis or Chlamydia psittaci were active while chlamydial lipopolysaccharide or major outer membrane preparations from C. trachomatis were not.
Similar scarring reactions in the conjunctivae were produced
in guinea pigs by a Triton X-100 extract of C. trachomatis or
of the GPIC strain of C. psittaci suggesting a genus-specific
component (Watkins et al., 1986). When the primary infection
was induced at another mucosal site delayed type hypersensitivity (DTH) reactions could still be elicited in the conjunctiva.
Later it was shown that a 60 kDa heat shock protein similar
to GroEL from E. coli was the active component (Morrison
et al., 1989a, b). In a monkey model of genital chlamydial
infection repeated inoculations produced tubal scarring probably due to a delayed hypersensitivity reaction (Patton et al.,
1990, 1994).
Chlamydial infections then seem to be able to sensitize
individuals for DTH-like reactions at various mucosal surfaces.
The scarring reaction observed in animals was genus-related
rather than species-specific. It is therefore conceivable that
C. pneumoniae infections might prime an immune response
in humans which may influence the development of tubal
infertility in response to a subsequent genital infection by
C. trachomatis.
Antibodies to heat shock proteins (hsp) have been shown
to be more common in infertile women compared to different
control groups (Wager et al., 1990; Brunham et al., 1992;
Toye et al., 1993; Arno et al., 1995; Dieterle and Wollenhaupt,
1996; Eckert et al., 1997; Claman et al., 1997). Chlamydial
hsp antibodies are also more common in patients with pelvic
inflammatory disease or in cases with ectopic pregnancies
(Peeling et al., 1997; Eckert et al., 1997; Sziller et al., 1998).
Some of the studies have demonstrated that the presence of
hsp60 antibodies is a predictor of tubal occlusion independent
of other chlamydial antibodies.
We (Osser et al., 1989) have previously confirmed the
association between C. trachomatis antibodies and tubal factor
infertility reported by several different groups. In this study
we re-examined the material with regard to the antibody
response to the hsp60 and hsp10 of C. trachomatis. The
correlation between such antibodies and species-specific antibodies to structural antigens of C. trachomatis and C. pneumoniae was analysed to be able to detect any joint effect between
these two infections with regard to hsp antibodies. Antibodies
to the lipopolysaccharide chlamydial antigen, which is shared
1969
K.Persson et al.
by the different species, were also analysed and the association
to hsp antibodies examined.
Materials and methods
As described elsewhere 163 women were identified with tubal factor
infertility (Osser et al., 1989). One age-matched pregnant woman
was selected as control for each of the 163 patients. Sera were
collected from both groups.
Chlamydial antibodies by micro-immunofluorescence (MIF)
The sera have been examined for antibodies to C. trachomatis
previously. At that time C. pneumoniae had not been recognized as
a common human pathogen. The sera were therefore re-tested for
antibodies to different species of Chlamydia. The micro-immunofluorescence test according to Wang (Wang and Grayston, 1970) was
used and performed in the following way.
Prototype strains were grown in yolk sacs of embryonated hens’
eggs. Suspensions of Chlamydia from infected yolk sacs were treated
with 0.1% formalin in PBS and then used as antigens. All antigens
were titrated using specific monoclonal antibodies to the different
species of Chlamydia to obtain optimal reactivity. The C. pneumoniae,
IOL-207 strain, and C. psittaci, 6BC strain, were used. A pool of
C. trachomatis serovars D through K was also included. One dot of
each antigen was placed in a cluster on microscopic slides. Each
slide had 12 such antigen clusters in two rows. Serum of different
dilutions was placed on the antigen clusters. End-point titrations of
the sera were performed. Geometric mean titres (GMT) were calculated for positive sera. Cut-off titres for positive sera were 1:64 for
both C. trachomatis and C. pneumoniae.
Antibodies to the chlamydial lipopolysaccharide antigen
Antibodies to the lipopolysaccharide (LPS) antigen of Chlamydia
were measured by a commercial kit (Medac, Hamburg, Germany).
The test employs a recombinant antigen of the chlamydial LPS in an
enzyme-linked immunosorbent assay (ELISA) format. IgG and IgA
antibodies were measured. Titres were calculated according to the
manufacturer’s suggestions. Positive titres for IgG were .150 and
for IgA .75.
