Detection of serum antibodies against Chlamydia pneumoniae by ELISA

Abstract Chlamydia pneumoniae causes pneumonia and other respiratory infections in children, adolescents and adults. We tried to evaluate the diagnostic value of detection of serum antibodies by ELISA for C. pneumoniae infections in Japanese children. Serum IgG, IgA and IgM antibodies to C. pneumoniae were determined by the microimmunofluorescence (MIF) test. Serum IgG and IgA antibodies were also determined by ELISA test kits. Results obtained by ELISA were compared with those obtained by MIF test. IgG antibody to C. pneumoniae was detected in 135 (39.5%) by ELISA and in 125 (36.5%) by MIF out of 342 sera from Japanese infants and children without respiratory infections (aged from 2 months old to 15 years old). IgA antibody to C. pneumoniae was detected in 129 (37.7%) by ELISA and in 117 (34.2%) by MIF out of 342 sera tested. Of 342 specimens 113 were IgG-positive by ELISA and MIF (sensitivity: 90.4%, specificity: 89.9%, r = 0.853). Of 342 sera 28 had IgG antibody titers of 1:256 and none had titers 1:512 or higher by MIF. Of 28 infants and children a total of nine were less than 4 years of age. On the other hand, of 342 specimens 99 were IgA-positive by ELISA and MIF (sensitivity: 84.6%, specificity: 86.7%, r = 0.769). Of 342 sera 16 had IgA antibody titers of 1:256 or higher by MIF. Of 16 infants and children, ten were less than 4 years of age. ELISA had excellent sensitivity and specificity relative to MIF test for detection of IgC and IgA antibodies to C. pneumoniae . It was suggested that C. pneumoniae infection in Japanese infants and children under 4 years of age was not infrequent.     

Chlamydia pneumoniae induces interleukin‐6 and interleukin‐10 in human gingival fibroblasts

Chlamydia pneumoniae is an obligate intracellular Gram‐negative bacterium with a unique biphasic developmental cycle that can cause persistent infections. In humans, Chlamydia causes airway infection and has been implicated in chronic inflammatory diseases, such as asthma and atherosclerosis. In addition, recent studies demonstrated that patients with severe periodontitis can harbor C. pneumoniae, which can increase the risk for a host inflammatory response with weighty clinical sequelae. Previous studies have established that periodontal pathogenic bacteria (i.e. Gram‐negative bacteria) can induce the synthesis and release of cytokines and other inflammatory mediators in human gingival fibroblasts. HGF are resident cells of the periodontium that respond to receptor stimulation by producing a variety of substances including cytokines and growth factors. Our results demonstrate that after 48 hr of incubation with viable C. pneumoniae HGF showed a proliferative response, as seen by both colorimetric MTT assay and direct cell count (30% and 35%, respectively). In addition, HGF incubated with viable or UV light‐inactivated C. pneumoniae organisms showed an increase in the levels of IL‐6 and IL‐10, but not IL‐4; on the contrary, HGF infected with heat‐killed bacteria did not show a significant production of any of the cytokines considered. In conclusion, the present study suggests that C. pneumoniae may modulate the expression of IL‐6 and IL‐10 by human gingival fibroblasts. Further studies are warranted to clarify the molecular mechanisms of C. pneumoniae in the regulation of cytokine expression by host cells and to elaborate the relevant clinical implications.     

Streptococcus pneumoniae heat shock protein 70 does not induce human antibody responses during infection

Mouse monoclonal antibodies (mAbs) were developed against Streptococcus pneumoniae in search for potential common pneumococcal proteins as vaccine antigens. mAb 230,B-9 (IgG1) reacted by immunoblotting with a 70-kDa protein which was isolated by immunoaffinity chromatography and subsequent preparative electrophoresis. N-terminal amino acid sequencing showed homology to that of heat shock protein 70 (hsp70). The hsp70 epitope reactive with mAb 230,B-9 was found in all the pneumococci examined as well as in other streptococci and enterococci. The epitope was not expressed in several other examined Gram-positive or -negative bacteria. Pneumococcal hsp70 has by other investigators been proposed to be a vaccine candidate. Binding experiments using flow cytometry showed that the epitope was not surface-exposed on live exponential phase grown S. pneumoniae. Human patient sera did not react with affinity-purified pneumococcal hsp70. Therefore the pneumococcal hsp70 does not seem to be of special interest in a vaccine formulation. The human sera contained antibodies to high molecular proteins co-purified with hsp70. Some of these proteins could be the pneumococcal surface protein A.     

