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ASIC SCIENCE: GYNECOLOGY
ssociation of tubal factor infertility with elevated antibodieso Chlamydia trachomatis caseinolytic protease Pllison K. Rodgers, MD; Jie Wang, MD; Yingqian Zhang, BS; Alan Holden, PhD; Blake Berryhill, MD;icole M. Budrys, MD; Robert S. Schenken, MD; Guangming Zhong, MD, PhD
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BJECTIVE: The objective of the study was to assess antibodiesgainst Chlamydia trachomatis heat shock proteins (HSP) in patientsith tubal factor infertility (TFI), infertility controls (IFC), and fertile con-
rols (FC). HSPs assist organisms in surviving caustic environmentsuch as heat.
TUDY DESIGN: Twenty-one TFI, 15 IFC, and 29 FC patients were en-olled after laparoscopic tubal assessment. The titers of antibodiesgainst C trachomatis organisms and 14 chlamydial HSPs were com-ared among the 3 groups.
ESULTS: TFI patients developed significantly higher levels of antibod-
ynecol 2010;203:494.e7-14.
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002-9378/$36.00 • © 2010 Mosby, Inc. All rights reserved. • doi: 10.1016
SP60 and caseinolytic protease (Clp) P, a subunit of the ATP-depen-ent Clp protease complex involved in the degradation of abnormalroteins.
ONCLUSION: In addition to confirming high titers of antibodies againsttrachomatis organisms and HSP60 in TFI patients, we identified a
ovel link of TFI with anti-ClpP antibodies. These findings may provideseful information for developing a noninvasive screening test for TFInd constructing subunit anti-C trachomatis vaccines.
ey words: antibodies to caseinolytic protease P, Chlamydia
es against C trachomatis and specifically recognizing chlamydial trachomatis, heat shock protein, tubal factor infertilityite this article as: Rodgers AK, Wang J, Zhang Y, et al. Association of tubal factor infertility with elevated antibodies to Chlamydia trachomatis ClpP. Am J Obstet
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hlamydia trachomatis is the mostcommon reported agent of sexually
ransmitted infections worldwide.1 Theate of C trachomatis infection in thenited States has increased significantlyver the last 2 decades.2 Infection with Crachomatis poses serious health risks,ncluding long-term reproductive tractequelae such as infertility, chronic pel-ic pain, ectopic pregnancy,3-6 and de-elopment of cervical cancer.7,8
The linkage of tubal factor infertilityTFI) to C trachomatis infection has beenxtensively studied. C trachomatis or-anisms can be isolated from a large por-ion of women with TFI3 and elevatednti–C trachomatis antibodies can be de-
rom the Departments of Obstetrics and Gynudrys, and Schenken) and Microbiology andniversity of Texas Health Science Center at Sf Immunology (Dr Wang), Xiangya Medicalhangsha, Hunan, China.
eceived April 10, 2009; revised May 20, 2010;
eprints: Guangming Zhong, MD, PhD, Departmf Texas Health Science Center at San Antonio,[email protected].
his study was supported in part by Grant R01Aealth.
ected in more than 70% of women withubal occlusion.9 Women with prior Crachomatis infection usually maintainigh titers of C trachomatis antibodies.10
lthough urogenital tract infections withtrachomatis is common and has been
ecognized as a significant cause of tubalnfertility, the pathogenic mechanisms of
trachomatis–induced tubal damage re-ain unknown and no effective vaccines
re available.It has been hypothesized that host
esponses triggered by chlamydial in-ection contribute to both protectivemmunity and pathogenesis. Antibodiesgainst the chlamydial major outerembrane protein (MOMP) are associ-
logy (Drs Rodgers, Holden, Berryhill,munology (Drs Wang, Zhang, and Zhong),Antonio, San Antonio, TX; and Departmentool, The Central South University,
epted June 1, 2010.
t of Microbiology and Immunology, University3 Floyd Curl Dr., San Antonio, TX 78229.
