Humoral immune response to the chlamydial heat shock proteins hsp60 and hsp70 in Chlamydia -associated chronic salpingitis with tubal occlusion

The aim of this study was to evaluate the prevalence of serumimmunoglobulin (Ig) G and IgA antibodies to recom-binant chlamydial60 kDa heat shock protein (C-hsp60) and to assess the prevalenceof serum IgG antibodies to recombinant chlamydial 70 kDa heatshock protein (C-hsp70) in Chiamydia-associated chronic salpingitisand/or salpingitis isthmica nodosa with tubal occlusion. Infertilepatients (n = 34) with Chlamydia-associated, histologicallydocumented chronic salpingitis and/or salpingitis isthmica nodosaand bilateral tubal occlusions (group I) were compared withinfertile patients (n = 19) without tubal occlusions (groupII). The prevalence of chlamydial antigen in endocervical, urethraland urine samples was low in both groups. The median chlamydialserum IgG and IgA antibody titres were significantly higherin group I than in group II (P < 0.0001 and P = 0.0002 respectively).Serum IgG antibodies to C-hsp60 and C-hsp70 were detected in24 out of 34 patients (71%) in group I compared with 10 outof 19 (53%) and nine out of 19 (47%) patients in group II (notsignificantly different). There was a significant difference(P = 0.035) between the prevalences of serum IgA antibodiesto C-hsp60 in groups I (seven out of 34 patients; 21%) and II(none of the 19 patients). The association between the presenceof serum IgA antibodies to C-hsp60 and Chlamydia-ossocmted chronicsalpingitis and/or salpingitis isthmica nodosa with tubal occlusionunderlines the significance of chlamydial 60 kDa heat shockprotein in the pathogenesis of tubal infertility.     

Antibodies to Chlamydia trachomatis heat shock proteins Hsp60 and Hsp10 and subfertility in general population at age 31

PROBLEM: To assess the association between antibodies to Chlamydia trachomatis heat shock proteins 60 and 10 (Hsp60 and Hsp10) and subfertility in a general population sample. METHOD OF STUDY: A nested case (n = 146)-control (n = 278) study in a population-based birth cohort. Serum immunoglobulin (Ig)G and IgA antibodies against C. trachomatis Hsp60 and Hsp10, explanatory factors, were measured by enzyme immunoassay, using recombinant proteins as antigens. The main outcome variable was subfertility (time to pregnancy > or =12 months). RESULTS: The prevalence and medians of serum IgA antibodies to Hsp60 and Hsp10 were significantly higher in the female partners of subfertile couple than in their fertile controls. On the contrary, among male partners of subfertile couple, especially among smokers serum antibody levels to Hsp antigens were lower than in the controls. CONCLUSION: The results indicate a serological association of antibodies to chlamydial Hsp antigens with female subfertility in a population-based sample.     

Chlamydia trachomatis heat shock protein-60 induced interferon-gamma and interleukin-10 production in infertile women

Chlamydia trachomatis-associated tubal factor infertility (TFI) involves enhanced humoral and cell-mediated immune response to the chlamydial 60 kDa heat shock protein (CHSP60). We evaluated the role of CHSP60-induced immune response in TFI by studying lymphocyte proliferation and cytokine (interferon (IFN)-gamma, interleukin (IL)-12 and IL-10) secretion in response to C. trachomatis elementary body (EB) and CHSP60 antigens in 57 women with TFI and in 76 women with other causes of infertility. Positive proliferative response of PBMC to CHSP60 was more common in the TFI group (20/57; 36%) than in the other groups (17/76; 22%) although the frequency or the median responses did not differ significantly (1.6, range 0.2-22.1 versus 1.4; 0.2-24.4). C. trachomatis EB induced significantly higher IFN-gamma and lower IL-10 secretion in the TFI group compared to the other groups. The EB and CHSP60 induced IL-12 secretion was similar in all study groups and correlated with IFN-gamma secretion in the other but not in the TFI group. The lack of correlation between EB-induced IL-12 and IFN-gamma production and simultaneously found prominent IL-10 secretion in response to CHSP60 in the TFI group suggests that the CHSP60 may have a specific role in regulating the immune reactions during chlamydial infection and may consequently contribute to the immunopathogenesis of TFI.     