Antibodies to heat shock proteins 60 and 10
The C. trachomatis genes for GroEL (Hsp60) and GroES (Hsp10)
were cloned into the expression vector pGEX3X, as previously
described (Cerrone et al., 1991; Larsen et al., 1994). The heat
shock protein was expressed as a fusion protein with glutathione-Stransferase and affinity-purified on glutathione-sepharose columns.
Likewise glutathione-S-transferase itself was also purified and used
as control protein.
An ELISA test was developed where StarWell MaxiSorp® 96-well
plastic plates (Nunc, Roskilde, Denmark) were coated with the hsp
fusion proteins or the control protein. An appropriate dilution of the
hsp60 was determined in preliminary tests using a monoclonal
antibody (Sigma, St Louis, MO, USA, H-3524 that cross-reacts with
hsp60 from both procaryotic and eucaryotic cells). Human sera diluted
1/100 were tested in microtitre plates coated with approximately
0.2 µg of the fusion hsp60 protein. An anti-human-IgG serum
conjugated with alkaline phosphatase was used together with a sodium
p-nitrophenylphosphate substrate to detect human antibodies. The
optical densities of the plates were measured in a spectrophotometer
at 405 nm. ELISA for hsp10 was performed in a similar way. A
positive serum for hsp60 and another for hsp10 were included in all
test runs and used as reference sera. The positive control serum was
assigned a relative titre of 100. Negative control sera were also
1970
Table I. Prevalence of antibodies to different chlamydial antigens in
patients and controls
Antibodies to
Patients (%)
Controls (%)
P value
MIF C. trachomatis
GMT
MIF C. pneumoniae
GMT
LPS (IgG)
GMT
LPS (IgA)
GMT
Hsp60
GMT
hsp10
GMT
144/163 (88)
199
84/163 (52)
23
141/163 (87)
993
128/163 (79)
172
103/163 (63)
24
73/163 (45)
27
78/163
19
95/163
28
77/163
341
74/163
104
33/163
9
39/163
15
,0.001a
,0.001b
NSa
NSb
,0.001a
,0.001b
,0.001a
,0.001b
,0.001a
,0.001b
,0.001a
,0.001b
(48)
(58)
(47)
(45)
(20)
(24)
compared by the χ2 test.
mean titres (GMT) by the Mann–Whitney U test.
NS 5 not significant; LPS 5 lipopolysaccharide; MIF 5 microimmunofluorescence; hsp 5 heat shock protein.
aProportions
bGeometric
included in each run. The titre was calculated as the difference
between the ODs for a serum between positive and negative antigens
in relation to that of the reference positive serum with a titre of 100.
Sera with a titre value of .10 were considered antibody positive and
sera with titre values of ,10 were considered negative.
Statistical methods
Student’s t-test was used to compare group mean values, but for
some variables, where severe skewness could not be eliminated even
after transformation, the Mann–Whitney rank sum test was used. The
χ2 test was used to compare proportions, and Spearman’s rank
correlation coefficient was used to investigate correlations among
variables. Logistic regression analysis was used to identify associations
between tubal factor infertility and different chlamydial antibodies.
Results
MIF antibodies to C. trachomatis and C. pneumoniae
Antibodies specific to C. trachomatis were detected more often
in patients than in controls (Table I) and the geometric mean
titre of such antibodies was significantly higher among infertile
women. In contrast, no differences either in frequency or in
titre were demonstrated between patients and controls for
antibodies to C. pneumoniae. Cross-reactivity was not detected
as C. trachomatis antibodies were equally common in patients
with and without concurrent antibodies to C. pneumoniae, 75/
84 (89%) and 69/79 (87%) respectively.
Chlamydial LPS antibodies
IgG and IgA antibodies to the chlamydial LPS antigen were
again significantly more common and with higher titres among
patients than controls. Chlamydial LPS antibodies can be
elicited by infections by either C. trachomatis or C. pneumoniae. In the whole study group, including patients and controls,
there was a good correlation between LPS-IgG antibodies and
MIF antibodies to C. trachomatis (rs 5 0.67, P , 0.001
Spearman’s correlation) but a poor correlation to C. pneumoniae antibodies (rs 5 0.28, P , 0.001). The same trends were
observed for LPS-IgA antibodies although the coefficients
were lower. The same pattern was observed when patients and
Chlamydia hsp response in tubal infertility
Table II. Logistic regression analysis of factors that might predict the
presence of antibodies to hsp60 and hsp10
hsp
Group
Antibodies
P value
OR
95% CI
hsp60
patients
C. trachomatis
C. pneumoniae
LPS-IgG
LPS-IgA
C. trachomatis
C. pneumoniae
LPS-IgG
LPS-IgA
C. trachomatis
C. pneumoniae
LPS-IgG
LPS-IgA
C. trachomatis
C. pneumoniae
LPS-IgG
LPS-IgA
0.004
NS
NS
NS
0.004
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
NS
1.93
1.25–2.97
controls
hsp 10
patients
controls
2.06
1.28–3.33
OR 5 odds ratio.