537 (to G.Z.) from the National Institutes of
2/j.ajog.2010.06.005
NOVEMBER 2010 Americ
ted with protective host immune re-ponses, which is consistent with the re-ent findings that immunization with aative MOMP-induced protection.11,12
n contrast, antibodies to chlamydialeat shock protein (HSP) 60 are associ-ted with pathologies,4,13-15 which mayrovide a partial explanation for thealf-century-old observation that wholehlamydial organism-based vaccinesesigned for preventing trachoma inhildren actually exacerbated patholo-ies.16-18 HSPs assist organisms in sur-iving stressful environments such ascidity or heat.
Our objective was to test whether hu-an antibodies against other C tracho-atis HSPs are also associated with
hlamydia-induced tubal pathologies byomparing all 14 chlamydial HSPs forheir reactivity with antibodies in pa-ients with TFI, infertility controls (IFC),nd fertile controls (FC).
ATERIALS AND METHODSuman antiseraollowing approval by the institutionaleview board at the University of Texasealth Science Center at San Antonio,
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1 TFI, 15 IFC, and 29 FC patients were
an Journal of Obstetrics & Gynecology 494.e7
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Research Basic Science: Gynecology www.AJOG.org
4
nrolled. All recruited women under-ent diagnostic laparoscopy with chro-otubation. Diagnosis of tubal infer-
ility was based on 1 of the followingndings: hydrosalpinx, fimbrial phimo-is, or peritubal adhesions. Women withrior tubal ligation or a history of pelvic
nfection or inflammation other thanelvic inflammatory disease such as ap-endicitis were excluded. IFC patientsere women with normal pelvic findings
nd tubal patency at laparoscopy. FC pa-ients had no history of infertility with ateast 1 live birth and normal pelvic find-ngs at time of tubal ligation. All partici-ants underwent a single blood draw. Se-um samples were stored at –20°C untilnalyzed.
ell culture and chlamydial infectioneLa cells (American Type Culture Col-
ection, Manassas, VA) were cultured inulbecco’s modified Eagle’s medium
DMEM; Gibco PRL, Rockville, MD)ith 10% fetal calf serum (FCS; GibcoRL) at 37°C with 5% carbon dioxideCO2) as previously described.19 C tra-homatis serovar D or C pneumoniaeR39 organisms were grown, purified,nd titrated as previously described.20,21
After titration, organisms were storedt – 80°C. For immunofluorescence as-ay, chlamydial organisms were used tonfect HeLa cells grown on glass cover-lips in 24-well plates. The subconfluenteLa cells were treated with DMEM
ontaining 30 �g/mL of diethylamino-thyl (DEAE)-dextran (Sigma, St Louis,
O) for 10 minutes. After removal ofEAE-dextran solution, chlamydial or-
anisms were added to the wells for 2ours at 37°C. The infected cells wereontinuously cultured in DMEM with0% FCS and 2 �g/mL of cycloheximideSigma).
For preparing whole-cell lysates, in-ection was carried out in tissue cultureasks. Infected cultures were processedr harvested 48 hours after infection ors indicated in individual experiments.
mmunofluorescence assayntichlamydial organism antibodies inuman sera were titrated using an im-unofluorescence assay as previously
escribed.22,23 Briefly, HeLa cells grown t
94.e8 American Journal of Obstetrics & Gynecolo
n coverslips were infected with C tra-homatis or C pneumoniae organisms,xed 48 hours after infection for C tra-homatis and 72 hours for C pneumoniaeith 2% paraformaldehyde, and perme-
bilized with 2% saponin. After block-ng, human antisera were added to thehlamydia-infected cell samples. Goatntihuman immunoglobulin (Ig) G con-ugated with Cy2 (green; Jackson Immu-oResearch Laboratories, Inc, Westrove, PA) was used to visualize human
ntibody binding and a Hoechst de-xyribonucleic acid (DNA) dye (blue;igma) to visualize HeLa and chlamydialNA. The highest dilution of a serum
hat still gave a positive reactivity was de-ned as the titer of the given serumample.
All human serum samples were seri-lly diluted, and the appropriate dilu-ions were repeated multiple times basedn the results obtained from prior dilu-ions to obtain a more accurate titer forach serum. For the time-course study,he C trachomatis–infected HeLa cellsere processed as described above at var-
ous time points after infection as indi-ated in the data figure.
