Chlamydial Heat Shock Protein Research Articles

Inhibitory actions of several natural products on proliferation of rat vascular smooth muscle cells induced by Hsp60 from Chlamydia pneumoniae J138.

Atherosclerosis is a vascular disorder involving inflammation, a narrowed vascular lumen in the entire tunica intima, and reduced elasticity of the arterial wall. It has been found that Hsp60 from Chlamydia pneumoniae, an obligate bacterial pathogen associated with atheroma lesions, mimics human Hsp60, thereby causing attacks by immune cells on stressed endothelial cells expressing endogenous Hsp60 on their surface. Furthermore, Hsp60 from C. pneumoniae has been shown to promote the growth of vascular smooth muscle cells (VSMCs). To explore probes that can be used for studying signal transduction elicited by the chlamydial Hsp60, we have tested several natural products for their inhibitory actions on the Hsp60-induced proliferation of rat arterial smooth muscle cells. Sesamol, vanillyl alcohol, and trans-ferulic acid exhibited moderate inhibitory actions on the Hsp60-induced cell proliferation; zerumbone, humulene, and caryophylene effectively inhibited it at low concentrations with IC(50) values of 529, 122, and 110 nM, respectively. The results indicated that the 11-membered alicyclic ring is favorable for interactions with receptors involved in the Hsp60-induced VSMC proliferation.

Association between tubal infertility, antibodies to Chlamydia trachomatis and the Chlamydia 60kDa heat shock protein (HSP) and a mannose binding lectin (MBL) gene polymorphism in women undergoing IVF

Association between tubal infertility, antibodies to Chlamydia trachomatis and the Chlamydia 60kDa heat shock protein (HSP) and a mannose binding lectin (MBL) gene polymorphism in women undergoing IVF

Sexually transmitted chlamydial infections and subfertility

Introduction: Sexually transmitted infections (STI) are on the rise in Europe and the United States. The major cause is changing sexual behavior. The median age of sexual debut continues to decrease, and the time window between age of first sexual intercourse and marriage has lengthened extending the period of experimentation and changing partners. The resurgence of STIs among young people during the last decade may also reflect declining awareness and fear of HIV and AIDS. STIs, most notably infections caused by Chlamydia trachomatis, are the major threat to reproductive health among women. Methods: To review the role of C. trachomatis (CT) in subfertility among women and to review the current strategies of STI prevention. Results: CT is the leading cause of permanent tubal damage. Tubal factor infertility (TFI) is the only preventable type of infertility. However, secondary prevention of CT infections (and other STIs) by so-called opportunistic screening has largely failed. This is frustrating since CT infection fills the prerequisites for disease prevention by screening. Furthermore, results with primary prevention through sexual or reproductive health education have been disappointing. The pathogenesis of CT-associated tubal damage is not yet fully understood, and a number of heavily interconnected factors appear to play a role. Immune response to chlamydial HSP60 is associated with long-term sequelae of chlamydial infections. Because of the complexity of the host immune response to CT, development of CT vaccine is a huge challenge. Preventing or limiting the long-term sequelae of chlamydial infection such as PID, adverse pregnancy outcome, and TFI would be a realistic goal for the first generation CT vaccine. Conclusions: New emphasis on CT control strategies is needed to shorten the period of infectiousness and to reduce risk-taking behavior among young people.

Serum antibody response to the heat shock protein 60 of Chlamydia trachomatis in women with developing cervical cancer

Objective The purpose of this study was to determine whether serum antibody response to the three versions of chlamydial heat shock protein 60 is associated with an increased risk for cervical cancer. Study design Women with cervical carcinoma were identified by linking the data files of three Nordic serum banks with cancer registries. Overall, 178 women with invasive cervical carcinoma were identified. For each case, the earliest prediagnostic serum sample was chosen, and three matched control subjects who were free of cancer at the time of the case diagnosis were selected randomly. Serum antibodies to the chlamydial heat shock protein 60 were measured by enzyme-linked immunosorbent assay and correlated with the risk of the subsequent development of cervical cancer. Results Antibodies to chlamydial heat shock protein 60–1 were associated with cervical squamous cell carcinoma among cases with long lag time (>3.5 years; odds ratio, 2.4; 95% CI, 1.1–5.1). Antibodies to chlamydial heat shock protein 60–2 or chlamydial heat shock protein 60–3 were not associated with cervical cancer risk. Conclusions The finding that chlamydial heat shock protein 60–1 antibodies are associated with an increased cervical cancer risk suggests that persistent Chlamydia trachomatis infection may contribute to cervical neoplasia.

