Chlamydial infection of polarized HeLa cells induces PMN chemotaxis but the cytokine profile varies between disseminating and non-disseminating strains.

While genital infections caused by Chlamydia trachomatis are generally asymptomatic, the density and pattern of inflammation varies considerably. The purpose of this study was to try to dissect the signalling in chlamydiae-infected epithelial cells that triggers innate responses and regulates polymorphonuclear neutrophil (PMN) chemotaxis. Polarized endocervical epithelial HeLa cells, grown in commercial inserts, were inoculated either with the non-disseminating (luminal) serovar E or the disseminating serovar L2. At 12-48 h after infection, the chambers were used in a quantitative chemotaxis assay, and cytokine production by infected cells was examined using cDNA microarray technology and confirmed by enzyme-linked immunosorbent assay (ELISA). Infection of HeLa cells with C. trachomatis E or L2 induced a strong and similar PMN chemotactic response, but larger amounts of interleukin (IL)-8 and IL-11 were released after infection with serovar L2. IL-6 was also produced in modest amounts after infection with either strain, but no IL-1alpha or tumour necrosis factor (TNF)-alpha was detected in any of the culture supernatants tested. IL-11 did not appear to influence the PMN response to chlamydial infection, but secretion of large amounts of this anti-inflammatory cytokine, mainly active on macrophages, in the very early stages of the infection may allow C. trachomatis to escape some innate defences to establish infection.

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.

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.

Chlamydial elementary bodies are translocated on the surface of epithelial cells.

Infection of eukaryotic cells by intracellular pathogens such as chlamydia requires attachment to the host cell surface. Chlamydia are thought to attach to the tips of microvilli in confluent monolayers of polarized cells. In vitro evidence obtained from migrating epithelial cells suggested that during healing the route of pathogen uptake might be different from that in intact epithelia. The small size of infectious chlamydial elementary bodies (approximately 0.3 microm in diameter) has made it difficult, however, to analyze the early stages of pathogen-host cell interaction in living cells by conventional microscopy. Contrast-enhanced video microscopy was therefore used to examine the earliest events of host-pathogen interaction and test the hypothesis that chlamydial uptake into the healing epithelia can involve translocation over the host cell surface. Observations made in this way were validated by scanning and immunofluorescence microscopy. These studies revealed two fates for chlamydiae taken onto the lamellipodial surface: 1) some chlamydiae were moved in a random fashion on the cell surface or were detached into the culture medium, whereas 2) other chlamydiae were translocated across the lamellipodium in a highly directed manner toward the microvillous perinuclear region. After internalization, these latter chlamydiae were found within intracellular inclusions, which demonstrated that this route of attachment and location of uptake resulted in productive growth.

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.

Delivery of azithromycin to Chlamydia trachomatis-infected polarized human endometrial epithelial cells by polymorphonuclear leucocytes.

An in-vitro model was designed to evaluate whether polymorphonuclear leucocytes (PMN) loaded with azithromycin could migrate and deliver the antibiotic in a bioactive form to chlamydia inclusions in polarized human endometrial epithelial (HEC-1B) cells infected with Chlamydia trachomatis. PMN chemotaxis through the extracellular matrix and between infected epithelial cells was readily observed if the HEC-1B cells had been infected with chlamydiae for 36 or 48 h. Inclusions in infected epithelial cells exposed to PMN loaded with azithromycin were initially distinguished by deformed reticulate bodies and an excessive amount of chlamydial outer membrane vesicles. As the amount of PMN-delivered antibiotic increased, chlamydial inclusions were filled with large cell envelope 'ghosts' which were the remnants of lysed reticulate bodies. The lethal effect of azithromycin was confirmed by a reduction in the viability of infectious progeny. Our results demonstrate that the damage to chlamydiae was due to transport and delivery of azithromycin by PMN to infected genital epithelial cells. When infected HEC-1B cells were exposed to PMN not loaded with the antibiotic, chlamydial morphology was not obviously affected yet few viable progeny could be recovered. In this case, PMN-induced damage to host epithelial cells probably interrupted chlamydial nutrient acquisition and subsequent maturation and formation of infectious progeny.

Accelerated development of genital Chlamydia trachomatis serovar E in McCoy cells grown on microcarrier beads.

Chlamydia trachomatis serovar E is a major cause of bacterially-acquired sexually transmitted infections. Stock cultures of these obligate intracellular bacteria are often propogated in McCoy cells. We recently reported that greater infectious titers of chlamydiae could be obtained if the McCoy cells were cultured on collagen-coated microcarrier beads versus plastic flasks, although the reason for the difference in efficiency was not clear. This study analyzed the development of C. trachomatis grown in McCoy cells by the two methods. Transmission electron microscopy analysis revealed an accelerated chlamydial development, with maturation of reticulate bodies into elementary bodies sooner in McCoy cells grown on the porous substratum. Comparison of particle counts versus infectivity titers indicated the production of fewer numbers of elementary bodies but which were highly infectious sooner from the infected McCoy cell-microcarrier bead cultures than from duplicate infected McCoy cell cultures grown in plastic tissue culture flasks.