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.     

Outer membrane complex proteins of Chlamydia pneumoniae

Abstract The protein composition of the outer membrane complex (OMC) of Chlamydia pneumoniae strain AR-39 was analyzed by metabolic labeling with [³⁵S]methionine and [³⁵S]cysteine. Cysteine-rich proteins with molecular masses of 98, 60 doublet, 39.5 (MOMP) and 15.5 kDa were found in the OMC of C. pneumoniae . The cysteine-rich proteins of the OMCs of the threee Chlamydia species showed specific reaction patterns by immunoassay and autoradiography to rabbit or turkey immune sera. Recognition of the MOMP and 60-kDa proteins of the three species was cross-reactive. However, the C. pneumoniae 98-kDa protein was recognized by anti- C. pneumoniae (AR-39) and anti- C. psittaci (TT3) immune sera. None of the immunee sera recognized the 12-kDa cysteine-rich complex.     

Ultrastructure of Chlamydia pneumoniae in cell culture

The electron microscopic appearance of Chlamydia pneumoniae elementary bodies with pear-shaped, loose outer membrane has been suggested as one criterion of its classification as a new chlamydial species. The study of the original strain TW 183 in LCL 929 and HL cells and a low-passage isolate of Kajaani-6 isolate in HL cells revealed spherical compact elementary bodies common to other chlamydia.     

Characterization of a hypervariable region in the genome of Chlamydophila pneumoniae

Chlamydophila pneumoniae displays surprisingly little genomic variation, as seen by comparisons of the published genomes from three different isolates and sequencing of four different genes from different isolates. We have in the present study, however, demonstrated genomic variation between 10 C. pneumoniae isolates in the 11690-bp region between the two outer membrane protein genes pmp1 and pmp2. This region of the C. pneumoniae CWL-029 isolate contains seven C. pneumoniae-specific open reading frames (hb1-7, encoding hydrophobic beta-sheet-containing proteins). We identified additionally 12 open reading frames in the C. pneumoniae CWL-029 genome encoding hypothetical proteins with similarity to the seven hypothetical Hb-proteins. Compared to other isolates, genomic variation is seen to cause frame-shifting of three of the 19 hb-open reading frames, which are proposed to be three full-length genes and eight frame-shifted pseudogenes. The hypothetical proteins encoded by these proposed genes contain an N-terminally located highly hydrophobic stretch of 50-60 residues. A similar motif is found in all identified Chlamydia inclusion membrane proteins and therefore the Hb-proteins are candidate inclusion proteins.     

Differential expression of Pmp10 in cell culture infected with Chlamydia pneumoniae CWL029

The complete genome of Chlamydia pneumoniae contains a total of 21 genes encoding polymorphic membrane proteins (Pmp). From this large Pmp family three genes, pmp8, pmp10 and pmp11, were cloned and antibodies against recombinant full-length Pmp proteins were produced. Indirect immunofluorescence microscopy of HEp-2 cells infected with C. pneumoniae CWL029 was performed with the Pmp antibodies in combination with a Chlamydia-specific anti-lipopolysaccharide (LPS) antibody. This double staining technique clearly showed that expression of Pmp10 was differential. Additional double staining with monoclonal antibodies to the surface of C. pneumoniae elementary bodies and the anti-LPS antibody resulted in identification of seven monoclonal antibodies that reacted identically to the Pmp10 antibody indicating that Pmp10 is an immunodominant protein. Finally, the molecular mechanism responsible for differential expression is suggested to be variation in the guanine residues in the polyG tract of pmp10.     

