Development of an immunochromatographic test for the detection of Mycoplasma pneumoniae GroES antigen.

Mycoplasma pneumoniae frequently causes community-acquired pneumonia in children; ß-lactam antibiotics are ineffective against this bacterium because of its lack of a cell wall. Hence, a rapid and simple detection method is required to ensure appropriate treatment. In this study, we developed a rapid and simple immunochromatography-based detection method using monoclonal antibodies that react with the co-chaperone GroES of M. pneumoniae. Mice were immunized with recombinant GroES, and hybridoma cells producing anti-GroES monoclonal antibodies were established. For the development of the immunochromatographic test, antibody pairs with superior reactivity and specificity were selected. The developed immunochromatographic test could detect 0.1 ng/mL of recombinant GroES within 20 min. Moreover, no cross-reaction was observed with other microorganisms, including six Mycoplasma species, 20 other bacterial species, and one yeast species. Macrolide-resistant and -susceptible M. pneumoniae clinical isolates were detected at approximately 104 to 105 colony-forming units/mL. The study indicates that immunochromatographic tests targeting GroES are useful for rapid and simple detection of M. pneumoniae.

Endogenous protein and enzyme fragments induce immunoglobulin E-independent activation of mast cells via a G protein-coupled receptor, MRGPRX2.

Mast cells play a central role in inflammatory and allergic reactions by releasing inflammatory mediators through 2 main pathways, immunoglobulin E-dependent and E-independent activation. In the latter pathway, mast cells are activated by a diverse range of basic molecules (collectively known as basic secretagogues) through Mas-related G protein-coupled receptors (MRGPRs). In addition to the known basic secretagogues, here, we discovered several endogenous protein and enzyme fragments (such as chaperonin-10 fragment) that act as bioactive peptides and induce immunoglobulin E-independent mast cell activation via MRGPRX2 (previously known as MrgX2), leading to the degranulation of mast cells. We discuss the possibility that MRGPRX2 responds various as-yet-unidentified endogenous ligands that have specific characteristics, and propose that MRGPRX2 plays an important role in regulating inflammatory responses to endogenous harmful stimuli, such as protein breakdown products released from damaged or dying cells.

The C-terminal disulfide bonds of Helicobacter pylori GroES are critical for IL-8 secretion via the TLR4-dependent pathway in gastric epithelial cells.

Helicobacter pylori GroES (HpGroES), a potent immunogen, is a secreted virulence factor that stimulates production of proinflammatory cytokines and may contribute to gastric carcinogenesis. HpGroES is larger than other bacterial orthologs because of an additional C-terminal region, known as domain B. We found that the HpGroES-induced IL-8 release by human gastric epithelial cells was dependent on activation of the MAPK and NF-κB pathways. HpGroES lacking domain B was unable to induce IL-8 release. Additionally, a TLR4 inhibitor significantly inhibited IL-8 secretion and reduced HpGroES-induced activation of MAPKs. Furthermore, HpGroES-induced IL-8 release by primary gastric epithelial cells from TLR4(-/-) mice was significantly lower than from wild-type mice. We also found that HpGroES bound to TLR4 in cell lysates and colocalized with TLR4 on the cell membrane only when domain B was present. We then constructed two deletion mutants lacking C-terminal regions and mutants with point mutations of two of the four cysteine residues, C111 and C112, in domain B and found that the deletion mutants and a double mutant lacking the C94-C111 and C95-C112 disulfide bonds were unable to interact with TLR4 or induce IL-8 release. We conclude that HpGroES, in which a unique conformational structure, domain B, is generated by these two disulfide bonds, induces IL-8 secretion via a TLR4-dependent mechanism.

Circulating heat shock protein 60 levels are elevated in HIV patients and are reduced by anti-retroviral therapy.

