Chlamydial Lipopolysaccharide Research Articles

Reactivity of a dual amplified chlamydia immunoassay with different serovars of Chlamydia trachomatis.

A study was undertaken with different serovars (D, E, F, L2, MoPn) of Chlamydia trachomatis to determine the analytical sensitivity of a new dual amplified immunoassay (IDEIA PCE Chlamydia) for detecting chlamydial lipopolysaccharide. IDEIA PCE Chlamydia incorporates a polymer conjugate consisting of multiple copies of antibody and enzyme molecules to provide signal amplification. The test was also assessed with different protein A producing strains of Staphylococcus aureus in order to assess whether the use of a multiple antibody conjugate increased nonspecific binding. The detection limits varied for each serovar with a detection limit of 38 IFU/ml obtained with serovar F and 237 IFU/ml obtained with serovar D. The incorporation of the polymer conjugate resulted in a 2-5 fold increase in analytical sensitivity compared to an earlier version of the test using a conventional conjugate. No increase in cross reactivity with protein A producing strains of S. aureus was obtained. The new dual amplified test format offers potential as a sensitive low-cost screening assay for C. trachomatis infections.

Structural Analysis of the Lipopolysaccharide fromChlamydia trachomatis Serotype L2

The lipopolysaccharide (LPS) of Chlamydia trachomatis L2 was isolated from tissue culture-grown elementary bodies using a modified phenol/water procedure followed by extraction with phenol/chloroform/light petroleum. From a total of 5 x 10(4) cm2 of infected monolayers, 22.3 mg of LPS were obtained. Compositional analysis indicated the presence of 3-deoxy-D-manno-oct-2-ulopyranosonic acid (Kdo), GlcN, phosphorus, and fatty acids in a molar ratio of 2.8:2:2.1:4.5. Matrix-assisted laser-desorption ionization mass spectrometry performed on the de-O-acylated LPS gave a major molecular ion peak at m/z 1781.1 corresponding to a molecule of 3 Kdo, 2 GlcN, 2 phosphates, and two 3-hydroxyeicosanoic acid residues. The structure of deacylated LPS obtained after successive treatment with hydrazine and potassium hydroxide was determined by 600 MHz NMR spectroscopy as Kdoalpha2-->8Kdoalpha2-->4Kdoalpha2-->6D-GlcpNbeta1 -->6D-GlcpNalpha 1,4'-bisphosphate. These data, together with those published recently on the acylation pattern of chlamydial lipid A (Qureshi, N., Kaltashov, I., Walker, K., Doroshenko, V., Cotter, R. J., Takayama, K, Sievert, T. R., Rice, P. A., Lin, J.-S. L., and Golenbock, D. T. (1997) J. Biol. Chem. 272, 10594-10600) allow us to present for the first time the complete structure of a major molecular species of a chlamydial LPS.

No association of Chlamydia with abortion.

Case reports and serological work have raised the possibility that chlamydias can infect the placenta and thus harm the fetus. We investigated the involvement of Chlamydia in a series of 195 unselected cases of spontaneous abortion or miscarriage. Formalin-fixed placental tissues from all cases were examined immunohistochemically, for the presence of chlamydial lipopolysaccharide, as well as histopathologically. A serum sample was collected from 187 of the patients for detection of anti-chlamydial antibodies by microimmunofluorescence. All placental sections were negative for chlamydial antigen. Serological findings indicated that 8 patients had been in contact with C. trachomatis, 15 patients with C. pneumoniae, and none with C. psittaci. A few cases of perivillitis or intervillitis were detected, but none exhibited the intracytoplasmic inclusions typical of C. psittaci. Although these results are negative a search for Chlamydia in abortion materials should be encouraged.

Coronary angioplasty induces rise in Chlamydia pneumoniae-specific antibodies.