CI 5 confidence interval.
Abbreviations as in Table I.
Table III. Correlations between antibodies to hsp60 and the other
chlamydial antibodies using the Spearman rank order correlation test
hsp60/
MIF C. pneumoniae
MIF C. trachomatis
LPS-IgG
LPS-IgA
hsp10
C. trachomatis/
LPS-IgG
LPS-IgA
hsp10
MIF C. pneumoniae
hsp10/
LPS-IgG
LPS-IgA
C. pneumoniae/
LPS-IgG
LPS-IgA
Patients: correlation
coefficient
Controls: correlation
coefficient
rs
P value
rs
P value
0.17
0.46
0.53
0.43
0.36
0.03
,0.001
,0.001
,0.001
,0.001
0.18
0.52
0.54
0.43
0.24
0.02
,0.001
,0.001
,0.001
0.002
0.49
0.27
0.24
0.10
,0.001
0.001
0.002
NS
0.61
0.37
0.11
0.07
,0.001
,0.001
NS
NS
0.21
0.13
0.007
NS
0.20
0.17
0.01
0.03
0.26
0.15
0.001
0.05
0.35
0.25
,0.001
0.001
Abbreviations as in Table I.
controls were analysed separately but the correlations were
generally less pronounced. The influence of prior C. trachomatis or C. pneumoniae on the incidence of LPS antibodies
was also analysed in a logistic regression model. Antibodies
to C. trachomatis and C. pneumoniae were both independent
predictors of LPS-IgG and LPS IgA antibodies although C.
trachomatis seemed to be the most important predictor in both
patients and controls.
As the LPS-IgA antibody response is generally considered
more transient than that of LPS-IgG the ratio between IgA
and IgG antibodies to chlamydial LPS was calculated. In the
patient group this ratio was 0.91 and in the controls it was
0.96, not statistically different (χ2 test). The GMT for LPSIgG among patients who had such antibodies was 1142 and
645 among controls, which was a statistically significant
difference (P , 0.001, Student’s t-test). The corresponding
LPS-IgA mean titres in patients and controls were 205 and
172 respectively, a difference not statistically significant.
Antibodies to chlamydial hsp60 and hsp10
The prevalence of antibodies to chlamydial hsp60 and hsp10
were higher in patients than in controls and with higher titres
in patients. The individual association of C. trachomatis and
C. pneumoniae antibodies to hsp antibodies was analysed by
a logistic regression model. The model included MIF antibodies
to C. trachomatis and C. pneumoniae and IgG and IgA
antibodies to the chlamydial LPS. The only factor that independently predicted the presence of antibodies to hsp60 or
hsp10 was MIF IgG antibodies to C. trachomatis. This was
the case in both patients and controls. These results are
summarized in Table II.
A synergistic effect between infections to C. pneumoniae
and C. trachomatis on the antibody response to hsp60 would
appear as an interaction between these factors in the logistic
model. To test for such an effect an interaction variable was
constructed in which the presence of antibodies to both
C. trachomatis and C. pneumoniae was noted as positive and
all other combinations as negative. This variable when included
in the analysis did not come out as a significant individual
predictor of antibodies to hsp60. A synergistic effect between
C. trachomatis and C. pneumoniae could therefore not be
detected.
The correlations between titres of hsp60 and the various
antibodies to Chlamydia were also examined. Antibodies to
hsp60 were correlated to C. trachomatis in patients (rs 5 0.46,
P , 0.001) and in controls (rs 5 0.52, P , 0.001) but not to
antibodies to C. pneumoniae (rs 5 0.17/0.18, P 5 0.03/0.02)
for patients and controls (Table III). The correlation between
antibodies to hsp60 and chlamydial LPS antibodies was as
good (rs 5 0.43–0.54, P , 0.001) as the correlation between
hsp60 and C. trachomatis antibodies (Table III).