T helper type-1 (Th1)/Th2 profiles of peripheral blood mononuclear cells (PBMC); responses to antigens of Chlamydia trachomatis in subjects with severe trachomatous scarring.

Increased stimulation of Th2 cytokines may contribute to the development of persistent ocular chlamydial infection, resulting in the blinding pathological changes of trachoma. Proliferation and cytokine production profiles of PBMC in response to stimulation with antigens of Chlamydia trachomatis were compared in 30 patients with severe conjunctival scarring due to trachoma and in 30 age-, sex- and location-matched controls. Interferon-gamma (IFN-gamma) and IL-4 were detected at the single-cell level by ELISPOT assay. Transcription of the genes encoding IFN-gamma, IL-4 and IL-10 was detected in mRNA isolated from parallel cultures of PBMC using reverse transcriptase-polymerase chain reaction (RT-PCR). Incubation with the chlamydial heat shock protein (hsp)60 resulted in increased numbers of IL-4-producing cells in PBMC isolated from patients with scarring disease and increased secretion of IFN-gamma from PBMC of control subjects. Incubation with the chlamydial major outer membrane protein (MOMP) increased the number of IFN-gamma-producing cells in the control group only. Messenger RNA encoding IL-4 was only detected in PBMC of patients with scarring disease after in vitro stimulation with chlamydial antigens, but IFN-gamma mRNA and IL-10 mRNA were also more frequently detected in this group. Thirty-eight subjects were HLA-DRB1 and -DQB1 typed. Associations were observed between certain HLA class II alleles and cellular immune responses to chlamydial antigens. No HLA associations were found with clinical status, and overall we found no evidence of strong associations and the type of immune response. These data are consistent with a role for Th2 cells and cytokines in the pathogenesis of trachomatous scarring.

Chlamydia pneumoniae stimulates proliferation of vascular smooth muscle cells through induction of endogenous heat shock protein 60.

Chlamydia pneumoniae infection has been linked with atherosclerosis. However, the mechanism responsible for the atherogenic effects of C pneumoniae remains unclear. Heat shock proteins (HSPs) have been found in atherosclerotic lesions. HSPs of HSP70 and HSP90 families are involved in the regulation of cell cycle progression and cell proliferation. We assessed the hypothesis that HSP60 is induced in vascular cells infected with C pneumoniae and stimulates cell proliferation. Rabbit vascular smooth muscle cells (VSMCs) and human umbilical vein endothelial cells (HUVECs) were infected with C pneumoniae. Western blot analysis demonstrated the induction of endogenous HSP60 expression in C pneumoniae-infected VSMCs. C pneumoniae infection significantly increased the number of VSMCs, and the mitogenic effect correlated with the expression level of endogenous HSP60. In contrast to VSMCs, C pneumoniae infection had no effect on the expression level of HSP60 and did not stimulate cell proliferation in HUVECs. Exogenous addition of recombinant chlamydial HSP60 had no mitogenic effect on VSMCs and HUVECs. However, overexpression of HSP60 within VSMCs by infection with adenovirus encoding human HSP60 resulted in a significant increase in cell numbers compared with uninfected VSMCs. These results suggest that overexpression of endogenous HSP60 may be a central intracellular event responsible for the mitogenic effects induced by C pneumoniae infection. In addition to C pneumoniae, other infectious agents and atherogenic risk factors may also stimulate VSMC proliferation and contribute to the lesion formation through the induction of HSP60.

Prevalence of Chlamydia trachomatis in subfertile couples

Prevalence of Chlamydia trachomatis in subfertile couples

Serologic evidence of past infection with Chlamydia trachomatis, in relation to ovarian cancer.