Chlamydia pneumoniae is frequently found in atherosclerotic lesions, and high titers of specific antibodies are associated with increased risk for acute myocardial infarction. However, a causative relation has not been established yet. We performed a prospective study of 93 patients undergoing percutaneous transluminal coronary angioplasty (PTCA) to investigate whether angioplasty influences Chlamydia-specific antibody titers and whether there is an association with restenosis. Blood samples were obtained before and 1 and 6 months after angioplasty. Antibodies against chlamydial lipopolysaccharide and against purified C. pneumoniae elementary bodies were measured by enzyme-linked immunosorbent assay (ELISA). After angioplasty, the prevalence of antibodies to lipopolysaccharide rose from 20 to 26% for immunoglobulin A (IgA), from 53 to 64% for IgG, and from 2 to 7% for IgM (P = 0.021, 0.004, and 0.046, respectively). There was a rapid increase of mean antibody titers of all antibody classes within 1 month of PTCA. During the following 5 months, antibody titers decreased slightly but were still higher than baseline values. Results of the C. pneumoniae-specific ELISA were essentially the same. The rise of anti-Chlamydia antibodies was not caused by unspecific reactivation of the immune system, as levels of antibodies against cytomegalovirus did not change. Neither seropositivity nor antibody titers were related to restenosis. However, increases in mean IgA and IgM titers were restricted to patients who had suffered from myocardial infarction earlier in their lives. In conclusion, we show that PTCA induces a stimulation of the humoral immune response against C. pneumoniae. These data support the idea that plaque disruption during angioplasty might make hidden chlamydial antigens accessible to the immune system.

Persistent chlamydial envelope antigens in antibiotic-exposed infected cells trigger neutrophil chemotaxis.

An in vitro coculture model system was used to explore conditions that trigger neutrophil chemotaxis to Chlamydia trachomatis infected human epithelial cells (HEC-1B). Polarized HEC-1B monolayers growing on extracellular matrix (ECM) were infected with C. trachomatis serovar E. By 36 h, coincident with the secretion of chlamydial lipopolysaccharide and major outer membrane protein to the surfaces of infected cells, human polymorphonuclear neutrophils (PMNL) loaded with azithromycin migrated through the ECM and infiltrated the HEC-1B monolayer. Bioreactive azithromycin was delivered by the chemotactic PMNL to infected epithelial cells in concentrations sufficient to kill intracellular chlamydiae. However, residual chlamydial envelopes persisted for 4 weeks, and PMNL chemotaxis was triggered to epithelial cells containing residual envelopes. Infected endometrial cells demonstrated up-regulation of ENA-78 and GCP-2 chemokine mRNA. Thus, despite appropriate antimicrobial therapy, residual chlamydial envelope antigens may persist in infected tissues of culture-negative women and provide one source for sustained inflammation.

A Chlamydia pneumoniae component that induces macrophage foam cell formation is chlamydial lipopolysaccharide.

Chlamydia pneumoniae infection is associated with atherosclerotic heart and vessel disease, but a causal relationship between this pathogen and the disease process has not been established. Recently, it was reported that C. pneumoniae induces human macrophage foam cell formation, a key event in early atheroma development, suggesting a role for the organism in atherogenesis. This study further examines C. pneumoniae-induced foam cell formation in the murine macrophage cell line RAW-264.7. Infected RAW cells accumulated cholesteryl esters when cultured in the presence of low-density lipoprotein in a manner similar to that described for human macrophages. Exposure of C. pneumoniae elementary bodies to periodate, but not elevated temperatures, inhibited cholesteryl ester accumulation, suggesting a role for chlamydial lipopolysaccharide (cLPS) in macrophage foam cell formation. Purified cLPS was found to be sufficient to induce cholesteryl ester accumulation and foam cell formation. Furthermore, the LPS antagonist lipid X inhibited C. pneumoniae and cLPS-induced lipid uptake. These data indicate that cLPS is a C. pneumoniae component that induces macrophage foam cell formation and suggest that infected macrophages chronically exposed to cLPS may accumulate excess cholesterol to contribute to atheroma development.

Characterization of a neutralizing monoclonal antibody directed at the lipopolysaccharide of Chlamydia pneumoniae.