Circulating heat shock protein 60 (Hsp60) and heat shock protein 10 (Hsp10) have been associated with pro- and anti-inflammatory activity, respectively. To determine whether these heat shock proteins might be associated with the immune activation seen in HIV-infected patients, the plasma levels of Hsp60 and Hsp10 were determined in a cohort of 20 HIV-infected patients before and after effective combination anti-retroviral therapy (cART). We show for the first time that circulating Hsp60 levels are elevated in HIV-infected patients, with levels significantly reduced after cART, but still higher than those in HIV-negative individuals. Hsp60 levels correlated significantly with viral load, CD4 counts, and circulating soluble CD14 and lipopolysaccharide levels. No differences or correlations were seen for Hsp10 levels. Elevated circulating Hsp60 may contribute to the immune dysfunction and non-AIDS clinical events seen in HIV-infected patients.

Molecular and functional characterisation of the heat shock protein 10 of Strongyloides ratti.

Strongyloides stercoralis and S. ratti are intestinal parasitic nematodes infecting rats and humans, respectively. Both present extraordinary life cycles comprising a free-living generation in addition to parasitic stages. In search of molecules possibly involved in parasite-host interaction, we performed mass spectrometry to identify excretory/secretory products of S. ratti. Amongst others we detected homologs of the heat shock proteins HSP10 and HSP60 (Sr-HSP10 and Sr-HSP60). HSPs are well known as chaperones involved in stress responses of cells, but recent studies suggest additional roles of small HSPs for parasite biology including immune modulation. To characterise Sr-HSP10, we cloned its full-length cDNA, analysed the genomic organisation, tested its presumptive role as an interaction partner of Sr-HSP60, studied its transcription in the parasite, and expressed the protein to test its immune responses. The cDNA contains an open reading frame of 330bp encoding a polypeptide of 110 amino acids with an approximate molecular weight of 10kDa. The Sr-HSP10 protein is highly homologous to that of the human pathogen S. stercoralis with only eight amino acid substitutions. Analysis of the genomic organisation of the Sr-HSP10 locus revealed that the gene is linked head-to-head to the gene encoding Sr-HSP60, and both share a bidirectional promoter. RT-PCR experiments indicated potential independent expression of the Sr-HSPs genes. In situ hybridisation results demonstrate Sr-HSP10 transcription in the gut area. Mammalian and yeast two-hybrid assays show dimerisation of Sr-HSP10, but no binding to recombinant Sr-HSP60. Immunisation experiments finally revealed a strong immunogenicity of Sr-HSP10 and provided evidence for a role in regulating the host-parasite interaction.

HSP 10 is a new autoantigen in both autoimmune pancreatitis and fulminant type 1 diabetes.

To search autoantigens in autoimmune pancreatitis (AIP), we have screened the human pancreas cDNA library with a patient's serum and obtained 10 positive clones. Seven out of 10 clones were amylase alpha-2A, the autoantibody to which was specifically detected in sera from patients with AIP and fulminant type 1 diabetes (FT1DM) [T. Endo, S. Takizawa, S. Tanaka, M. Takahashi, H. Fujii, T. Kamisawa, T. Kobayashi, Amylase alpha-2A autoantibodies: novel marker of autoimmune pancreatitis and fulminant type 1 diabetes mellitus, Diabetes 58 (2009) 732-737]. Sequencing of 1 out of remaining 3 positive clones revealed that it was identical to heat shock protein 10 (HSP 10) cDNA. Using a recombinant HSP 10, we have developed enzyme-linked immunosorbent assay (ELISA) system for detecting autoantibodies against HSP 10. We found that autoantibody against HSP 10 was also produced with high frequency in sera from patients with AIP (92%) and FT1DM (81%), but not in chronic alcoholic pancreatitis (8%) or healthy volunteers (1.4%). These results suggest that an autoantibody against HSP 10 is also a new diagnostic marker for both AIP and FT1DM.

Tolerization with Hsp65 induces protection against adjuvant-induced arthritis by modulating the antigen-directed interferon-gamma, interleukin-17, and antibody responses.