Pelvic inflammatory disease has been inconsistently linked with ovarian cancer. We measured antibodies to Chlamydia trachomatis, to chlamydial heat shock protein (CHSP) 60, and to CHSP10, in 117 women with ovarian cancer and in 171 age- and ethnicity-matched population-based control subjects from Oahu, Hawaii. IgG antibodies to serovar D of chlamydia elementary bodies (EB) and IgG antibodies to CHSP60-1, CHSP60-2, CHSP60-3, and CHSP10 were detected using an ELISA assay. The probability of having ovarian cancer was 90% greater in women with the highest, compared with the lowest (optical density, >or =0.40 vs. <0.10), levels of chlamydia-EB antibodies (P=.05). There was also a monotonic trend (P=.09) in ovarian cancer risk associated with CHSP60-1 but not with CHSP60-2, CHSP60-3, or CHSP10. These data suggest that past or chronic persistent infection with chlamydia may be a risk factor for ovarian cancer.

Role of Chlamydia pneumoniae-infected macrophages in atherosclerosis developments of the carotid artery.

Chlamydia pneumoniae (C. pneumoniae) infection has been recently accepted as an important cause of atherosclerosis. However, the precise mechanisms remain unclear. The present study was aimed to clarify the distribution link among C. pneumoniae, chlamydial HSP 60, and activated macrophages. Atheromatous carotid plaques were obtained from 40 consecutive carotid endarterectomies (CEA). The specimens were prepared for HE and elastica-van Gieson staining. Parallel sections were stained immunocytochemically with monoclonal antibodies for a C. pneumoniae-specific antigen, chlamydial HSP 60, activated macrophages, and smooth muscle cells. Immunoreactivity for the C. pneumoniae-specific antigen was observed within the endothelial cells, activated macrophages, and smooth muscle cells in 36 of 40 specimens (90%). Chlamydial HSP 60 was found in all specimens positive for the C. pneumoniae-specific antigen, and mainly co-localized with the C. pneumoniae-specific antigen within the activated macrophages. The present results suggest that C. pneumoniae is a key microbial organ that causes atheroma developments in the carotid artery. Chlamydia pneumoniae-infected macrophages may come into the arterial intima and mediate inflammatory and autoimmune processes through the production of chlamydial HSP 60, leading to atherosclerosis.

Antibodies to Chlamydia trachomatis heat-shock protein 60 kDa and detection of Mycoplasma genitalium and ureaplasma urealyticum are associated independently with chronic nongonococcal urethritis.

Chronic nongonococcal urethritis (NGU) is a well-recognized clinical problem in genitourinary medicine clinics, but its etiology and optimal management are poorly understood. The authors showed previously that antibody to chlamydial hsp60 is associated with urethritis 30 to 92 days after treatment of acute NGU (chronic NGU) and that the detection of ureaplasmas or is associated with chronic NGU in which symptoms or signs are present. The aim was to determine whether these associations are independent of each other. This was a longitudinal prospective follow-up study over a 3-month period of 86 men with acute NGU. Men were tested for, ureaplasmas, and antibody to chlamydia hsp60 at presentation and during follow-up. The detection of either ureaplasmas or (OR, 29.45; 95% CI, 1.78-487) and the occurrence of hsp60 antibody at 30 to 92 days' follow-up (OR, 26.45; 95% CI, 1.34-523) were associated independently with the development of chronic NGU 30 to 92 days after treatment of acute NGU. The presence of chlamydial hsp60 antibody at 30 to 92 days was not associated with the development of chronic NGU in which symptoms or signs were present (P= 0.363). Chlamydial hsp60 antibody associated, however, with chronic NGU in which there were no symptoms or signs (P = 0.01). The results suggest that the immune response to chlamydial hsp60 may have a role in the etiology of chronic NGU in asymptomatic men who have no discharge on examination. The clinical relevance of this is unknown.

Endotoxin Contamination in Recombinant Human Heat Shock Protein 70 (Hsp70) Preparation Is Responsible for the Induction of Tumor Necrosis Factor α Release by Murine Macrophages