Chlamydia trachomatis and chlamydial heat shock protein 60-specific antibody and cell-mediated responses predict tubal factor infertility

BACKGROUND: To evaluate the role of Chlamydia trachomatis-induced humoral and cell-mediated immune (CMI) responses in predicting tubal factor infertility (TFI). METHODS: Blood samples were taken from 88 women with TFI and 163 control women. C. trachomatis and chlamydial heat shock protein 60 (CHSP60)-specific immunoglobulin G (IgG) antibodies were analysed using enzyme-linked immunosorbent assay (ELISA) kits. Proliferative reactivity of peripheral blood mononuclear cells was studied in vitro against Chlamydia elementary body (EB) and recombinant CHSP60 antigens. RESULTS: C. trachomatis-specific IgG antibodies were found more frequently (43.2 versus 13.5%), and the antibody levels were higher in the TFI cases than in the controls (P < 0.001). C. trachomatis EB-induced lymphocyte responses were positive in 81.8% of the TFI cases and 58.9% of the controls (P < 0.001). Similarly, CHSP60-induced lymphocyte responses were found in 45.5% of the TFI cases and 30.7% of the controls (P < 0.001). CHSP60 antibody test was the best single test predicting TFI. Compared to cases with all four markers negative, the estimated risk for TFI was 4.1 (95% CI 1.4-11.9) among those with one positive marker and 19.9 (95% CI 6.9-57.4) among those with three to four positive markers. CONCLUSION: Our results show that TFI prediction model can be improved by combining tests for humoral and CMI response to chlamydial antigens.     

Previously undetected Chlamydia trachomatis infection, immunity to heat shock proteins and tubal occlusion in women undergoing in-vitro fertilization.

The relationship between a previously undetected Chlamydia trachomatis infection, tubal infertility, immunity to heat shock proteins and subsequent in-vitro fertilization (IVF) outcome was evaluated. Women with tubal occlusion, with or without hydrosalpinges, and no history of C. trachomatis infection were tested for circulating antibodies to the human 60-kDa heat shock protein (Hhsp60), the C. trachomatis 10-kDa heat shock protein (Chsp10) and C. trachomatis surface antigens prior to their initial IVF cycle. Sera were obtained from 50 women whose male partners were infertile, 58 women with tubal occlusion but no hydrosalpinx and 39 women with tubal occlusions plus hydrosalpinx. Clinical pregnancies were documented in 68% of the women with male factor infertility. This was higher than the 43.1% rate in women with tubal occlusions (P = 0.04) and the 41% rate in women with hydrosalpinx (P = 0.02). C. trachomatis antibodies were present in one (2%) women with male factor infertility as opposed to 15 (25.9%) women with tubal occlusion (P = 0.003) and 13 (33%) with hydrosalpinx (P < 0.0001). Antibodies to Chsp10 were more prevalent in women with hydrosalpinx (46.8%) than in women with male factor infertility (P < 0.0001, 6%) or tubal occlusion (P = 0.0009, 15.5%). Hhsp60 antibodies were equally more prevalent in women with tubal occlusion plus (46.8%) or minus hydrosalpinx (41.4%) than in women with male factor infertility (P < 0.0002). Hhsp60 was more prevalent in those women positive for Chsp10 (P = 0.02) or C. trachomatis (P = 0.04) antibodies than in women lacking these antibodies. There was no relationship between any of the antibodies measured in sera and IVF outcome.     