OBJECTIVE: Pretreatment of Lewis rats with soluble mycobacterial Hsp65 affords protection against subsequent adjuvant-induced arthritis (AIA). This study was aimed at unraveling the mechanisms underlying mycobacterial Hsp65-induced protection against arthritis, using contemporary parameters of immunity. METHODS: Lewis rats were given 3 intraperitoneal injections of mycobacterial Hsp65 in solution prior to the initiation of AIA with heat-killed Mycobacterium tuberculosis. Thereafter, mycobacterial Hsp65-specific T cell proliferative, cytokine, and antibody responses were tested in tolerized rats. The roles of anergy and the indoleamine 2,3 dioxygenase (IDO)-tryptophan pathway in tolerance induction were assessed, and the frequency and suppressive function of CD4+FoxP3+ Treg cells were monitored. Also tested was the effect of mycobacterial Hsp65 tolerization on T cell responses to AIA-related mycobacterial Hsp70, mycobacterial Hsp10, and rat Hsp65. RESULTS: The AIA-protective effect of mycobacterial Hsp65-induced tolerance was associated with a significantly reduced T cell proliferative response to mycobacterial Hsp65, which was reversed by interleukin-2 (IL-2), indicating anergy induction. The production of interferon-gamma (but not IL-4/IL-10) was increased, with concurrent down-regulation of IL-17 expression by mycobacterial Hsp65-primed T cells. Neither the frequency nor the suppressive activity of CD4+FoxP3+ T cells changed following tolerization, but the serum level of anti-mycobacterial Hsp65 antibodies was increased. However, no evidence was observed for a role of IDO or cross-tolerance to mycobacterial Hsp70, mycobacterial Hsp10, or rat Hsp65. CONCLUSION: Tolerization with soluble mycobacterial Hsp65 leads to suppression of IL-17, anergy induction, and enhanced production of anti-mycobacterial Hsp65 antibodies, which play a role in protection against AIA. These results are relevant to the development of effective immunotherapeutic approaches for autoimmune arthritis.

In infertile women, cells from Chlamydia trachomatis infected sites release higher levels of interferon-gamma, interleukin-10 and tumor necrosis factor-alpha upon heat-shock-protein stimulation than fertile women.

BACKGROUND: The magnitude of reproductive morbidity associated with sexually transmitted Chlamydia trachomatis infection is enormous. Association of antibodies to chlamydial heat shock proteins (cHSP) 60 and 10 with various disease sequelae such as infertility or ectopic pregnancy has been reported. Cell-mediated immunity is essential in resolution and in protection to Chlamydia as well as is involved in the immunopathogenesis of chlamydial diseases. To date only peripheral cell mediated immune responses have been evaluated for cHSP60. These studies suggest cHSPs as important factors involved in immunopathological condition associated with infection. Hence study of specific cytokine responses of mononuclear cells from the infectious site to cHSP60 and cHSP10 may elucidate their actual role in the cause of immunopathogenesis and the disease outcome. METHODS: Female patients (n = 368) attending the gynecology out patient department of Safdarjung hospital, New Delhi were enrolled for the study and were clinically characterized into two groups; chlamydia positive fertile women (n = 63) and chlamydia positive infertile women (n = 70). Uninfected healthy women with no infertility problem were enrolled as controls (n = 39). cHSP60 and cHSP10 specific cytokine responses (Interferon (IFN)-gamma, Interleukin (IL)-10, Tumor Necrosis Factor (TNF)-alpha, IL-13 and IL-4) were assessed by ELISA in stimulated cervical mononuclear cell supernatants. RESULTS: cHSP60 and cHSP10 stimulation results in significant increase in IFN-gamma (P = 0.006 and P = 0.04 respectively) and IL-10 levels (P = 0.04) in infertile group as compared to fertile group. A significant cHSP60 specific increase in TNF-alpha levels (P = 0.0008) was observed in infertile group as compared to fertile group. cHSP60 and cHSP10 specific IFN-gamma and IL-10 levels were significantly correlated (P < 0.0001, r = 0.54 and P = 0.004, r = 0.33 respectively) in infertile group. CONCLUSION: Our results suggest that exposure to chlamydial heat shock proteins (cHSP60 and cHSP10) could significantly affect mucosal immune function by increasing the release of IFN-gamma, IL-10 and TNF-alpha by cervical mononuclear cells.

Evidence for persistent Chlamydia pneumoniae infection of human coronary atheromas.