Identification of protective epitopes is one of the first steps in the development of a subunit vaccine. One approach to accomplishing this is to identify structures or epitopes by using monoclonal antibodies (MAb) that can attenuate infectivity in vitro and in vivo. To date attempts to use this approach with Chlamydia pneumoniae have failed. This report is the first description of a MAb directed to the lipopolysaccharide (LPS) of Chlamydia that neutralizes both in vitro and in vivo the infectivity of C. pneumoniae. MAb CP-33, an immunoglobulin G2b (IgG2b), was identified from a fusion using splenocytes from mice immunized with C. pneumoniae TW-183. By Western blot analysis, MAb CP-33 exhibited genus-specific reactivity in that it recognized the LPSs of C. pneumoniae, Chlamydia trachomatis, and Chlamydia psittaci. MAb CP-33 did not react with 15 genera of gram-negative and gram-positive bacteria and Candida albicans. By using isolated LPS of Re mutants of Escherichia coli, Salmonella enterica serovar Minnesota, and recombinants expressing the 3-deoxy-D-manno-oct-2-ulosonic acid (Kdo) transferase gene kdtA of C. trachomatis, MAb CP-33 was shown to require for binding the presence of the genus-specific trisaccharide epitope alphaKdo(2-->8)alphaKdo(2-->4)alphaKdo. By employing synthetic oligosaccharides and neoglycoconjugates in an enzyme immunoassay (EIA) and EIA inhibition, it was further shown that MAb CP-33 differed from the extensively investigated prototype chlamydial LPS MAb S25-23. Most likely, MAb CP-33 recognizes a conformational epitope in which the alphaKdo(2-->8)alphaKdo(2-->4)alphaKdo trisaccharide is an essential structural component. When tested in an in vitro neutralization assay, MAb CP-33 gave a 50% neutralization titer of 8 ng/ml against C. pneumoniae TW-183. However, this MAb did not neutralize other C. pneumoniae strains, C. trachomatis, or C. psittaci. C. pneumoniae TW-183 was treated with either MAb CP-33 or a control IgG and then used to inoculate mice by the respiratory route. Five days after inoculation, there was a difference between the mice inoculated with the control IgG-treated inoculum and those inoculated with the MAb CP-33-treated organisms as to the number of mice infected as well as the number of inclusion-forming units recovered from lung cultures (P < 0.05). In summary, a Chlamydia-specific LPS MAb was able to neutralize in vitro the infectivity of C. pneumoniae TW-183.

Localization of Chlamydia trachomatis heat shock proteins 60 and 70 during infection of a human endometrial epithelial cell line in vitro.

Unlike chlamydial lipopolysaccharide, which is released from the developing inclusion to the surface of infected genital epithelial cells, both Chlamydia trachomatis heat shock protein (hsp) 60 and 70 antigens remained confined within the inclusion during the course of the chlamydial developmental cycle. Exposure of the infected cells to penicillin to induce a persistent infection or to a lipophilic microbicide did not potentiate secretion or exocytosis of the chlamydial hsp.

Studies of persistent infection by Chlamydia trachomatis serovar K in TPA-differentiated U937 cells and the role of IFN-gamma.

Inoculation of phorbol ester-differentiated U937 cells as a model for human macrophages with Chlamydia trachomatis of the urogenital serovar K resulted in a persistent infection, with maximal growth at day 7, until day 10 post-infection. At these times inclusion bodies were present in 0.5-2% of the cells. Typical inclusion bodies containing elementary bodies and reticulate bodies were observed by electron microscopy. Furthermore, single chlamydial particles resembling atypical elementary or intermediate bodies were identified in the cytoplasm in > 80% of the host cells. IFN-gamma exerts antichlamydial activity in epithelial and fibroblastoid cells, but the infection of U937 cells by C. trachomatis was not affected by IFN-gamma. The activity of the tryptophan-degrading enzyme indoleamine 2,3-dioxygenase (IDO) was not detected in untreated or in IFN-gamma-treated or chlamydiae-infected or mock-infected U937 cells. The presence of atypical persisting chlamydiae and the lack of IDO expression in U937 cells indicates that the development of these atypical bacteria is independent from IFN-gamma-mediated tryptophan deprivation and other IFN-gamma-mediated effects. Evaluation of persistently infected cells revealed that the expression of the chlamydial major outer-membrane protein, heat-shock protein (hsp60) and lipopolysaccharide (LPS) antigens was not significantly altered in the course of the culture. An intense staining of the LPS on the surface of the host cells was demonstrated by immunofluorescence. The data show that phorbol ester-differentiated U937 cells restrict chlamydial growth strongly but not completely through a mechanism distinct from IDO-mediated tryptophan deprivation. The mechanisms of persistence of chlamydiae in monocytes, which differ considerably from those described for other cells, require further investigation.

IgG subclass-specific antibodies in Chlamydia pneumoniae infections.