The processed samples were coimmu-ostained with a mouse anti-HSP60unpublished data) or anti-caseinolyticrotease (Clp) P (unpublished data) plusabbit anti–C trachomatis serovar D or-anisms. The primary antibody bindingas visualized with a goat antimouse IgG
onjugated with Cy3 (red) and a goat an-irabbit IgG conjugated with Cy2 (green;oth from Jackson ImmunoResearchaboratories), respectively, and DNA byHoechst DNA dye.Images were acquired with an Olympus
X70 fluorescence microscope equippedith multiple filter sets (Olympus, Mel-ille, NY) as previously described.23 All mi-roscopic images were processed using andobe Photoshop program (Adobe Sys-
ems, San Jose, CA).
hlamydial fusion protein–arrayedicroplate enzyme-linked
mmunosorbent assay (ELISA)he glutathione S-transferase (GST) fu-
ion protein ELISA for detecting humanntibody recognition of chlamydial pro-
eins was carried as previously de- sgy NOVEMBER 2010
cribed.23 The bacterial lysates contain-ng individual chlamydial GST fusionroteins were added to 96-well mi-roplates precoated with glutathionePierce, Rockford, IL).
The GST fusion protein lysates in-luded all 14 chlamydial HSP familyembers: GST-CT110 (GroEL, HSP60);ST-CT111 (GroES, HSP10); GST-T113 (ClpB, ClpB-related ATP-depen-ent protease); GST-CT286 (ClpC, Clprotease ATP-binding subunit); GST-T341 (DnaJ protein); GST-CT395
GrpE, HSP70 cofactor); GST-CT396DnaK, HSP70); GST-CT407 (DksA,robable DnaK suppressor); GST-T431 (ClpP, ATP-dependent ClpP en-opeptidase); GST-CT604 (GroEL,SP60); GST-CT705 (ClpX, ATP-de-
endent ClpX-related protease; GST-T706 (ClpP, ATP-dependent ClpP
ndopeptidase subunit); GST-CT709MreB, Rod shape determining protein
reB/HSP70 sugar kinase); and GST-T755 (HSP60).Lysates containing GST alone, as neg-
tive, and GST-chlamydial protease-likectivity factor, as positive controls, werelso included. After blocking, human an-isera preabsorbed with a bacterial ly-ates containing GST alone were reactedith the plate-immobilized fusion pro-
eins. The human antibody reactivityas detected with a goat antihuman-IgG
onjugated with horseradish peroxidaseHRP; Jackson ImmunoResearch Labo-atories) plus the substrate 2,2’-azino-i(2-ethylbenzothiazoline-6-sulforiccid) diammonium salt (ABTS; Sigma).he optical density (OD) was measuredt 405 nm using a microplate readerMolecular Devices Corp, Sunnyvale,A).To confirm the antibody-binding
pecificity, all antisera were further ab-orbed with lysates made from eithereLa cells alone or C trachomatis serovar–infected HeLa cells prior to reactingith the fusion protein-coated plates.he antibody binding that remainedositive after HeLa-alone lysate absorp-ion but significantly reduced by chla-
ydia-HeLa lysate absorption was con-
idered true positive.
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www.AJOG.org Basic Science: Gynecology Research
estern blotestern blot with GST fusion proteins
s antigens was carried out as previouslyescribed.20 GST fusion proteins (GST-SP60, GST-HSP10, GST-ClpP) were
urified from the corresponding bacte-ial lysates using glutathione agaroseeads as previously described.24 The pu-ified fusion proteins were resolved on aodium dodecyl sulfate-polyacrylamideel and transferred to a nitrocelluloseembrane. Membrane-immobilized pro-
eins were reacted with human seraooled from each patient group and pre-bsorbed with bacterial lysates contain-ng GST alone. Human antibody bindingas detected with a goat antihuman IgG-RP secondary antibody and visualizedith an enhanced chemiluminescenceit (Santa Cruz Biotechnology, Inc,
TABLE 1Titers of human antibodies against
Variable
Antibo
TFI(n � 2
Mean 90,19...................................................................................................................
SD 123,70...................................................................................................................
ANOVA P � .0...................................................................................................................
Student t test TFI vs ITFI vs FIFC vs
...................................................................................................................
Categorization of serum samples into negative..........................................................................................................