热休克蛋白与抗感染免疫

热休克蛋白(heatshockproteins,HSPs)又称为应激蛋白,由多基因家族及相关基因产物诱导产生,参与高度保守的细胞防御机制.作为细胞内分子,HSPs参与蛋白成熟必要的生化途径,对于正常细胞自稳状态的维持起重要作用.除上述分子伴侣功能外,HSPs还具有另一种重要的生物特性--免疫原性.在感染引起的免疫性疾病的发生和发展中,HSPs具有防止感染应激引起的细胞损害和促进受损细胞恢复等重要作用.作为感染过程中体液和细胞免疫的重要靶目标,HSPs用于感染性疾病的疫苗研究日益受到人们的关注.     

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.

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.     

Circulating antibodies to a conserved epitope of the Chlamydia trachomatis 60-kDa heat shock protein is associated with decreased spontaneous fertility rate in ectopic pregnant women treated by salpingectomy

PROBLEM: This prospective study was aimed to evaluate whether non-invasive clinical and serologic parameters of tubal disease are predictive for subsequent spontaneous conception and pregnancy outcome after first episode of ectopic pregnancy (EP). METHOD OF STUDY: Overall, 144 women aged <35 years were enrolled. Outcome of subsequent spontaneous conception was analyzed after 3 years and compared with clinical parameters and antibody responses to Chlamydia trachomatis and epitopes of the 60-kDa chlamydial heat shock protein (CHSP-60). RESULTS: Antibody response to a conserved epitope of CHSP-60 (amino acids, aa 260-277) was independently correlated with both decreased spontaneous conception and term delivery rates (adjusted odds ratios, OR: 3.6 and 5.4, respectively). CONCLUSION: Presence of circulating antibodies to a conserved epitope of the CHSP-60 is associated with a lower spontaneous conception rate, and increased likelihood of adverse pregnancy outcome in women treated by salpingectomy for first episode of EP.     

沙眼衣原体主要外膜蛋白核酸疫苗诱导小鼠产生特异性细胞和体液免疫应答

目的构建沙眼衣原体主要外膜蛋白核酸疫苗,并观察其诱导小鼠产生的体液免疫和细胞免疫。方法将核酸疫苗(pcDNA3.1MOMP)或对照空质粒(pcDNA3.1)注射于4～6周龄小鼠后腿股四头肌,每次剂量为100mg。间隔2周加强免疫2次。末次免疫后,ELISA法测定脾淋巴细胞培养上清液中IFNγ及小鼠血清中抗MOMP水平;MTT法测定脾淋巴细胞特异性增殖反应。结果小鼠接种核酸疫苗后,能产生特异性抗体,第3次免疫后抗体最高滴度达1∶1024,培养上清液中IFNγ达(532.0±45.4)pg/mL;实验组小鼠脾淋巴细胞刺激指数为3.94±0.25,其抗原特异性反应明显高于对照组。结论沙眼衣原体主要外膜蛋白核酸疫苗能刺激机体产生特异的细胞免疫和体液免疫。     

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.     

E型沙眼衣原体主外膜蛋白DNA疫苗和重组蛋白疫苗联合免疫诱导出的免疫效应

目的 研究沙眼衣原体主要外膜蛋白(MOMP)DNA疫苗和重组蛋白(rMOMP)疫苗联合免疫小鼠诱导出的免疫效应.方法 3～4周BALB/c雌鼠60只,分5组,每组12只,于第0、2、4周通过双侧股四头肌肌注免疫相应的疫苗.通过血清IgG抗体和IFN-γ含量、阴道冲洗液sIgA抗体含量、脾淋巴细胞增殖指数、迟发型超敏反应、阴道脱落细胞种植等指标进行小鼠免疫效应的测定.结果 DNA蛋白联合组小鼠血清IgG抗体水平A405值为0.629±0.052;sIgA,A450值为0.379±0.052;脾淋巴细胞增殖指数为5.682±0.484;淋巴细胞培养上清液中IFN-γ含量为(1265±128)ps/ml.DNA蛋白联合组小鼠的各项指标均好于EB阳性对照组除外的其他组(P<0.01).结论 沙眼衣原体DNA疫苗和蛋白疫苗联合免疫可以增强免疫保护效应.     