The correlation between antibodies to hsp10 and all other
antibodies was generally rather low even to hsp60 (Table III).
Predictors of tubal factor infertility
The prevalence of hsp antibodies and MIF antibodies as well
as LPS IgG and IgA antibodies was higher in patients with
tubal factor infertility than in fertile control women. The
frequency of antibodies to C. pneumoniae did not differ
between these groups. The individual association between the
different antibodies and tubal factor infertility was tested in a
logistic regression model where patients and controls were
grouped together. Only hsp60 antibodies and MIF antibodies
to C. trachomatis came out as independent predictors of tubal
factor infertility while LPS IgG and IgA did not. The odds
ratio for hsp60 was 3.54 (CI 1.92–5.86) and for C. trachomatis
antibodies 1.36 (CI 1.10–1.69).
Discussion
In this study we confirmed our previously reported results that
antibodies to C. trachomatis detected by MIF are more common
in women with tubal factor infertility than in age-matched
1971
K.Persson et al.
pregnant women. This is also in agreement with results from
other studies (Punnonen et al., 1979; Jones et al., 1982; Moore
et al., 1982; Cevenini et al., 1982; Gump et al., 1983; Kane
et al., 1984). Antibodies to C. pneumoniae can also be
separately distinguished by MIF. The prevalence of C. trachomatis antibodies was independent of concurrent C. pneumoniae antibodies. The prevalence of antibodies to C. pneumoniae
was similar in both patients and controls. Individuals who had
been exposed to C. pneumoniae were therefore not more likely
to develop tubal factor infertility than those who had not.
These results differ from those of one other study that addressed
this issue (Freidank et al., 1995). That study reported that
antibodies to C. pneumoniae were more common in patients
with bilateral tubal occlusion than in patients with unilateral
tubal damage or in women with patent Fallopian tubes. There
is no obvious explanation for the differing results in that study
and ours.
The results of our study, however, tend to argue against a
synergistic effect of C. pneumoniae and C. trachomatis infections for the development of tubal damage causing infertility.
Had such an effect existed a higher frequency of C. pneumoniae
antibodies would have been expected among the patients with
tubal infertility as prior C. pneumoniae infection would have
been a risk factor for tubal damage at subsequent C. trachomatis
infection.
The relation between antibodies to C. pneumoniae and the
antibody response to hsp60 was also examined. The hsp60 has
been associated with tarsal scarring in animal models of
trachoma and antibodies to the hsp60 have been found more
often in patients with tubal factor infertility, ectopic pregnancy
and pelvic inflammatory disease than in control women (Wager
et al., 1990; Brunham et al., 1992; Toye et al., 1993; Arno
et al., 1995; Dieterle and Wollenhaupt, 1996; Claman et al.,
1997; Peeling et al., 1997; Eckert et al., 1997; Sziller et al.,
1998). Our results are in agreement with these previous studies
as we detected a higher prevalence and a higher mean titre of
antibodies to hsp60 in patients than in controls. The relation
between hsp60 antibodies and MIF and LPS antibodies was
examined in a logistic regression model. Only MIF antibodies
to C. trachomatis were associated with hsp 60 antibodies in
patients and controls while antibodies to C. pneumoniae and
chlamydial LPS were not. This association suggests that
infection by C. trachomatis but not by C. pneumoniae is
important for the hsp60 antibody response. We also examined
how the different antibodies predicted tubal factor infertility.
Only MIF antibodies to C. trachomatis and hsp60 antibodies
independently predicted tubal factor infertility where hsp60
was the stronger predictor. This was true even when only
individuals who were antibody positive for C. trachomatis
were included in the analysis. These findings agree with what
have been reported by others as well (Wager et al., 1990; Toye
et al., 1993; Claman et al., 1997; Peeling et al., 1997).
In most studies MIF antibodies to C. trachomatis have been
measured in addition to hsp60 antibodies. In this study we
also tested for IgG and IgA antibodies to the common
chlamydial LPS. Both C. trachomatis and C. pneumoniae can
elicit such antibodies. LPS antibodies were more prevalent
among patients than controls and correlated with antibodies
1972
to hsp60 and C. trachomatis but not with antibodies to
C. pneumoniae. An association between LPS antibodies and
antibody reactivity to hsp60 has been reported in one other
study (Domeika et al., 1998). Thus the LPS antibody response
in our patients seems to be mainly due to C. trachomatis.