To date, structures representing developmental stages of Chlamydia pneumoniae, especially persistent forms of this intracellular bacteria, have not been described in human atherosclerotic tissues using specific antibody labeling and transmission electron microscopy. Staining of atherosclerotic tissue from five patients seeking heart transplantation with gold-labeled antibodies specific for up-regulated chlamydial heat shock proteins, GroEL and GroES, and visualisation via transmission electron microscopy revealed intracellular, atypical, round to oval structures of variable diameter. These structures resembled reticulate bodies of Chlamydia, were surrounded by membranes and were located within smooth muscle cells, macrophages or fibroblasts. By using double immunogold electron microscopy technique (GroEL and GroES in combination with chlamydial LPS/MOMP antibodies), we demonstrated these structures were of chlamydial origin. In the current study, we demonstrated the presence of aberrant bodies of C. pneumoniae in vivo in archival coronary atheromatous heart tissues by the immunogold electron microscopy technique.

Activation of human CD4+ T cells by targeting MHC class II epitopes to endosomal compartments using human CD1 tail sequences.

Distinct CD4(+) T-cell epitopes within the same protein can be optimally processed and loaded into major histocompatibility complex (MHC) class II molecules in disparate endosomal compartments. The CD1 protein isoforms traffic to these same endosomal compartments as directed by unique cytoplasmic tail sequences, therefore we reasoned that antigen/CD1 chimeras containing the different CD1 cytoplasmic tail sequences could optimally target antigens to the MHC class II antigen presentation pathway. Evaluation of trafficking patterns revealed that all four human CD1-derived targeting sequences delivered antigen to the MHC class II antigen presentation pathway, to early/recycling, early/sorting and late endosomes/lysosomes. There was a preferential requirement for different CD1 targeting sequences for the optimal presentation of an MHC class II epitope in the following hierarchy: CD1b > CD1d = CD1c > > > CD1a or untargeted antigen. Therefore, the substitution of the CD1 ectodomain with heterologous proteins results in their traffic to distinct intracellular locations that intersect with MHC class II and this differential distribution leads to specific functional outcomes with respect to MHC class II antigen presentation. These findings may have implications in designing DNA vaccines, providing a greater variety of tools to generate T-cell responses against microbial pathogens or tumours.

Seroprevalence of antibodies to conserved regions of Chlamydia trachomatis heat shock proteins 60 and 10 in women in India.

Persistent, untreated chlamydial infection causes chronic stimulation of the host immune system against immunogenic antigens such as chlamydial heat shock proteins (cHSP) 60 and 10. In order to find the seroprevalence of antibodies to cHSPs, enzyme-linked immunosorbent assay (ELISA) is performed using specific peptide sequences to measure antibody response against major outer membrane protein (MOMP), cHSP60 and cHSP10 in patient sera. In this study, 255 patients attending the gynaecology out-patient department (March 2004 to August 2005) of Safdarjung Hospital were enrolled. Of these patients, 107 were diagnosed with cervicitis while 52 had pelvic inflammatory disease (PID)/infertility. Chlamydia trachomatis infection in endocervical specimens is diagnosed by a direct fluorescence assay (DFA) and the polymerase chain reaction (PCR). In 75 (29.4%) of the C. trachomatis-positive women, 50 (66.7%) were ELISA positive for MOMP 48 (64.0%) were positive for cHSP60 and 46 (61.3%) were positive for cHSP10. The anti-MOMP index correlated positively with anti-cHSP60 (R = 0.522, P < 0.01) and anti-cHSP10 (R = 0.286, P < 0.05). Antibody titre for MOMP was significantly higher than that for cHSP60 (1:5; P < 0.01 and 1:25; P < 0.05). Moreover, patients with PID/infertility showed significantly higher antibody titres for cHSP60 and cHSP10 when compared to patients with cervicitis at dilutions of 1 in 50, 1 in 250, 1 in 1250 (P < 0.001) and at 1 in 6250 (P < 0.01).

Mucosal and peripheral immune responses to chlamydial heat shock proteins in women infected with Chlamydia trachomatis.