Negative titers (�1:10) 1 (5%..........................................................................................................
Low titers (1:10-1:10,000) 6 (29%..........................................................................................................
High titers (�1:10,000) 14 (67%...................................................................................................................
�2 test P � .0...................................................................................................................
Logistic regression TFI vs I..................
TFI vs I..................
TFI vs F..................
FC vs I..................
FC vs I...................................................................................................................
Serum samples from women with TFI, IFC, or FC were 2-fold sthat still gave a positive reactivity was defined as the serum titgiven serum sample. ANOVA was used to analyze the overall dbut not C pneumoniae (P � .45) organisms. The significant dicategories (negative, low, and high) based on antibody titers, thagainst C trachomatis (P � .009) but not C pneumoniae (P �TFI and IFC, TFI and FC, and the IFC and FC groups. The numbgroups, although there are also differences between the IFC aANOVA, analysis of variance; FC, fertile controls; IFC, infertility
Rodgers. Tubal factor infertility and anti-ClpP antibodies
anta Cruz, CA). a
ata analysesata were analyzed using SPSS version
5.0 software (IBM, Chicago, IL). As areliminary step, titer values were logransformed to produce a normal distri-ution and analyses were performed onransformed values. Analysis of varianceas used to assess anti-C trachomatis and
nti-C pneumoniae antibodies to evalu-te overall mean differences among the 3roups of patients.The Student t test was utilized to com-
are differences between groups. Becausehe antibody titers had large variationsithin a given group, the serum titers were
valuated by ranges of less than 1:10 (neg-tive), 1:10 to 1:10,000 (low), and greaterhan 1:10,000 (high). The �2 and Fisher’sxact tests were used to compare TFI, IFC,nd FC overall antibodies to C trachomatis
trachomatis and C pneumoniaeto C trachomatis
IFC(n � 15)
FC(n � 29)
4488 36,994.........................................................................................................................
10,110 83,570.........................................................................................................................
.........................................................................................................................
P � .012P � .075P � .142.........................................................................................................................
w, and high titer groups.........................................................................................................................
2 (13%) 1 (3%).........................................................................................................................
12 (80%) 15 (52%).........................................................................................................................
1 (7%) 13 (45%).........................................................................................................................
.........................................................................................................................
High vs negative P � .04.........................................................................................................................
High vs low P � .004.........................................................................................................................
High vs low P � .03.........................................................................................................................
High vs negative P � .04.........................................................................................................................
High vs low P � .03.........................................................................................................................
y diluted starting with 1:10 and reacted with HeLa cells infected wach serum sample was titrated 3 times, and the average from thences among the 3 groups. There is a statistically significant diffece was determined between the TFI and IFC groups by Student t
test still revealed a significant difference in the number of sera in dirganisms. Further logistic regression analyses of the anti–C trach
f individuals with high anti–C trachomatis antibody titers in the TFC groups.trols; TFI, tubal factor infertility.
J Obstet Gynecol 2010.
nd antibodies to C pneumoniae. 1
NOVEMBER 2010 Americ
Finally, we evaluated pairwise differ-nces between TFI vs IFC, TFI vs FC, andC vs IFC in C trachomatis using logisticegression. ELISA results were analyzedlso using �2 and Fisher’s exact tests asppropriate.
ESULTS
hen C trachomatis–infected cells weresed as antigens to titrate the patienterum antibodies, the TFI group hadigher titers than the IFC and FC groupsTable). The titers of anti-C trachomatisntibodies were significantly greater inatients with TFI. Because the antibodyiters had large variations within a givenroup, the serum titers were evaluated byanges of less than 1:10 (negative), 1:10o 1:10,000 (low), and greater than
Antibodies to C pneumoniae
TFI(n � 21)
IFC(n � 15)
FC(n � 29)
56,010 32,027 56,429..................................................................................................................
75,580 41,930 65,140..................................................................................................................
P � .45..................................................................................................................
..................................................................................................................
..................................................................................................................
3 (14%) 4 (27%) 0 (0%)..................................................................................................................
3 (14%) 1 (7%) 5 (17%)..................................................................................................................
15 (71%) 10 (67%) 24 (83%)..................................................................................................................
P � .09..................................................................................................................
..................................................................................................................