Using commercially available recombinant human heat shock protein 70 (rhHsp70), recent studies have shown that rhHsp70 could induce the production of tumor necrosis factor alpha (TNFalpha) by macrophages and monocytes in a manner similar to lipopolysaccharide (LPS) e.g. via CD14 and Toll-like receptor 4-mediated signal transduction pathway. In the current study, we demonstrated that a highly purified rhHsp70 preparation (designated as rhHsp70-1) with a LPS content of 1.4 pg/microg was unable to induce TNFalpha release by RAW264.7 murine macrophages at concentrations up to 5 microg/ml. In contrast, a less purified rhHsp70 preparation (designated as rhHsp70-2) at 1 microg/ml with a LPS content of 0.2 ng/microg was able to induce TNFalpha release to the same extent as that induced by 0.2 ng/ml LPS. Failure of rhHsp70-1 to induce TNFalpha release was not because of defective physical properties since rhHsp70-1 and rhHsp70-2 contained identical hsp70 content as determined by SDS gels stained with Coomassie Blue and Western blots probed with an anti-rhHsp70 antibody. Both rhHsp70 preparations also had similar enzymatic activities as judged by their ability to remove clathrin from clathrin-coated vesicles. Removal of LPS from rhHsp70-2 by polymyxin B-agarose column or direct addition of polymyxin B to the incubation medium essentially eliminated the TNFalpha-inducing activity of rhHsp70-2. The addition of LPS at the concentration found in rhHsp70-2 to rhHsp70-1 resulted in the same TNFalpha-inducing activity as observed with rhHsp70-2. The TNFalpha-inducing activities of rhHsp-2, LPS alone, and LPS plus rhHsp70-1 were all equally sensitive to heat inactivation. These results suggest that rhHsp-70 does not induce TNFalpha release from murine macrophages and that the observed TNFalpha-inducing activity in the rhHsp70-2 preparation is entirely due to the contaminating LPS.

Chlamydia infection and subfertility

In the majority of women chlamydia infections remain asymptomatic but they may increase the risk for tubal factor subfertility. Pelvic inflammatory disease (PID) and its chronic sequelae are associated with chlamydial IgG antibody formation in serum, and a correlation between the height of antibody titres and the presence of tubal factor subfertility has been established. The predictive value of chlamydia antibody testing (CAT) is limited however. Several factors affecting sensitivity and specificity of CAT have been identified. Because it is assumed that the presence of chlamydial heat shock proteins (HSPs) may be indicative of chronic inflammation, chlamydial HSP60 antibody testing has been evaluated in its prediction of tubal factor subfertility.

The role of serology, antibiotic susceptibility testing and serovar determination in genital chlamydial infections

Systemic and local antibodies regularly develop in genital infections caused by Chlamydia trachomatis. Such antibodies cannot be used as a sign of current infection as they often persist for years after the infection has resolved. Chlamydial antibodies have, however, been extremely useful for demonstrating associations between C. trachomatis and clinical conditions such as ectopic pregnancy and tubal factor infertility. In particular, antibodies to the chlamydial heat shock protein 60 predict the presence of tubal scarring. C. trachomatis has been divided into 15 (or 18) serovars, with many genotypes within each serovar. Differences in pathogenicity between serovars have been reported but no general pattern has emerged. Genotyping is a powerful epidemiological tool but is not yet ready for routine clinical use. C. trachomatis infections can be successfully treated by tetracycline or macrolides. Some resistant strains have been reported, causing treatment failures, and the problem of emerging antibiotic resistance cannot be neglected.

Adjuvant modulation of the immune responses and the outcome of infection with Chlamydia pneumoniae.

Immunization with different adjuvants resulted in antithetic outcomes of infection with Chlamydia pneumoniae. Immunization with the outer major protein-2 from C. pneumoniae (OMP-2) emulsified in Freund's complete adjuvant (FCA) thus increased the susceptibility of mice to infection with the bacteria. The detrimental effect was not observed upon inoculation of irrelevant antigens or major outer membrane protein (MOMP) in FCA, but was also observed after immunization with FCA-chlamydial heat shock protein-60 (HSP-60). The harmful effect of FCA-OMP-2 depended on the presence of both CD4+ and CD8+ cells and was mediated by IL-10, as shown using gene-ablated mice. The increased susceptibility to infection caused by FCA-OMP-2 immunization was long-lasting and observed in mice infected 4 months after the last dose of immunogen. In contrast, partial protection against C. pneumoniae was observed when FCA was replaced with oligodeoxynucleotides containing immunostimulatory CpG motifs mixed with Freund's incomplete adjuvant (FIA-IS-CpG). These polar outcomes of infection related to the cytokine pattern: antigen-stimulated spleen cells from FCA-OMP-2-immunized mice showed higher IL-10/IFN-gamma ratios than FIA-IS-CpG-OMP-2-immunized animals. In agreement, sera from FCA-OMP-2 showed higher anti-OMP-2 IgG1/IgG2a ratios than FIA-IS-CpG-OMP-2-immunized animals. Finally, OMP-2 also generated a protective response when delivered by a eukaryotic expression vector in tandem with CTLA4, a procedure that targeted OMP-2 to antigen-presenting cells.