Most of the studies on 60-kDa and 10-kDa chlamydial heat shock proteins (HSPs) to date have been carried out with blood lymphocytes or serum antibody responses, which do not provide a clear picture of the actual pathogenesis as they do not differentiate primary infection from recurrent infection. Thus, in the present study induction of the immune response was evaluated by studying lymphoproliferation of both cervical and peripheral lymphocytes to synthetic peptides of cHSP60, cHSP10 and major outer membrane protein (MOMP) antigen. In addition, cervical antibody prevalence to MOMP antigen, cHSP60 and cHSP10 and cytokine levels in cervical washes was also determined. Positive proliferative responses of cervical lymphocytes to cHSP10 peptide were significantly higher (P < 0.05) in women with recurrent infections and that to MOMP antigen were significantly higher in primary infection. On proliferation of PBMCs with the above antigens, no significant difference was observed between primary and recurrent infection. Prevalence of cervical IgG and IgA antibodies to Chlamydia trachomatis was significantly higher (P < 0.05) during primary infection than recurrent infections. In contrast, prevalence of IgG and IgA antibodies to cHSP10 and IgG antibodies to cHSP60 was higher during recurrent infections than primary infections. Interferon (IFN)-gamma levels were significantly higher in cervical washes of women with recurrent infection and correlated strongly with cHSP60 antibody titres. Our data thus suggest that mucosal responses are more appropriate in understanding the pathogenesis of chlamydial infection and IFN-gamma could be involved in the modulation of immune responses towards chlamydial infection directly, by causing acute inflammation, or indirectly through modulation of HSP expression.

Local markers for prediction of women at higher risk of developing sequelae to Chlamydia trachomatis infection.

PROBLEM: Chlamydial infections are often associated with various fertility-related disorders. Serological prediction of these has limitations, as they do not differentiate between past and current infections. Thus, we looked for local markers that could predict more precisely women at higher risk of developing severe complications. METHOD OF STUDY: A total of 320 Chlamydia trachomatis positive women with or without fertility disorders were tested for the prevalence of immunoglobulin A antibodies to synthetic peptides of chlamydial heat-shock protein 60 (cHSP60) and cHSP10 along with cervical interferon-gamma (IFN-gamma) and serum C-reactive protein (CRP) levels. RESULTS: Positive IFN-gamma level was the single best predictor for fertility disorder [odds ratio (OR) 15.4]. The predictive value of IFN-gamma could be significantly improved only by the addition of CRP test (OR 37.9). CONCLUSION: Positive IFN-gamma levels in cervical washes along with elevated CRP levels could be used to predict women who are at higher risk of developing fertility disorders.

Comparative immunoproteomics of identification and characterization of virulence factors from Helicobacter pylori related to gastric cancer.

Helicobacter pylori is an important risk factor of gastric cancer (GC). Although many H. pylori virulence factors have been reported, the pathogenic mechanism by which H. pylori infection causes GC remains unclear. The aims of this study were to identify GC-related antigens from H. pylori and characterize their roles in the development of GC. As GC and duodenal ulcer (DU) are considered clinically divergent, we compared two-dimensional immunoblots of an acid-glycine extract of H. pylori probed with serum samples from 15 patients with GC and 15 with DU to find GC-related antigens, which were subsequently identified by mass spectrometry. Many protein spots were recognized by more than one serum, and 24 of these were better recognized by GC sera. The proteins showing higher frequency of recognition in GC group are threonine synthase, rod shape-determining protein, S-adenosylmethionine synthetase, peptide chain release factor 1, DNA-directed RNA polymerase alpha subunit, co-chaperonin GroES (monomeric and dimeric forms), response regulator OmpR, and membrane fusion protein. Of these proteins, GroES was identified as a dominant GC-related antigen with a much higher seropositivity of GC samples (64.2%, n = 95) compared with 30.9% for gastritis (n = 94) and 35.5% for DU (n = 124). GroES seropositivity was more commonly associated with antral GC than with non-antral GC (odds ratio = 2.7; 95% confidence interval, 1.1-6.7). In peripheral blood mononuclear cells, GroES stimulated production of interleukin (IL)-8, IL-6, granulocyte macrophage colony-stimulating factor, IL-1beta, tumor necrosis factor-alpha, cyclooxygenase-2, and prostaglandin E(2). Moreover when incubated with gastric epithelial cells, GroES induced expression of IL-8, cell proliferation, and up-regulation of c-jun, c-fos, and cyclin D1 but caused down-regulation of p27(Kip1). We conclude that GroES of H. pylori is a novel GC-associated virulence factor and may contribute to gastric carcinogenesis via induction of inflammation and promotion of cell proliferation.