IgG subclass-specific antibodies to Chlamydia pneumoniae and chlamydial lipopolysaccharide (LPS) were analysed in paired sera obtained from 15 patients with primary C. pneumoniae pneumonia and from 16 pneumonia patients with reinfection, as well as in single sera of 40 subjects with possible chronic C. pneumoniae infection and 40 healthy controls. The microimmunofluorescence (MIF) method was used to measure total IgG, IgM and IgG subclass-specific antibodies to C. pneumoniae protein antigens and enzyme immunoassay (EIA) to measure antibodies against the LPS antigen. By MIF, IgG1 antibodies to C. pneumoniae were demonstrated in all individuals of the 3 patient groups and also in all healthy controls. IgG2 subclass antibodies were not found by MIF. IgG3 antibodies were detected in 40% of patients with primary infection, in 31% of patients with reinfection, in 25% of those with chronic infection and in 8% of the controls. IgG4 antibodies were associated with acute C. pneumoniae infection and were found in 13% of primary infections and 31% of reinfections. The subclass pattern of LPS antibodies resembled that of protein antibodies measured by MIF: IgG1 was the most common subclass among the antibodies to LPS.

Bewertung vier unterschiedlicher serologischer Verfahren in der gynäkologischen Chlamydiendiagnostik

Genitale Chlamydieninfektionen persistieren bei Frauen und Männern über Jahre. Aufgrund der geringen Erregerzahl im Urogenitaltrakt kann der direkte Nachweis von Chlamydia trachomatis schwierig sein. In solchen Fällen stellt die Serologie eine hilfreiche diagnostische Ergänzung dar bei klinischem Verdacht auf eine Infektion oder Sterilität durch Tubenverschluß. Es wurden 178 Sterilitätspatientinnen (63 mit pelviskopisch nachgewiesenen verschlossenen, 115 mit offenen Tuben), 103 Frauen mit positivem Zervixabstrich, 100 Wöchnerinnen (6% positiver Zervixabstrich) und 100 Blutspender serologisch untersucht. Dabei wurden folgende Tests eingesetzt: LMikroimmunfluoreszenztest (MIF, Craystone), 2. ein kommerzieller Test mit Lipopolysaccharid (LPS) extrahierten Elementarkörperchen als Antigen, 3. ein käuflicher Chlamydienzelleinschluß-Test, 4. ein kommerzieller Test mit rekombinantem Chlamydien-LPS. In dieser Testreihenfolge lagen die positiven Vorhersagewerte für Tubenverschluß bei 71 %, 65%. 66%, 52% und für positiven Chlamydiennachweis im Zervixabstrich der Wöchnerinnen bei 40%, 40%, 21 % und 13%. Prinzipiell waren nur die Chlamydia trachomatis speziesspezifischen Tests (MIF-Test und LPS extrahierter Elementarkörperchen-Test) für die gynäkologische Chlamydiendiagnostik geeignet. Aufgrund der Kreuzreaktion mit Chlamydia pneumoniae sollte der Chla-mydienzelleinschluß-Test auf Hochprävalenzkollektive wie Sterilitätspatienten beschränkt bleiben. Der gattungsspezifische LPS-Test erwies sich als nicht hilfreich für den Gynäkologen. Eine Unterscheidung zwischen abgelaufener oder noch aktiver Chlamydia-trachomatis Infektion war mit keinem serologischen Test möglich.

Evaluation of the Vidas Chlamydia test to detect and verify Chlamydia trachomatis in urogenital specimens.