..................................................................................................................
..................................................................................................................
..................................................................................................................
..................................................................................................................
ither C trachomatis or C pneumoniae. The highest dilutiondependent titrations was used as the geometric titer of ae in titers of antibodies against C trachomatis (P � .018)(P � .012). When the serum samples were divided into 3t categories among the 3 groups of patients for antibodies
tis antibodies revealed significant differences between thep is significantly higher than those in either the IFC or FC
Cdies
1)
9......... .........
0......... .........
18......... .........
FC,C,
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)......... .........
)......... .........
)......... .........
09......... .........
FC......... .........
FC......... .........
C......... .........
FC......... .........
FC......... .........
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:10,000 (high).
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494.e10 American Journal of Obstetrics & Gynecology NOVEMBER 2010
Further logistic regression (Table)nalyses revealed significant differencesetween TFI and IFC in the high com-ared with both negative and low titers,FI and FC in the high compared withegative titers, and IFC and FC in theigh compared with both negative and
ow titers. The number of individualsith high anti–C trachomatis antibody
iters in TFI group is significantly morehan those in either the IFC or FCroups.These results have demonstrated an
ssociation of TFI with anti–C trachoma-is antibodies, which is consistent witharious previous observations.25 The an-i–C pneumoniae antibody titers amonghe 3 groups were not significantly dif-erent (Table 1). The high titers of anti–Cneumoniae antibodies in most of theatients in all 3 groups did not interfereith the measurements of anti-C tracho-atis antibodies because high titers of
nti–C trachomatis antibodies were de-ected only in most TFI patients.
Serum samples from 16 TFI, 7 IFC,nd 13 FC patients with high anti–C. tra-homatis antibodies (�1:1000) were fur-her evaluated in a fusion protein ELISAFigure 1). Antibodies against ClpP wereignificantly higher in the TFI group asompared with the control groups. An-ibodies against the remaining 12 HSPsncluding HSP10 were not significantlyifferent among the groups.We further confirmed the specificity
f the human antibody binding to ClpPusion proteins using an absorption ap-roach (Figure 2). In addition to the pre-bsorption with bacterial lysates con-aining GST alone, the human sera fromhe TFI group were further absorbedith either C trachomatis–infected oreLa-alone lysates prior to reacting with
he fusion proteins in the ELISA assay.Absorption with C trachomatis–in-
ected HeLa lysate but not the HeLa-lone lysate completely removed ClpP-eactive antibodies from all 4 TFIntisera, demonstrating that the recog-ition of ClpP by the TFI antisera waspecific. Binding of TFI sera to ClpP wasonfirmed on Western blot (data nothown).
Protein expression of ClpP and HSP60
FIGURE 1Reactivity of human antibodies with chlamydialfusion proteins arrayed to microplate wells
he bacterial lysates containing individual chlamydial GST fusion proteins or GST alone (listed alonghe X-axis) were directly added to glutathione-coated microplates. Human antisera from 3 groups ofatients (listed along the Y-axis) were first preabsorbed with bacterial lysates containing GST alonend then reacted with the plate-immobilized chlamydial fusion proteins. The human antibody bindingas detected with a goat antihuman IgG conjugated with HRP plus the soluble substrate ABTS (Sigma,t Louis, MO) and measured in OD values at 405 nm. A reaction with an OD value of 2 SD about theean was considered positive as indicated with horizontal bars. The number of positive individuals
rom different groups of patients was compared with Pearson’s �2 test. The number of sera thatositively recognized HSP60 (P � .001) or ClpP (P � .03) was significantly higher in the TFI grouphen compared with either the IFC or the FC groups.
lpP, caseinolytic protease P; FC, fertile controls; GST, glutathione S-transferase; HSP, heat shock proteins; IFC, infertility controls; OD,ptical density; SD, standard deviation; TFI, tubal factor infertility.
odgers. Tubal factor infertility and anti-ClpP antibodies. Am J Obstet Gynecol 2010.
as assessed over time in cell culture fol-
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www.AJOG.org Basic Science: Gynecology Research
owing chlamydial infection (Figure 3).SP60 was detected as early as 12 hours
fter infection, whereas ClpP was ex-ressed 24 hours after infection. Bothroteins were restricted to the intracellu-
ar chlamydia inclusions and persistedhroughout the infection cycle.