T-h-2 immunity and CD3+CD45RBlow-activated T cells in mice immunized with recombinant bacillus Calmette-Guérin expressing HIV-1 principal neutralizing determinant epitope.

The genetic engineering of Mycobacterium bovis-bacillus Calmette-Guérin to express foreign epitopes is an attractive strategy in the field of epitope vaccines. We constructed an 'epitope-trap vector' with Mycobacterium tuberculosis chaperonin-10 as a carrier antigen and used it to express the HIV-1 principal neutralizing determinant epitope. We also identified a new chaperonin-10 promoter that was hyperexpressive compared with the heat shock protein-65 promoter. Splenocytes from recombinant bacillus Calmette-Guérin-immunized mice showed enhanced lymphocyte proliferation and interleukin-4 (but not interferon-gamma) secretion. The recombinant bacillus Calmette-Guérin-immunized group also exhibited mild delayed-type hypersensitivity reaction and a high frequency of CD3+CD45RBlow-activated T cells, together with high titer of antiprincipal neutralizing determinant immunoglobulin G antibodies. Thus, this epitope delivery system induced strong epitope-specific T-h-2 polarization.

HSP-10 in ovarian cancer: expression and suppression of T-cell signaling.

OBJECTIVE: Suppressed T-cell activation is a hallmark of advanced ovarian cancer. Studies in pregnancy have demonstrated similar T-cell dysfunction mediated, at least in part, by HSP10, identified as "early pregnancy factor." This pilot study addresses the presence of HSP10 in the circulation of ovarian cancer patients and assesses its role in suppressing CD3-zeta. METHODS: Sera were obtained from ovarian cancer patients (n = 10) and age-matched noncancer-bearing female controls (n = 9). HSP10 presence was determined semiquantitatively by Western immunoblotting in sera, ascites, and ovarian tumor cell conditioned media. The consequences of HSP10 on CD3-zeta suppression were defined using a Jurkat cell bioassay, using unfractionated patient sera, sera with HSP10 removed by immunoprecipitation and the immunoprecipitate. RESULTS: HSP10 was detected in both sera and ascites of patients with ovarian cancer; however, it was not detectable in controls. HSP10 was also detected in the culture media of ovarian tumor cells. Sera containing HSP10 suppressed T-cell CD3-zeta expression, which correlated with HSP10 levels (r2 = 0.839). When HSP10 was removed from the sera, the ability to suppress CD3-zeta was diminished and the immunoprecipitated material was capable of suppressing CD3-zeta. CONCLUSIONS: HSP10 appears to be produced and released from ovarian tumor cells and is detectable in the peripheral blood and ascites of patients. This circulating HSP10 appears to suppress T-cell expression of CD3-zeta, a key component of T-cell activation. Our findings indicate that, as in pregnancy, production and release of HSP10 may be a critical factor in the suppression of T-cell activation, allowing the tumor to escape immune surveillance.

The unusual chaperonins of Mycobacterium tuberculosis.

Heat shock proteins (Hsps), also known as molecular chaperones, are a diverse set of proteins that mediate the correct folding, assembly, transport and degradation of other proteins. In addition, Hsps have been shown to play a variety of important roles in immunity, thereby representing prominent antigens in the humoral and cellular immune response. Chaperonins form a sub-group of molecular chaperones that are found in all domains of life. Chaperonins in all bacteria are encoded by the essential groEL and groES genes, also called cpn60 and cpn10 arranged on the bicistronic groESL operon. Interestingly, Mycobacterium tuberculosis contains two copies of the cpn60 genes. The existence of a duplicate set of cpn60 genes in M. tuberculosis, however, has been perplexing. Cpn10 and Cpn60s of M. tuberculosis have been shown to be highly antigenic in nature, eliciting strong B- and T-cell immune responses. Recent work has shown intriguing structural, biochemical and signaling properties of the M. tuberculosis chaperonins. This review details the recent developments in the study of the M. tuberculosis chaperonins.