Effect of clinically relevant culture conditions on antimicrobial susceptibility of Chlamydia trachomatis.

Infection of polarized human endometrial-gland epithelial cells obtained at hysterectomy with Chlamydia trachomatis serovar E may provide a relevant in vitro model for studies of pharmacokinetics in genital chlamydial infections. The minimal bactericidal concentration of azithromycin against C. trachomatis was lower in this model than in studies with nonpolarized cells (0.125 and 0.5 mg/L, respectively). Polarized cells also internalized more azithromycin over 24 hours. The results indicate that the eradication of chlamydial infections may be difficult to prove by antigen detection methods: the persistence of chlamydial envelope material within the intracellular vacuoles of azithromycin-treated cells may lead to a false-positive diagnosis of persisting chlamydial infection.

In-vitro activity of azithromycin on Chlamydia trachomatis infected, polarized human endometrial epithelial cells.

The in-vitro activity of azithromycin on Chlamydia trachomatis infected human endometrial epithelial cells, both primary and transformed cells growing in a polarized and non-polarized orientation, was analyzed. Addition of azithromycin two hours after adsorption inoculation with continued exposure until 72 h gave an MIC90 and MBC90 of 0.063 and 0.5 mg/L, respectively. In addition, the MBC results were more pronounced in infected cells growing in a polarized orientation. Numerous small fluorescent 'spots' (presumed small abnormal inclusions) were visible in the infected cells exposed to MIC concentrations of azithromycin. Immuno-transmission electron microscopy examination revealed intracellular inclusions filled with chlamydial envelope ghosts. Since standard diagnostic antigen detection methods use anti-envelope antibodies, the aberrant envelope-filled inclusions might be interpreted as viable inclusions by fluorescent microscopy and result in high false positive readings. To simulate treatment of an infected patient, azithromycin was added at 18 h to infected cells containing many reticulate bodies and exposure continued for 54 h after which killing of chlamydiae was seen. The use of polarized human cells may offer a more relevant in-vitro model system for examining the efficacy of antimicrobial action.

Eukaryotic cells grown on microcarrier beads offer a cost-efficient way to propagate Chlamydia trachomatis.

The use of microcarrier cell culture as a method for the in vitro propagation of the obligate intracellular bacterial parasite, Chlamydia trachomatis, is described. The microcarrier beads proved to be a more cost-effective means to propagate C. trachomatis than traditional tissue culture flasks or roller bottles without sacrificing yields or infectivity. In addition, microcarrier cell culture was found to be a much simpler technique to study the intracellular development of these bacteria.

Recombinant Escherichia coli clones expressing Chlamydia trachomatis gene products attach to human endometrial epithelial cells.

To identify Chlamydia trachomatis genes involved in attachment to host cells, a chlamydial genomic library was screened on the basis of binding characteristics by two methods. In the whole-cell screen, individual recombinant Escherichia coli clones were assayed for adherence to eukaryotic cells. In the membrane-binding screen, each recombinant colony of E. coli was treated with CHCl3 and assayed for binding to purified, 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS)-solubilized, 35S-labeled eukaryotic membrane material. Initial screening with McCoy cells was refined by using HEC-1B cells, a human endometrial epithelial cell line, which discriminate among recombinants adhering to McCoy cells. Some recombinants demonstrate significantly greater adherence to HEC-1B cells than to McCoy cells and appear, by transmission electron microscopy, to associate with electron-dense areas of the epithelial cell plasma membrane, resembling coated pits. Recombinants positive by one or both screening methods were examined by Southern and Western (immunoblot) analyses, which revealed the presence of chlamydial sequences inserted in the plasmids and the expression of novel 18-, 28-, and approximately 82 kDa, and perhaps of 18 Maxicell analysis of selected recombinants confirmed that the proteins of 28 and approximately 82 kDa, and perhaps of 18 kDa, are plasmid encoded. Antiserum generated against the recombinant approximately 82-kDa protein reacted in Western analysis with a similar-sized protein from C. trachomatis serovar E elementary bodies (EB) and reticulate bodies, serovar L2 EB, and C. psittaci EB. E. coli JM109(pPBW58) contains a 6.7-kb plasmid insert which encodes proteins of all three sizes. Under a number of different conditions in the whole-cell attachment assay--i.e., at 4 degrees C, in Ca(2+)- and Mg(2+)-free medium, in the presence of trypsin or dextran sulfate, and with rabbit aortic endothelial cells--the binding specificity of JM109(pPBW58) parallels that of C. trachomatis EB. Finally, the adherence phenotype of E. coli JM109(pPBW58) correlates directly with the presence of the recombinant plasmid; the phenotype is lost concurrently with loss of the recombinant plasmid, and the into E. coli JM109. The role of the 18-, 28-, and approximately 82-kDa proteins in mediating attachment, whether they act in concert as a complex or individually, has yet to be determined.