The Vidas Chlamydia test (CHL) is an automated enzyme-linked immunofluorescence assay for the detection of Chlamydia trachomatis. Positive and equivocal results are confirmed with a blocking assay. A mouse monoclonal antibody directed against the chlamydial lipopolysaccharides was used for the test. The CHL assay is widely used in Europe, but U.S. experience with it is limited. Three clinical test sites (The Arlington Hospital, Arlington, Va., Indiana University, Indianapolis, and the University of California, San Francisco) compared CHL with tissue culture (TC) for the identification of chlamydia in urogenital specimens (2,453 females and 850 males). True positives (TP) were defined as either TC positive or TC negative and CHL positive by a positive direct fluorescent-antibody assay or PCR test. Overall prevalence was 5.5% for females, 10.3% for male urethral swabs, and 10.7% for combined male TC urethral swabs and CHL with first catch urine (FCU) specimens. Compared to TP, CHL and TC had sensitivities of 89.6 and 94.1% with female cervical swabs and 90.9 and 86.4% with male urethral swabs, respectively. CHL sensitivity was 81.2 for male FCU specimens and 77.7% for matching male TC swabs. There were relatively few false-positive results, with all specificities being >99.4%. With the blocking assay, Vidas CHL specificity was >99.7%. However, male FCU specimen sensitivity was compromised because 9.2% (7 of 76) of the TP were initially positive but were not confirmed. An improvement in the Vidas blocking assay is needed before we can recommend its use with male urine. Alternatively, one could argue that the specificity of the test is so high that a confirmatory assay is not needed. For male and female swabs, the Vidas CHL assay has a performance that is similar to that of TC.

Effect of immunoglobulin G isotype on the infectivity of Chlamydia trachomatis in a mouse model of intravaginal infection.

It has been previously shown with an in vitro neutralization system that monoclonal antibodies (MAbs) to the major outer membrane protein (MOMP) of Chlamydia trachomatis, depending on the isotype of the MAb and the host cell used, can either neutralize or enhance the infectivity of this organism. MAbs to variable domain 4 (VD 4) of MOMP have been described that neutralize the infectivity of C. trachomatis when tested in a system in which either the host cell does not have detectable Fc gammaRIII receptors or complement is added to block the interaction of the MAb with the receptor. However, if Fc gammaRIII receptors are available, immunoglobulin G2b (IgG2b) MAbs to the VD 4 are able to enhance the infectivity of this pathogen. Two MAbs that recognize the sequence TLNPTIA in VD 4 of the MOMP but differ in isotype, E4 (IgG2b) and E21 (IgG1), were used to test whether in vivo the isotype of the MAb modulates the outcome of a vaginal infection in a murine model. A third MAb, CP33 (IgG2b), that recognizes the chlamydial lipopolysaccharide but does not neutralize infectivity of C. trachomatis, was also tested. Elementary bodies (EBs) of C. trachomatis, serovar E (BOUR), were pretreated with the three MAbs and were used to inoculate the vaginas of C3H/HeJ mice which had been pretreated with progesterone. Subsequently mice were monitored over a 5-week period with vaginal cultures. In the groups that were inoculated with EBs pretreated with MAbs directed to VD 4 of MOMP, there was a significant decrease (P < 0.05) in the number of mice infected. Only 30% of the mice were infected in the MAb E4-treated group, and 10% were infected in the MAb E21 group. This was in contrast to the groups inoculated with EBs pretreated with MAb CP33 and control untreated EBs, which resulted in 100 and 79% of the mice infected, respectively. Therefore, in this setting in which EBs were introduced in vivo coated with MAb, there was no enhancement of infection by IgG2b MAbs; rather, the results paralled the in vitro neutralization results, in which cells lacking Fc gammaRIII receptors were employed. Mice were also given the MAbs, as well as purified IgG as a control, by intraperitoneal injection before and after intravaginal inoculation with C. trachomatis. Despite relatively high levels of MAbs in serum and detectable levels of MAbs in the vagina at the time of infection, there was only modest protection in animals receiving MAb E21, with 60% of the mice infected in contrast to 90% of the mice receiving MAb E4, MAb CP33, and IgG. However, by the second week of infection compared to controls, there was a significant increase (P < 0.05) in the amount of chlamydiae recovered from the vaginas of mice that had received the two IgG2b MAbs, E4 and CP33. In summary, the presence of IgG2b MAbs directed to surface components of C. trachomatis at certain times during the course of infection may play a role in enhancing the infectivity of this pathogen.

Polymerase chain reaction for detection of Chlamydia trachomatis in conjunctival swabs

AIMS/BACKGROUNDOcular Chlamydia trachomatis infection in the west occurs as ophthalmia neonatorum, acquired from the mother, or adult paratrachoma which is also associated with current genital tract infection. Accurate rapid laboratory...