Tubal occlusion might occur after a long-standing chronic
Chlamydia infection of the Fallopian tubes or after relapsing
or repeated infections, as would seem to be the case in
advanced trachoma. Weström reported, however, that the
severity of acute salpingitis as noted at laparoscopic examination could predict the fertility outcome (Weström, 1980). Tubal
damage with fertility implications may therefore occur during
the acute phase of chlamydial salpingitis whether or not chronic
infection will follow. IgA antibodies are of shorter duration
than IgG antibodies. This is the case particularly with LPSIgA antibodies. The presence of such antibodies might therefore
indicate continuing active infection. Although the prevalence
of IgA antibodies to chlamydial LPS was higher in patients
than in controls this was not the case after adjustment for
different exposure rates in the two groups. Thus the ratios of
IgA and IgG LPS antibodies were similar in patients and
controls and with similar mean titres. These serological results
did not suggest persistent infection.
C. trachomatis infection of the Fallopian tubes leading
to tubal occlusion and infertility elicit a strong antibody
response to several different chlamydial antigens. In agreement
with similar studies we found that hsp60 and MIF antibodies
to C. trachomatis independently predicted the presence of
tubal factor infertility. Although animal models of trachoma
have suggested that the immune response leading to scarring
is a genus specific reactivity and not species specific we could
find no evidence of C. pneumoniae being involved in the
development of tubal factor infertility.
Acknowledgement
The production of heat shock protein was supported by a grant from
the EU network, ERBCHRXCT920040.
References
Arno, J.N., Yuan, Y., Cleary, R.E. and Morrison, R.P. (1995) Serologic
responses of infertile women to the 60-kd chlamydial heat shock protein
(hsp60). Fertil. Steril., 64, 730–735.
Brunham, R.C., Peeling, R., Maclean, I. et al. (1992) Chlamydia trachomatisassociated ectopic pregnancy: serologic and histologic correlates. J. Infect.
Dis., 165, 1076–1081.
Cerrone, M.C., Ma, J.J. and Stephens, R.S. (1991) Cloning and sequence of
the gene for heat shock protein 60 from Chlamydia trachomatis and
immunological reactivity of the protein. Infect. Immun., 59, 79–90.
Cevenini, R., Possati, G. and La Placa, M. (1982) Chlamydia trachomatis
infection in infertile women. In Mårdh, P-A., Holmes, K.K., Oriel, J. et al.
(eds), Chlamydial Infections. Elsevier Biomedical Press, New York, pp.
189–192.
Claman, P., Honey, L., Peeling, R.W. et al. (1997) The presence of serum
antibodies to the chlamydial heat shock protein (CHSP60) as a diagnostic
test for tubal factor infertility. Fertil. Steril., 67, 501–504.
Dieterle, S. and Wollenhaupt, J. (1996) Humoral immune response to the
chlamydial heat shock proteins hsp60 and hsp70 in chlamydia-associated
chronic salpingitis with tubal occlusion. Hum. Reprod., 11, 1352–1356.
Domeika, M., Domeika, K., Paavonen, J. et al. (1998) Humoral immune
response to conserved epitopes of Chlamydia trachomatis and human
60-kDa heat-shock protein in women with pelvic inflammatory disease.
J. Infect. Dis., 177, 714–719.
Chlamydia hsp response in tubal infertility
Eckert, L.O., Hawes, S.E., Wölner-Hansen, P. et al. (1997) Prevalence and
correlates of antibody to chlamydial heat shock protein in women attending
sexually transmitted disease clinics and women with confirmed pelvic
inflammatory disease. J. Infect. Dis., 175, 1453–1458.
Freidank, H.M., Clad, A., Herr, A.S. et al. (1995) Immune response to
Chlamydia trachomatis heat shock protein in infertile female patients and
influence of Chlamydia pneumoniae antibodies. Eur. J. Clin. Microbiol.
Infect. Dis., 14, 1063–1069.
Gump, D.W., Gibson, M. and Ashikaga, T. (1983) Evidence of prior pelvic
inflammatory disease and its relationship to Chlamydia trachomatis antibody
and intrauterine contraceptive device use in infertile women. Am. J. Obstet.