Serological markers of persistent C. trachomatis infections in women with tubal factor subfertility

BACKGROUND: Persistent C. trachomatis infections are assumed to increase the risk of tubal pathology. We studied whether serological markers, assumed to be associated with persistent C. trachomatis infections, could identify subfertile women at risk of tubal pathology. METHODS: Sera of 313 subfertile women, who all underwent a laparoscopy with tubal testing to assess the grade of tubal pathology, were tested for the presence of immunoglobulin (Ig) G and IgA antibodies to C. trachomatis, IgG antibodies to chlamydia heat shock protein 60 (cHSP60) and C-reactive protein (CRP). RESULTS: C. trachomatis IgA, cHSP60 IgG and CRP, all serological markers of persistent infections, were significantly more prevalent in women with tubal pathology as compared to those without tubal pathology. The predictive value of the currently used screening test for tubal pathology (IgG to C. trachomatis) could be significantly improved by adding the CRP test. CONCLUSIONS: In subfertile women with tubal pathology, serological markers of persistent C. trachomatis infections are significantly more common as compared to women without tubal pathology. C. trachomatis IgG-positive subfertile women with slightly elevated (< 10 mg/l) CRP levels are at highest risk of persistent C. trachomatis infections and tubal pathology.     

沙眼衣原体感染后热休克蛋白与稽留流产相关性研究

目的探讨沙眼衣原体(CT)感染后热休克蛋白-60(HSP-60)与稽留流产的关系。方法采用荧光定量聚链酶反应(PCR)方法,对72例稽留流产患者和正常人工流产患者进行沙眼衣原体检测,将正常人工流产CT-DNA阴性者随机选择72例作为对照组。再采用免疫组化方法检测蜕膜组织内HSP-60表达,采用HPLAS-1000高清晰度彩色病理图文报告管理系统检测单位面积中各项免疫组化阳性颗粒平均光密度值。结果HSP-60阳性细胞光密度值,A组:116.36±22.63,B组:139.67±32.26,对照组:221.12±29.87;A组HSP-60表达强于对照组,对比有明显差异(P<0.001)A组与B组对比无明显差异(P>0.005)。结论沙眼衣原体感染后,HSP-60蜕膜组织内表达强度与稽留流产呈正相关性;可认为是稽留流产发生的重要原因之一。     

Antibody to the Chlamydia trachomatis 60 kDa heat shock protein in follicular fluid and in vitro fertilization outcome

PROBLEM: The association between 60 kDa Chlamydia trachomatis heat shock protein (CHSP60) antibodies and the etiology and outcomes of in vitro fertilization (IVF) outcomes is not well known. METHOD OF STUDY: A retrospective study with a double-blind analysis of follicular fluid from 253 IVF patients for IgG antibodies to CHSP60. RESULTS: The CHSP60 antibodies were detected in 74.1% of women without embryo implantation and in 47.9% of women with 1-3 implantations per IVF cycle (P = 0.0004). CHSP60 antibodies were detected in 69.5% of women with tubal occlusion and 49.7% of women with other causes of infertility (P = 0.01). CHSP60 antibody detection was unrelated to maternal age, number of oocytes collected, or percentage of oocytes fertilized. CONCLUSION: Detection of IgG antibody to CHSP60 may indicate persistence of C. trachomatis in the upper genital tract with low implantation rates resulting from a chronic inflammatory reaction. Alternatively, as human hsp60 is expressed in early stage embryogenesis, a cross-reacting antibody may induce destruction of the embryo.     