OMMENTeat shock proteins are stress response
roteins that increase expression withtress such as temperature changes andypoxia. HSPs are evolutionarily highlyonserved and found in bacteria and hu-ans.26-28 Antibody responses to chla-ydial HSP60 and HSP10 have been
inked to chlamydia-induced patholo-ies.29 However, it was unknownhether the antibody responses to any of
FIGURE 2Absorption of human sera with endthe binding of human antibodies to
he bacterial lysates containing individual chlamicroplates, and the ELISA was carried out as deere preabsorbed with bacterial lysates containin
o reacting with the chlamydial fusion proteins oGST-CT706) and the binding of the 4 sera to boot HeLa alone lysates.lpP, caseinolytic protease P; ELISA, enzyme-linked immunosorb
odgers. Tubal factor infertility and anti-ClpP antibodies. Am
he remaining 12 chlamydial HSPs m
re also associated with chlamydialathogenesis.We demonstrated that TFI patients
isplayed significantly higher levelsf anti–C trachomatis antibodies,hereas there was no significant differ-
nce in the anti–C pneumoniae anti-ody titers between TFI and controlatients, which is consistent with whatas been previously reported.10 Cneumoniae is a ubiquitous human re-piratory pathogen. Although infec-ion with C pneumoniae has been asso-iated with both airway allergiciseases and cardiovascular patholo-ies, C pneumoniae infection has noteen linked to tubal factor infertility.ndeed, we found that there were noignificant differences in anti–C pneu-
enous C trachomatis antigens blocklamydial fusion proteins
l GST fusion proteins or GST alone (as listed alonbed in the legend for Figure 1. The 4 human antiST alone and then further absorbed with either He microplate. Please note that none of the 4 seST-CT431 and GST-CT858 was completely blo
ssay; GST, glutathione S-transferase; TFI, tubal factor infertility.
bstet Gynecol 2010.
oniae antibodies among the 3 groups. c
NOVEMBER 2010 American
This observation has not only con-rmed the lack of association of C pneu-oniae infection with TFI but also
uggested that coinfection with C pneu-oniae did not significantly affect the
etection specificity when measuringnti–C trachomatis antibodies despitehe fact that C pneumoniae and C tracho-
atis share a very similar genome.Using fusion protein ELISA, we both
onfirmed the association of the anti-hlamydial HSP60 antibodies with TFInd found a new link of TFI to humanntibodies against C trachomatis ClpP.lpP is a proteolytic subunit of the ATP-ependent Clp protease complex. Thendopeptidase Clp is also called Ti endo-eptidase or ATP dependent endopepti-ase Ti, which is found in prokaryotes,
e left side of the figure) were allowed to bind tofrom the TFI group as listed on top of the figure
a alone or chlamydia-infected HeLa lysates prioround to the other subunit of the ClpP complex
d by absorption with the chlamydia-infected but
og sch
ydia g thscri serag G eLn th ra bth G cke
ent a
J O
hloroplasts, and mitochondria and
Journal of Obstetrics & Gynecology 494.e11
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Research Basic Science: Gynecology www.AJOG.org
4
lays an important role in the degrada-ion of abnormal proteins. The remain-ng 12 HSPs were not associated with TFIn our patients.