Chlamydial heat-shock protein-60 antibody and correlation with Chlamydia pneumoniae in atherosclerotic plaques.

A study was performed to determine whether serum antibody to Chlamydial heat-shock protein-60 (CHSP-60) and C-reactive protein (CRP) were associated with the presence of Chlamydia pneumoniae in atheromatous plaques in 75 patients. The mean (+/-SD) ELISA optical density (OD) of anti-CHSP-60 was 0.19+/-0.15 in 54 patients with detectable C. pneumoniae antigen, versus an OD of 0.11+/-0.08 in 21 patients without detectable C. pneumoniae I antigen (P=.008). Higher anti-CHSP-60 at an OD > or =0.12 was present in 38 (70.4%) of patients with detectable C. pneumoniae in atheromas, compared with 5 (23.8%) of patients without C. pneumoniae antigen (P<.001; 2-tailed test). The mean CRP concentration was 7.4+/-10.3 mg/L in patients with detectable C. pneumoniae antigen, versus 5.7+/-6.1 mg/L in those without (P=.556). Immune response to CHSP-60 may play a role in atherogenesis, but CRP serum levels does not appear to be related to C. pneumoniae infection.

Serum immunoglobulin G antibodies to chlamydial heat shock protein 60 but not to human and bacterial homologs are associated with coronary artery disease.

Evidence for an association between Chlamydia pneumoniae infection and coronary artery disease (CAD) has been reported by numerous studies, cross-reactive heat shock protein (Hsp) antibody responses have been causally linked to CAD, and the severity of chlamydial disease pathogenesis correlates with Hsp serology. Our aim was to determine if chlamydial Hsp (cHsp) antibody responses are predictive of CAD. Patients were recruited in a case-control study: 250 cases had angiographically significant CAD (stenosis > or =70%), and 250 controls had normal coronary arteries (stenosis <10%). Serum immunoglobulin G reactivity to Hsp10 and Hsp60 antigens (chlamydial, Escherichia coli, and human), and C pneumoniae whole organisms were measured by ELISA. Univariate analysis confirmed that classical CAD risk factors were predictors of CAD. Univariate analysis showed that cHsp60 (P= 0.001, OR 3.9), cHsp10 (P=0.045, OR 3.8), E coli Hsp60 (P=0.04, OR 1.5) and C pneumoniae (P=0.03, OR 1.8) ELISA optical density (OD) values were significantly different between cases and controls. Multivariate analysis found that only upper-quintile cHsp60 seroreactivity remained a significant predictor of CAD after controlling for classical CAD risk factors and seroreactivity to the other antigens (cHsp60 OD, P=0.005, OR 3.9 per OD unit; cHsp60 quintile, 5 versus 1 to 4; P=0.01, OR 2.1). The presence of elevated anti-cHsp60 immunoglobulin G antibodies, but not anti-human or anti-E coli homologs, was independently associated with CAD. This finding argues against previous suggestions that cross-reactive or autoimmune Hsp60 responses may contribute to disease progression. High anti-cHsp60 antibody response appears to identify the subset of patients with chlamydial infection and significant CAD.

Chlamydial heat shock protein 60 and lipopolysaccharide: potential virulence determinants in atherogenesis.

Chlamydia pneumoniae infection is associated with atherosclerosis and may be an emerging risk factor in coronary artery disease. C. pneumoniae can infect, multiply within and modulate the function of all atheroma cell types. Specific chlamydial virulence determinants have been identified that permit interaction with host cells and dysregulate cell function. In particular, chlamydial heat shock protein 60 and lipopolysaccharide may modulate cell function to dysregulate lipid metabolism, induce inflammatory cytokine cascades and trigger production of cross-reactive antibodies that initiate and promote atherogenesis. This paper reviews chlamydial heat shock protein 60 and lipopolysaccharide as potential virulence determinants in atherogenesis.