Lipopolysaccharide biosynthesis genes in koala type I Chlamydia: cloning and characterization

We showed in 1988 that there are two strains of Chlamydia psittaci which infect the koala (Phascolarctos cinereus). In order to further investigate the role of these chlamydial strains in pathogenesis, we have attempted to identify genes of koala type I strain chlamydia which are involved in the immunogenic response. Transformation of Escherichia coli with a plasmid containing a 6.3-kb fragment (pKOC-10) of C. psittaci DNA caused the appearance of a specific chlamydial lipopolysaccharide (LPS) epitope on the host strain. The smallest DNA fragment capable of inducing the expression of chlamydial LPS was an XbaI fragment, 2.4 kb in size (pKOC-5). DNA sequence analysis of the complete fragment revealed regions of high identity, at the amino acid level, to the gseA genes of C. pneumoniae, C. psittaci 6BC and C. trachomatis, and the kdtA gene of E. coli which code for transferases catalysing the addition of 3-deoxy-D-manno-octulosonic acid (Kdo) residues to lipid A. Two open reading frames (ORFs) of 1,314 and 501 nucleotides in size, within the 2.4-kb fragment, were evident, and mRNA species corresponding to these ORFs were detected by Northern analysis. Both ORF1 and ORF2 are required for the appearance of chlamydia-specific LPS on the surface of recombinant E. coli.

The microbicidal agent C31G inhibits Chlamydia trachomatis infectivity in vitro.

Safe and effective vaginal microbicidal compounds are being sought to offer women an independent method for protection against transmission of sexually acquired pathogens. The purpose of this study was to examine the efficacy of two formulations of one such compound, C31G, against Chlamydia trachomatis serovar E alone, its host epithelial cell (HEC-1B) alone, and against chlamydiae-infected HEC-1B cells. Preexposure of isolated, purified infectious chlamydial elementary bodies (EB) to C31G, at pHs 7.2 and 5.7, for 1 h at 4 degrees C resulted in reduced infectivity of EB for HEC-1B cells. Examination of the C31G-exposed 35S-EB on sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiographs and by Western blotting revealed a C31G concentration-dependent and pH-dependent destabilization of the chlamydial envelope, resulting in the release of chlamydial lipopolysaccharide and proteins. Interestingly, when the host human genital columnar epithelial cells were infected with chlamydiae and then exposed to dilute concentrations of C31G which did not alter epithelial cell viability, chlamydial infectivity was also markedly reduced. C31G gained access to the developing chlamydial inclusion causing damage to or destruction of metabolically active reticulate bodies as well as apparent alteration of the inclusion membrane, which resulted in premature escape of chlamydial antigen to the infected epithelial surface. These studies show that the broad-spectrum antiviral and antibacterial microbicide C31G also has antichlamydial activity.

Structure of the Monophosphoryl Lipid A Moiety Obtained from the Lipopolysaccharide of Chlamydia trachomatis

Monophosphoryl lipid A was prepared from the lipopolysaccharide of Chlamydia trachomatis, converted to the methyl ester, and fractionated by reverse-phase high-performance liquid chromatography. The peak fractions were collected and analyzed by mass spectrometry. Matrix-assisted laser desorption/ionization and liquid secondary ion mass spectrometry of the first of two major high-performance liquid chromatographic fractions showed multiple quasi-molecular ions of MNa+ at m/z 1780, 1794, 1808, 1822, and 1836. The positive-ion liquid secondary ion mass spectrometry spectrum also showed a minor series of peaks at m/z 1916, 1930, 1944, 1958, and 1971, consistent with the formation of matrix adducts with 3-nitrobenzyl alcohol. Oxonium ions representing the distal subunit were observed at m/z 1057, 1071, 1085, 1099, and 1113. The second fraction was similarly analyzed and found to contain structural homologs of the first fraction. Based on this study, the major lipid A component of chlamydial lipopolysaccharide is a glucosamine disaccharide that contains five fatty acids and a phosphate in the distal segment. Three fatty acyl groups are in the distal segment, and two are in the reducing end segment. The acyloxyacyl group is located in the distal segment in amide linkage. Two structural series, differing by 14 atomic mass units in the reducing subunit, were observed. Chlamydial lipid A is complex and consists of at least 20 homologous structural components. The relatively low potency of Chlamydia trachomatis lipopolysaccharide in activating lipopolysaccharide-responsive cells might be related to the unusual fatty acid composition of the lipid A moiety.

Demonstration of Chlamydia pneumoniae in the walls of abdominal aortic aneurysms.