Gynecol., 146, 153–159.
Jones, R.B., Ardery, B.R., Hui, S.L. and Cleary, R.E. (1982) Correlation
between serum antichlamydial antibodies and tubal factor as a cause of
infertility. Fertil. Steril., 38, 553–558.
Kane, J.L., Woodland, R.M., Forsey, T. et al. (1984) Evidence of chlamydial
infection in infertile women with and without Fallopian tube obstruction.
Fertil. Steril., 42, 843–848.
Larsen, B., Birkelund, S., Mordhorst, C.H. et al. (1994) The humoral immune
response to Chlamydia trachomatis in patients with reactive arthritis. Br. J.
Rheumatol., 33, 534–540.
Moore, D.E., Foy, H.M., Daling, J.R. et al. (1982) Increased frequency of
serum antibodies to Chlamydia trachomatis in infertility due to distal tubal
disease. Lancet, 2, 574–577.
Morrison, R.P., Belland, R.J., Lyng, K. and Caldwell, H.D. (1989a) Chlamydial
disease pathogenesis: The 57-kD chlamydial hypersensitivity antigen is a
stress response protein. J. Exp. Med., 170, 1271–1283.
Morrison, R.P., Lyng, K. and Caldwell, H.D. (1989b) Chlamydial disease
pathogenesis: ocular hypersensitivity elicited by a genus-specific 57 kd
protein. J. Exp. Med., 169, 663–675.
Osser, S., Persson, K. and Liedholm, P. (1989) Tubal infertility and silent
chlamydial salpingitis. Hum. Reprod., 4, 280–284.
Patton, D.L., Wölner-Hansen, P., Cosgrove, S.J. and Holmes, K.K. (1990) The
effects of Chlamydia trachomatis on the female reproductive tract of the
Macaca nemestrina after a single tubal challenge following repeated cervical
inoculations. Obstet. Gynecol., 76, 643–650.
Patton, D.L., Sweeney, Y.T. and Kuo, C.C. (1994) Demonstration of delayed
hypersensitivity in Chlamydia trachomatis salpingitis in monkeys: a
pathogenic mechanism of tubal damage. J. Infect. Dis., 169, 680–683.
Peeling, R.W., Kimani, J., Plummer, F. et al. (1997) Antibody to Chlamydial
hsp60 predicts an increased risk for chlamydial pelvic inflammatory disease.
J. Infect. Dis., 175, 1153–1158.
Punnonen, R., Terho, P., Nikkanen, V. and Meurman, O. (1979) Chlamydial
serology in infertile women by immunofluorescence. Fertil. Steril., 31,
656–659.
Sziller, I., Witkin, S.S., Ziegert, M. et al. (1998) Serological responses of
patients with ectopic pregnancy to epitopes of Chlamydia trachomatis 60
kDa heat shock protein. Hum. Reprod., 13, 1088–1093.
Taylor, H.R., Johnson, S.L., Schachter, J. et al. (1987) Pathogenesis of
trachoma: the stimulus for inflammation. J. Immunol., 138, 3023–3027.
Toye, B., Laferrière, C., Claman, P. et al. (1993) Association between antibody
to the chlamydial heat shock protein and tubal infertility. J. Infect. Dis.,
168, 1236–1240.
Wager, E.A., Schachter, J., Bavoil, P. and Stephens, R.C. (1990) Differential
human serologic response to two 60 000 molecular weight Chlamydia
trachomatis antigens. J. Infect. Dis., 162, 922–927.
Wang, S.P. and Grayston, J.T. (1970) Immunologic relationship between
genital TRIC, lymphogranuloma venereum and related organisms in a
new microtiter indirect immunofluorescence test. Am. J. Ophthalmol., 70,
367–374.
Watkins, N.G., Hadlow, W.J., Moos, A.B. and Caldwell, H.D. (1986) Ocular
delayed hypersensitivity: a pathogenetic mechanism of chlamydial
conjunctivitis in guinea pigs. Proc. Natl Acad. Sci. USA, 83, 7480–7484.
Weström, L. (1980) Incidence, prevalence, and trends of acute pelvic
inflammatory disease and its consequences in industrialised countries. Am.
J. Obstet. Gynecol., 138, 880–892.
Received on November 17, 1998; accepted on April 21, 1999
1973