Value of written health information in the general practice setting.

The value of a library for patients as a way of providing written health information in the general practice setting has been investigated using a questionnaire. In 15 months, 243 books were borrowed from one library. Each book contained a questionnaire and 163 questionnaires were returned (67%). It was found that 106 respondents (65%) would not have sought the information elsewhere and 159 respondents (98%) found the books they had read to be very useful or of some use. The perceived level of anxiety after reading was raised in nine respondents (6%) but reduced in 71 (44%). Patients read books on 53 separate topics overall, although 73 respondents (45%) read on 10 recurring topics. A patients' library thus enables most patients to gain useful information from their general practice without increasing their anxiety.

Entry of genital Chlamydia trachomatis into polarized human epithelial cells.

To study the initial invasion process(es) of genital chlamydiae, a model system consisting of hormonally maintained primary cultures of human endometrial gland epithelial cells (HEGEC), grown in a polarized orientation on collagen-coated filters, was utilized. After Chlamydia trachomatis inoculation of the apical surface of polarized HEGEC, chlamydiae were readily visualized, by transmission electron microscopy, in coated pits and coated vesicles. This was true for HEGEC maintained in physiologic concentrations of estrogen (proliferative phase) and of estrogen plus progesterone (secretory phase), despite the finding that association of chlamydiae with secretory-phase HEGEC is significantly reduced (P = 0.025; A.S. Maslow, C.H. Davis, J. Choong, and P.B. Wyrick, Am. J. Obstet. Gynecol. 159:1006-1014, 1988). In contrast, chlamydiae were rarely observed in the clathrin-associated structures if the HEGEC were cultured on plastic surfaces. The same pattern of coated pit versus noncoated pit entry was reproducible in HeLa cells. The quantity of coated pits associated with isolated membrane sheets derived from HeLa cells, grown on poly-L-lysine-coated cover slips in medium containing the female hormones, was not significantly different as monitored by radiolabeling studies and by laser scanning microscopy. These data suggest that culture conditions which mimic in vivo cellular organization may enhance entry into coated pits for some obligate intracellular pathogens.

In vitro activity of nonoxynol-9 on McCoy cells infected with Chlamydia trachomatis.

Nonoxynol-9, a nonionic detergent and active ingredient in spermicidal contraceptives, has been reported to have anti-chlamydial properties. However, in this study exposure of elementary bodies of Chlamydia trachomatis serovar E to nonoxynol-9 (12.5-10,000 micrograms/ml) had no effect on chlamydial infectivity. In contrast, uninfected McCoy cells incubated with increasing concentrations of nonoxynol-9 over 72 h displayed dose-related cytotoxicity. When infected McCoy cells were exposed to nonoxynol-9, the developing chlamydial inclusions did not stain with iodine even though they were similar in number and appearance to the inclusions in unexposed, infected monolayers. Transmission electron microscopy of nonoxynol-treated, infected cells revealed apparent damage to the inclusion membrane and reticulate bodies within. The infectivity of the chlamydiae in the iodine-negative inclusions on subpassage was only 0.3%. We conclude that the primary action of nonoxynol-9 is on the McCoy cell and that there may be secondary effects on the intracellular parasite.

Examination of various cell culture techniques for co-incubation of virulent Treponema pallidum (Nichols I strain) under anaerobic conditions.

Treponema pallidum (Nichols virulent) was incubated with and without cells in cell culture medium reduced to -275 mV Ecal, pH 7.3, under deoxygenated conditions. Five to ten percent of the treponemes attached to cells and remained motile for at least 120 h in cell-treponeme systems of co-incubation. Virulent treponemes could be detected after 120 to 144 h in the supernatant fluids of cell-treponeme co-incubation cultures and in cell-free tubes containing medium harvested from aerobically cultivated mammalian cells. Medium supplemented with ox serum ultrafiltrate, pyruvate, and sodium thioglycolate and gas mixtures containing H2 and CO2 enhanced treponemal survival. Increases in treponemal numbers were observed using dark-field microscopy but were not substantiated using the rabbit lesion test. Continuous passage of the treponeme was not achieved in vitro.

Retention of motility of Treponema pallidum (Nichols virulent strain) in an anaerobic cell culture system and in a cell-free system.

Optimum parameters for retention of motility of Treponema pallidum (Nichols virulent strain) were found by anaerobic co-incubation of the treponeme with rat glial cells and anaerobic incubation in spent medium obtained from glial cells originally grown aerobically.