11788例儿童血清肺炎支原体抗体检测结果分析

目的了解宁波儿童肺炎支原体（MP）感染情况,为儿童MP感染的临床诊断与防治提供依据。方法采用被动凝集法测定11788例14岁以下有呼吸道感染症状儿童的血清MP抗体,并根据患儿性别、年龄和不同季节对MP的阳性率进行统计学分析。结果11788例有呼吸道感染症状患儿中,MP阳性3081例,阳性率为26．14％,不同性别、年龄和季节MP抗体的阳性率差异有统计学意义（P〈0．05）。女性儿童阳性率高于男性儿童,并随着年龄的增加阳性率有上升趋势。结论MP在呼吸道感染中发病率较高,春冬季节、女性儿童和学龄期儿童易感。     

儿童哮喘与肺炎支原体感染关系的探讨

目的:探讨儿童哮喘发作与肺炎支原体(MP)感染之间的关系,并分析合并MP感染的患儿的临床表现。方法:将79例2-14岁急性哮喘发作的患儿依据病史分做两组:第一次哮喘发作的35人(始发哮喘组),已经有哮喘病史的44人(复发哮喘组)。采用被动冷凝集法检测两组患儿肺炎支原体抗体(MP-IgM)。结果:始发哮喘组和复发哮喘组分别有16例(45.7%)和10例(22.7%)患儿MP-IgM阳性(P0.05)。始发哮喘组与复发哮喘组MP-IgM阳性的患儿发热和肺部啰音发生率明显高于MP-IgM阴性的患儿(P0.05),血清IgE水平也明显高于MP-IgM阴性的患儿(P0.05)。结论:MP感染与儿童哮喘发作关系密切,合并MP感染的哮喘患儿发热或肺部啰音发生率明显高于未合并MP感染的哮喘患儿。     

Reversibility of heat shock in Chlamydia trachomatis

The heat shock effect on chlamydia development was studied. We report here that the reversibility of the heat shock response did not depend on the stage of chlamydial morphogenesis at which transfer to high temperature occurred, and the infectivity of the particles produced was not affected significantly, so long as the heat shock exposure was not prolonged. Exposure to heat shock for more than 9 h resulted in stagnation of the growth cycle, appearance of aberrant reticulate body particles and loss of infectivity. SDS-PAGE analysis of proteins synthesized under prolonged heat shock showed increased relative abundance of heat shock proteins in common with other procaryotic organisms.     

外周血单个核细胞中肺炎衣原体的分离、培养及连续传代

开发一种分离并增殖肺炎衣原体(Cpn)的简易方法极具意义。该方法先分离血液标本中的外周血单个核细胞(PBMC),用PEG使Cpn-Ag阳性的PBMC裂解释放出Cpn,然后与人喉表皮癌细胞(Hep-2)一起离心,继续培养Hep-2细胞,再将Hep-2细胞冻融破碎后,放入到新的Hep-2细胞中进行离心,以完成Cpn的1次传代,然后以同样的方法进行2-4次传代。分别用微量免疫荧光法(MIF)及PCR法检测Hep-2细胞中的Cpn-Ag和CpnDNA,并用FITC标记的属特异性衣原体脂多糖单克隆抗体检测实验分离以及进口菌株传代后的包涵体形成单位数量。结果显示MIF法检测Cpn感染后的Hep-2细胞,其胞内Cpn-Ag强阳性;MIF法检测1次传代及2次传代的Hep-2细胞,其胞内Cpn-Ag亦均强阳性,3次传代为阳性,而4次传代为阴性;PCR法检测2次传代后CpnDNA阳性。该简化方法可以实现PBMC中Cpn的分离,分离菌株传代4次后出现退化(优于进口菌株),但该方法仍可实现进一步的培养及传代。     

Proteolytic cleavage of the Chlamydia pneumoniae major outer membrane protein in the absence of Pmp10

The genome of the obligate intracellular bacteria Chlamydia pneumoniae contains 21 genes encoding polymorphic membrane proteins (Pmp). While no function has yet been attributed to the Pmps, they may be involved in an antigenic variation of the Chlamydia surface. It has previously been demonstrated that Pmp10 is differentially expressed in the C. pneumoniae CWL029 isolate. To evaluate whether the absence of Pmp10 in the outer membrane causes further changes to the C. pneumoniae protein profile, we subcloned the CWL029 isolate and selected a clone with minimal Pmp10 expression. Subsequently, we compared the proteome of the CWL029 isolate with the proteome of the subcloned strain and identified a specific cleavage of the C-terminal part of the major outer membrane protein (MOMP), which occurred only in the absence of Pmp10. In contrast, when Pmp10 was expressed we predominantly observed full-length MOMP. No other proteins appeared to be regulated according to the presence or absence of Pmp10. These results suggest a close association between MOMP and Pmp10, where Pmp10 may protect the C-terminal part of MOMP from proteolytic cleavage.     