Combined detection of Chlamydia trachomatis-specific antibodies against the 10 and 60-kDa heat shock proteins as a diagnostic tool for tubal factor infertility: Results from a case-control study in Cameroon.

A case-control study was conducted to determine the diagnostic value of Chlamydia trachomatis-associated anti-Chsp10 and/or anti-Chsp60 antibodies in the detection of secondary infertility. There were significant associations between C. trachomatis infection and infertility (p<0.01), and between C. trachomatis-specific anti-Chsp10 or anti-Chsp60 antibodies and secondary infertility (p<0.001). A significant correlation was found between anti-Chsp10 and anti-Chsp60 titers (p<0.01). The detection of either C. trachomatis-associated anti-Chsp10 or anti-Chsp60 antibodies cumulatively allowed specific diagnosis of secondary infertility (57.4% sensitivity, 75.5% specificity).

Low prevalence of antibodies against heat shock protein 10 of Chlamydophila pneumoniae in patients with coronary heart disease.

In this study the prevalence of antibodies against the heat shock protein 10 (HSP10) of Chamydophila pneumoniae (CP) (as assessed by ELISA) in patients with coronary heart disease (CHD) and seropositive or seronegative to CP, as assessed by microimmunofluorescence (MIF), was investigated. The controls were age- and sex-matched healthy subjects. The HSP10 preparation used throughout this study was a 6-his-tagged recombinant protein preliminarily shown to be immunogenic in mice. Low level IgG reactivity against CP-HSP10 was detected in 19 out of 200 and 5 out of 100 CHD patients and controls, respectively. No IgM or IgA isotypes were found. Furthermore, there was no difference in the frequency or level of anti-HSP10 IgG between CP-positive and CP-negative sera either in patients (11/140=7.9% vs. 8/60=13%) or in healthy subjects (3/40=7.5% vs. 2/60=3.3%). Overall, our data indicate that CP-HSP10, at variance with CP-HSP60, to which it is genetically and physiologically linked, should not be regarded as a major expressed immunogen or a marker of infection by CP in CHD patients.

Association of uterine and salpingeal fibrosis with chlamydial hsp60 and hsp10 antigen-specific antibodies in Chlamydia-infected koalas.

Infection by Chlamydia pneumoniae or Chlamydia pecorum commonly causes chronic, fibrotic disease of the urogenital tracts of female koalas. Studies of humans have associated titers of serum immunoglobulin G (IgG) against chlamydial hsp60 and hsp10 antigens with chronic infection, salpingeal fibrosis, and tubal infertility. To determine whether a similar relationship exists in Chlamydia-infected koalas, samples were collected opportunistically from 34 wild female koalas and examined by gross pathology and histopathology, PCR, and immunohistochemistry for Chlamydia spp. and enzyme-linked immunosorbent assay for serological responses to chlamydial hsp10 and hsp60 antigens. Greater anti-hsp titers occurred in Chlamydia-infected koalas with fibrous occlusion of the uterus or uterine tube than in other Chlamydia-infected koalas (for hsp10 IgG, P = 0.005; for hsp60 IgG, P = 0.001; for hsp10 IgA, P = 0.04; for hsp60 IgA, P = 0.09). However, as in humans, some koalas with tubal occlusion had low titers. Among Chlamydia-infected koalas with tubal occlusion, those with low titers were more likely to have an active component to their ongoing uterine or salpingeal inflammation (P = 0.007), such that the assay predicted, with 79% sensitivity and 92% specificity, tubal occlusion where an active component of inflammation was absent. Findings of this study permit advancement of clinical and epidemiological studies of host-pathogen-environment interactions and pose intriguing questions regarding the significance of the Th1/Th2 paradigm and antigen-presenting and inflammation-regulating capabilities of uterine epithelial cells and the roles of latency and reactivation of chlamydial infections in pathogenesis of upper reproductive tract disease of koalas.