The role of heat shock proteins in reproduction

Heat shock proteins (HSP) were first identified in cells after exposure to elevated temperature. Subsequently HSP have been identified as a critical component of a very complex and highly conserved cellular defence mechanism to preserve cell survival under adverse environmental conditions. HSP are preferentially expressed in response to an array of insults, including hyperthermia, free oxygen radicals, heavy metals, ethanol, amino acid analogues, inflammation and infection. HSP interact with intracellular polypeptides and prevent their denaturation or incorrect assembly. In addition HSP are also involved in several processes essential for cellular function under physiological conditions. HSP production is enhanced during in-vitro embryo culture and they are among the first proteins produced during mammalian embryo growth. The spontaneous expression of HSP as an essential part of embryo development is well documented and the presence or absence of HSP influences various aspects of reproduction in many species. Finally, HSP are immunodominant antigens of numerous microbial pathogens, e.g. Chlamydia trachomatis, which have been recognized as the main cause of tubal infertility. Many couples with fertility problems have had a previous genital tract infection, have become sensitized to microbial HSP, and a prolonged and asymptomatic infection may trigger immunity to microbial HSP epitopes that are also expressed in man. Antibodies to both bacterial and human HSP are present at high titres in sera and hydrosalpinx fluid of many patients undergoing in-vitro fertilization (IVF). In a mouse in-vitro embryo culture model, these antibodies impaired the mouse embryo development at unique developmental stages. Recent studies indicate an association between a previous infection, immunity to HSP and reproductive failure.     

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.     

The immune responses to human and microbial heat shock proteins in periodontal disease with and without coronary heart disease

The human 60 kDa and microbial 65 kDa heat shock proteins (HSP) have been implicated in the pathogenesis of chronic periodontitis (P) and coronary heart disease (CHD). We have studied four male non-smoking cohorts of 81 subjects, matched for age. Group (a) consisted of a healthy group with minimal gingivitis (n = 18), group (b) were patients with P (n = 23), group (c) patients with CHD and minimal gingivitis (n = 20) and group (d) patients with CHD and P (n = 20). T cells separated from peripheral blood were found to be primed to both microbial HSP65 and human HSP60 but significant CD4, human leucocyte antigen (HLA) class II-restricted proliferative responses were found only with the human HSP60 in patients with P (P < 0.001) and CHD without (P < 0.001) or with (P < 0.00001) periodontitis. Dose-dependent inhibition of T cell proliferative responses was carried out to determine the receptors involved in recognition of HSP60 and HSP65. Monoclonal antibodies to CD14 showed inhibition of T cell proliferation stimulated by both HSP60 and HSP65, consistent with the role of CD14 as a receptor for these HSPs in P and CHD. The toll-like receptor 2 (TLR-) and TLR-4 were then studied and these showed that TLR-4 was recognized by microbial HSP65, whereas TLR-2 was recognised by human HSP60 in both P and CHD. However, a dissociation was found in the HSP60 and TLR4 interaction, as TLR4 appeared to have been recognized by HSP60 in P but not in CHD. The results suggest an autoimmune or cross-reactive CD4(+) class II-restricted T cell response to the human HSP60 in P and CHD. Further studies are required to determine if there is a common epitope within HSP60 that stimulates T cell proliferation in P and CHD.     

热休克蛋白在子宫内膜异位症中的表达及意义

目的 探讨热休克蛋白27（HSP27）和HSP70在子宫内膜异位症发病中的作用。方法 采用免疫组化链霉菌抗生素蛋白－过氧化物酶染色法（SP法），检测子宫内膜异位症中56例异位内膜与在位内膜（30例）中HSP27和HSP70的表达。采用原位杂交，检测HSP70mRNA的水平，并与正常子宫内膜相比较。结果 免疫组化结果显示，异位内膜组织中HSP27与HSP70均呈高表达，与正常内膜组的表达差异显著（P＜0．01），而且失去其在正常内膜组织中表达的周期性变化。卵巢子宫内膜异位症组（OEM）与子宫腺肌症组（AM）组中，HSP27和HSP70的表达无显著差异（P＞0．05）。原位杂交结果显示，HSP70 mRNA（分别为P＜0．01和P＜0．05）。HSP27与HSP70的表达水平，同OEM的严重性无关。结论 异位内膜组织中的HSP70基因被激活，转录增加。HSP27和HSP70呈高表达，可能在EM的发病中起重要作用。