Regulation of asthmatic airway relaxation by estrogen and heat shock protein 90

We tested the hypothesis that asthmatic mouse airways exhibit impaired relaxation to NO donors. Mouse tracheal rings were incubated overnight in serum from asthmatic human subjects or from nonasthmatic controls. The next day, cumulative concentration-response curves (CCRC) to sodium nitroprusside (SNP) and nitroglycerine (NTG) were obtained. Both SNP and NTG relaxed the pre-constricted normal tracheal rings. Tracheal rings exposed to serum from asthmatic patients exhibited a more than a threefold increase in the EC50 of SNP and NTG. Pre-incubation of tracheal rings with heat shock protein 90 inhibitors decreased the relaxation of both normal and asthmatic tracheal rings to SNP and NTG. Pre-incubation with estradiol did not affect normal tracheal ring relaxation but exhibited an increase in asthmatic tracheal ring relaxation, which was abolished by an estrogen receptor (ER) antagonist. ER subtype-selective agonists, but not GPR30 agonists, mimicked the action of estradiol on tracheal ring relaxation. Co-incubation of rings with radicicol and estradiol produced an ER-dependent increase in the relaxation response to SNP of both normal and asthmatic ASM. Estrogen-induced relaxation of ASM was abolished by overnight incubation with radicicol and this was associated with reduced expression of ERβ. These data suggest that asthmatic ASM is considerably less responsive to NO-donors and that both estrogen and hsp90 play important roles in ASM relaxation.     

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).     

Alveolar epithelial cells type II are major target cells for C. pneumoniae in chronic but not in acute respiratory infection

Pulmonary presence of Chlamydia pneumoniae is associated with acute and chronic infections. We show that unapparent chlamydial infection in four out of 31 chronic obstructive pulmonary disease (COPD) patients (12.9%) is characterized by a significant increase in infected alveolar epithelial cells type II (18.2 +/- 3.5% vs. 2.3 +/- 0.9; IHC/ISH) compared to a newly established model of acute chlamydial infection (ACIM) in vital lung specimens from pulmonary lobectomy. Expression of cHSP60 demonstrated pathogen viability and virulence in the ACIM. We conclude that target cells differ in acute and chronic chlamydial infection and suggest the ACIM as a novel tool to analyze the host-pathogen-interactions in acute respiratory infections.     

Identification of an in vivo CD4+ T cell-mediated response to polymorphic membrane proteins of Chlamydia pneumoniae during experimental infection

Chlamydia pneumoniae is an obligate intracellular bacterium that causes upper and lower respiratory tract infection in humans. C. pneumoniae harbors the polymorphic membrane protein (Pmp) family with 21 different proteins with a molecular mass around 100 kDa. The Pmps are species-specific, abundant and, together with major outer membrane protein and outer membrane protein 2, the dominant proteins in the C. pneumoniae outer membrane complex. Nevertheless, it is unknown whether Pmps are recognized by the cell-mediated immune response. To address this issue, C57BL/6J mice were infected intranasally with C. pneumoniae and the immune response to primary infection was investigated. We demonstrate, as expected, that the primary response is of the Th1 type by IgG2a- and IgG1-specific sELISA (Medac) on serum. In vivo-primed spleen lymphocytes were found to be reactive to Pmp8, Pmp20 and Pmp21 in an interferon-gamma ELISpot assay. The responses were shown to be mediated by CD4(+) T cells. To our knowledge, this is the first identification of antigens recognized by CD4(+) T cells during murine C. pneumoniae infection.