The mechanisms on how the HSPsnd their antibodies contribute to theubal pathologies are still unknown.ome have proposed that the largemounts of bacterial HSPs secreted dur-ng infection can lead to an autoimmuneesponse, resulting in tubal patholo-ies.30,31 Although immune dominant Bell epitopes of chlamydia HSP60 haseen mapped,32,33 potential autoreactivepitopes have been identified30 and thessociation between chlamydial HSP60-nduced circulating autoantibodies andubal pathologies has been estab-ished,34-36 there is still a lack of directemonstration for a role of the HSP60utoreactive epitopes in chlamydialathogenesis.Antibody responses might just indi-
ate the presence of chlamydial antigensn the host, and it is the chlamydial anti-en-induced inflammatory37,38 and cel-ular immune responses39 that may be
ainly responsible for causing the pa-hologies. Chlamydia HSP60 is a power-ul inflammatory stimulus that can acti-ate both macrophages and epithelial
FIGURE 3Expression of CT110 (HSP60) and C(ClpP) during C trachomatis infecti
eLa cells grown on coverslips were infected withimes after infection as listed on the top of themmunofluorescence labeling with mouse antibodolabeled with an anti-MOMP antibody (green) aetected as early as 12 hours (yellow � red overly 24 hours (white arrows).lpP, caseinolytic protease P; HSP, heat shock proteins; MOMP,
odgers. Tubal factor infertility and anti-ClpP antibodies. Am
ells to secrete inflammatory cyto- t
94.e12 American Journal of Obstetrics & Gyneco
ines.38 HSP60 can also induce T cell re-ponses,39 which can be pathogenic, de-ending on the phenotype, time, andxtent of the responses.40-42
ClpP is a proteolytic subunit of theTP-dependent Clp protease complex.he Clp proteases represent a distinctive
amily of energy-dependent serine pro-eases that are highly conserved through-ut bacteria and eukaryotes.43 Chlamyd-
al ClpP share 45% amino acid sequencedentity with its homolog in humanshttp://blast.ncbi.nlm.nih.gov/Blast.cgi).equence alignment analysis led to thedentification of 5 distinct regions eachith more than 5 identical amino ac-
ds between chlamydial and humanlpPs. These 5 stretches of sequencesay serve as potential cross-reactive lin-
ar epitopes. It is possible that some ofhe antichlamydial ClpP human anti-odies may recognize the cross-reactivepitopes and attack human ClpP in theubal tissues.
Although our sample size is limited,he significantly elevated anti-ClpP anti-odies in TFI patients may serve as a po-ential marker for aiding in diagnosis ofhlamydia-induced tubal damage. Diag-ostic laparoscopy with chromotuba-
ion is the gold standard for evaluating
31
trachomatis serovar D organisms, and at variousure, the infected cultures were processed foragainst HSP60 or ClpP (red ). The samples werea DNA dye (blue). Please note that HSP60 wasing with green), whereas ClpP was detected only
r outer membrane protein.
bstet Gynecol 2010.
ubal patency in an infertility evaluation. p
logy NOVEMBER 2010
Hysterosalpingogram (HSG) is less in-asive in evaluating tubal patency, butSG does have inherent risks of perito-itis or endometritis. Furthermore, aetaanalysis suggests that HSG has a
ensitivity of only 65% and specificity of3% in diagnosing tubal occlusion.44
hus, there is an urgent need for devel-ping a more reliable and noninvasivearker for diagnosing tubal infertility.he observation that detection of anti–C
rachomatis antibody titers can be asood as HSG in diagnosing tubal occlu-ion45 suggests that chlamydial protein-pecific antibodies can be explored forredicting TFI.Efforts have been made to use anti-
odies against chlamydial HSPs forcreening for TFI.45-47 In the populationecruited into the current pilot study, thenti-HSP60 antibodies can be used toetect TFI with 56% sensitivity and 95%pecificity (Figure 1). Inclusion of anti-lpP antibody detection would increase
he sensitivity of this screening test to9%. The negative predictive value of us-
ng ClpP plus HSP60 is 79% and the posi-ive predictive value is 92%. Thus, anti-lpP antibody may prove to be a valuablearker for improving both detection sen-
itivity and specificity for the antibody-ased diagnosis of tubal occlusion.The discovery of a unique marker for
etecting TFI using the limited numberf chlamydial fusion proteins has en-ouraged us to expand the scope of ourssay to include the entire genome. Webtained fusion protein clones coveringll open reading frames encoded by Crachomatis genome and plasmid and aren the process of preparing a whole-ge-ome scale proteome ELISA for screen-
ng the TFI patient sera as more patientera are obtained. We hope to use thehole genome scale approach to identify
dditional unique markers for TFI sohat we can further improve the detec-ion specificity and sensitivity of the an-ibody-based diagnostic approach. f
CKNOWLEDGMENTe acknowledge Jani Jensen, MD (Mayolinic, Rochester, MN) for her work in establish-
ng both this project and the collaboration be-ween departments as well as enrolling our initial
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www.AJOG.org Basic Science: Gynecology Research
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