Seroepidemiologic studies have indicated an association between chronic Chlamydia pneumoniae infection and coronary heart disease. The organism, which is a common respiratory pathogen, has been demonstrated in atherosclerotic lesions of the aorta and coronary arteries. Abdominal aortic aneurysms are frequently associated with atherosclerosis, and inflammation may actually be an important factor in aneurysmal dilatation. Hence it could be assumed that C. pneumoniae may play a role in maintaining an inflammation and triggering the development of aortic aneurysms. Specimens from abdominal aortic aneurysm were examined for the presence of C. pneumoniae by immunohistochemical analysis, the polymerase chain reaction amplifying omp 1 gene, transmission electron microscopy, and culture methods with histologically atherosclerosis-negative human aortic tissues used as a control group. Chlamydial lipopolysaccharide and C. pneumoniae specific antigens were found by immunohistochemistry in 12 and 8 of 12 aneurysm specimens, respectively, and C. pneumoniae DNA could be demonstrated in 6 of 6 aneurysm specimens studied. Furthermore electron microscopy revealed the presence of Chlamydia-like elementary bodies in three of four aneurysm specimens tested. None of the control samples gave positive reaction in the polymerase chain reaction, and C. pneumoniae antigens were not detected in any of them. C. pneumoniae is frequently found in the vessel wall of abdominal aortic aneurysm. The potential etiopathogenetic role of C. pneumoniae in the development of these aneurysms remains to be studied.

Ultrastructural and molecular analyses of the persistence ofChlamydia trachomatis(serovar K) in human monocytes

Previous studies have suggested that monocytes may play a role in the dissemination ofChlamydia trachomatis, and in establishment of persistent infection with this bacterium. Infection of cultured human peripheral blood monocytes withC. trachomatisserovar K produced persistent, non-productive infection. Transmission electron microscopy of such infected cultures revealed single or multipleChlamydiain monocyte inclusions over a culture period of 10 days. Those inclusions were aberrant, and normal reticulate bodies within the inclusions were not observed. Immunoelectron microscopy showed the chlamydial major outer membrane protein and lipopolysaccharide to be associated with the bacterial plasma membrane. Lipopolysaccharide was also identified in the monocyte cytoplasm. Molecular analyses of primary chlamydial rRNA transcripts demonstrated that the organism is viable and metabolically active within monocyte inclusions. However, attempts to overcome chlamydial growth arrest by incubation ofChlamydia-infected monocytes with tryptophan, and antibodies against alpha interferon, gamma interferon, or tumor necrosis factor, were all ineffective, suggesting that known mechanisms of growth inhibition do not hold in human monocytes. These observations indicate that infection of human peripheral blood monocytes withC. trachomatismay be involved in the genesis/maintenance of extra-urogenital inflammation, since non-culturable, metabolically active bacteria persist in those cells.

Structural requirements of synthetic oligosaccharides to bind monoclonal antibodies against Chlamydia lipopolysaccharide.

Monoclonal antibodies were generated against a synthetic glycoconjugate containing the trisaccharide alpha-Kdo-(2-->8)-alpha-Kdo-(2-->4)-alpha-Kdo (Kdo, 3-deoxy-D-manno-2-octulopyranosonic acid) which represents the genus-specific epitope of the lipopolysaccharide from the obligatory intracellular human pathogen Chlamydia. Antibodies of all immunoglobulin G isotypes were obtained and characterized by enzyme immunobinding and inhibition assays using the immunizing antigen as well as chemically synthesized derivatives of the Kdo trisaccharide. The latter contained (1) one of the three residues in beta- instead of alpha-linkage, (2) a Kdo residue the carboxyl group of which had been reduced to a CH2OH group (Kdo(C1-red)), or (3) changing the linkage of the terminal Kdo from 2-->8 to 2-->4. Only one compound, namely, alpha-Kdo-(2-->8)-alpha-Kdo(C1-red)-(2-->4)-alpha-Kdo exhibited binding to and inhibition of Kdo trisaccharide-specific antibodies, whereas all other compounds were not active. Structural and conformational investigations using NMR spectroscopy at high field on the allyl glycosides of the oligosaccharides 6-12 confirmed the conformational similarities between those structures 4, 5, and 10 which were able to bind to the antibodies investigated.