Role of chlamydial heat shock protein 60 in the stimulation of innate immune cells by Chlamydia pneumoniae.

Chlamydia pneumoniae stimulates potently maturation of and cytokine secretion by bone marrow-derived dendritic cells (BMDDC). BMDDC responses depend mainly on Toll-like receptor (TLR)2 and to a minor extent on TLR4. We demonstrate here using C. pneumoniae in an infectious model with the replication-permissive epithelial cell line HEp2 that HSP60 is produced in substantial amounts in chlamydial inclusions during infection. Electron microscopy of chlamydial inclusions revealed that HSP60 was mainly associated with reticulate bodies, but was also located in between the different chlamydial developmental forms. Supernatants of permissive HEp2 cells infected with C. pneumoniae contained soluble chlamydial HSP60 as demonstrated by Western blotting and were able to stimulate BMDDC of wild-type mice. The stimulatory capacity of culture supernatants correlated with the presence of chlamydial HSP60. In contrast, BMDDC from TLR4-mutant mice crossed to TLR2-deficient mice were not stimulated by the culture supernatant, indicating that chlamydial HSP60 but not cytokines, possibly secreted by infected HEp2 cells, are responsible for the observed stimulation of BMDDC. Purified recombinant HSP60 from C. pneumoniae stimulated BMDDC in a TLR2- and TLR4-dependent fashion similar to the whole microorganism. In summary, these data suggest chlamydial HSP60 as an important mediator of inflammatory responses during infection with C. pneumoniae.

HLA DQ alleles and interleukin-10 polymorphism associated with Chlamydia trachomatis-related tubal factor infertility: a case-control study

The relationship between Chlamydia trachomatis tubal factor infertility (TFI) and the host's immunoregulatory genes was studied. Cell-mediated immune responses to C. trachomatis and chlamydial heat shock protein (CHSP60) were determined by lymphocyte proliferation assay. HLA-DQ alleles and interleukin-10 (IL-10) promoter polymorphism (-1082 A/G) were analysed in 52 TFI cases and in 61 controls by PCR. HLA-DQB1 or DQA1 alleles did not significantly differ between the TFI group and the control group. However, DQA1*0102 and DQB1*0602 alleles together with IL-10 -1082AA genotype were found significantly more frequently in the TFI patients than in the controls (0.18 and 0.02 respectively; P = 0.005). Five (22%) of the 23 patients who had a positive lymphocyte proliferative response to CHSP60 were positive also for IL-10 -1082AA and for the HLA-DQA1*0102 and HLA-DQB1*0602 alleles. Our results reveal an association of a cellular immune response to CHSP60, HLA class II alleles and IL-10 promoter genotypes in patients with chlamydial TFI.

Chlamydia trachomatis in subfertile women undergoing uterine instrumentation: an alternative to direct microbial testing or prophylactic antibiotic treatment.

Chlamydia trachomatis is the major cause of tubal occlusion, and is also associated with IVF failure and spontaneous abortion. These infections are asymptomatic in most individuals and can persist in the genital tract for long periods of time in a form resistant to immune destruction. A significant percentage of couples seeking treatment for infertility might, therefore, harbour C. trachomatis in their genital tract. An unresolved question is what to do about this possible chlamydial persistence. Cervical, endometrial and semen samples can be tested for C. trachomatis and only positive individuals treated. Alternatively, all couples undergoing infertility treatment can receive prophylactic antibiotics. We advocate a third option, to screen and treat only individuals who are positive for systemic and/or local anti-chlamydial antibody production. Detection of species-specific C. trachomatis antibodies in peripheral blood will determine which individuals have been exposed to this organism and who, therefore, may be at risk for harbouring persistent forms. Identification of IgA antibodies in genital tract secretions may be an even better indicator of the presence of C. trachomatis in the genital tract. Circulating antibodies to the chlamydial 60kDa heat shock protein (hsp60) is a specific indicator of tubal occlusion and, furthermore, correlates with the continued presence of this micro-organism in the genital tract of non-human primates. Screening for both cervical IgA antibodies to C. trachomatis and serum IgG anti-chlamydial hsp60 appears to provide the best indication as to which women may be harbouring C. trachomatis.