Chlamydia trachomatis infection of human fallopian tube organ cultures

The pathogenic events that precede Chlamydia trachomatis salpingitis in the human fallopian tube have not been fully described. We used a model of human fallopian tubes in organ culture (HFTOC) infected with strain E/UW-5/CX of C. trachomatis to study these events. The model supported sustained C. trachomatis infection as demonstrated by recovery of viable C. trachomatis from medium and tissue over 5-7 d. However, the level of infectivity was low. Maximal infection occurred at 72 h after initial inoculation. In contrast to gonococcal infection of the HFTOC, C. trachomatis did not damage overall ciliary function of HFTOC. However, a local direct cytotoxic effect characterized by loss of microvilli and disruption of cell junctions was noted when multiple chlamydial elementary bodies attached to mucosal cells. Beginning at 24 h, and continuing throughout the course of C. trachomatis infection of HFTOC, ruptured epithelial cells releasing elementary bodies were noted. Chlamydial inclusions were seen in the mucosa by 72 h in approximately 6% of both ciliated and nonciliated epithelial cells. Mucosal inclusions contained all forms of the C. trachomatis developmental cycle. These data suggest that factors present in the human fallopian tube may limit susceptibility to chlamydial infection but support the use of the HFTOC model in the study of the pathogenesis of C. trachomatis salpingitis.     

CCDC65 Mutation Causes Primary Ciliary Dyskinesia with Normal Ultrastructure and Hyperkinetic Cilia

Background

Primary ciliary dyskinesia (PCD) is a genetic disorder characterized by impaired ciliary function, leading to chronic sinopulmonary disease. The genetic causes of PCD are still evolving, while the diagnosis is often dependent on finding a ciliary ultrastructural abnormality and immotile cilia. Here we report a novel gene associated with PCD but without ciliary ultrastructural abnormalities evident by transmission electron microscopy, but with dyskinetic cilia beating.

Methods

Genetic linkage analysis was performed in a family with a PCD subject. Gene expression was studied in Chlamydomonas reinhardtii and human airway epithelial cells, using RNA assays and immunostaining. The phenotypic effects of candidate gene mutations were determined in primary culture human tracheobronchial epithelial cells transduced with gene targeted shRNA sequences. Video-microscopy was used to evaluate cilia motion.

Results

A single novel mutation in CCDC65, which created a termination codon at position 293, was identified in a subject with typical clinical features of PCD. CCDC65, an orthologue of the Chlamydomonas nexin-dynein regulatory complex protein DRC2, was localized to the cilia of normal nasal epithelial cells but was absent in those from the proband. CCDC65 expression was up-regulated during ciliogenesis in cultured airway epithelial cells, as was DRC2 in C. reinhardtii following deflagellation. Nasal epithelial cells from the affected individual and CCDC65-specific shRNA transduced normal airway epithelial cells had stiff and dyskinetic cilia beating patterns compared to control cells. Moreover, Gas8, a nexin-dynein regulatory complex component previously identified to associate with CCDC65, was absent in airway cells from the PCD subject and CCDC65-silenced cells.

Conclusion

Mutation in CCDC65, a nexin-dynein regulatory complex member, resulted in a frameshift mutation and PCD. The affected individual had altered cilia beating patterns, and no detectable ultrastructural defects of the ciliary axoneme, emphasizing the role of the nexin-dynein regulatory complex and the limitations of certain methods for PCD diagnosis.     

Functional activity of ciliated outgrowths from cultured human nasal and tracheal epithelia

Primary cultures of respiratory epithelium were produced as outgrowths from human fetal and adult tracheal and nasal polyp explants. Video recordings of the epithelial cell outgrowths were carried out after 5 days of culture and the ciliary beating frequency was analyzed by using a video technique. Uniform fields of differentiated ciliated cells were observed near the edge of the explant. In the transition region of the outgrowth from the explant to the outgrowth periphery, isolated ciliated cells were present, as well as cells with fused cilia. The ciliary beating frequency of the outgrowth of well-differentiated ciliated cells (13.5 +/- 1.4 Hz) was significantly higher (p less than 0.001) than the beating frequency of both the explant (11.9 +/- 0.7 Hz) and the ciliated cells with fused cilia (9.8 +/- 1.7 Hz). The same differentiation stages and functional activities were observed in the outgrowth cultures, whatever their origin. These in vitro models are comparable with each other and therefore could be useful for studying the ciliogenesis and functional activity of the human respiratory epithelium.     

Proliferation,differentiation and ciliary beating of human respiratory ciliated cells in primary culture

Summary The growth, differentiation, ciliary beating pattern and frequency of human respiratory ciliated cells in primary culture were studied by scanning and transmission electron microscopy and by videomicroscopy. The epithelial cells were obtained as outgrowth from explants of adult nasal polyps. When the explants were grown on type-I and type-IV collagen substrates in a standard serum-free, hormone-supplemented medium, a high percentage of ciliated cells (range 29±5% to 37±6%) was present within 2 days of culture. After 5 days of culture, the percentage of ciliated cells near the explant was 51±5%. Most of the cultured ciliated cells (85%) were characterized by individual cilia showing a coordinated movement during the beat cycle and a beating frequency (13.3±1.3 Hz) similar to that reported in vivo. In the other 15% of the ciliated cells, the dyskinetic cilia were aggregated into clumps and characterized by a rigid and planar bending movement and a lower (P<0.01) beating frequency (10.7±1.4 Hz). It is suggested that the latter type of cell, already described during fetal development, might be an intermediate type of ciliated cell which appears temporarily during the surface respiratory epithelial differentiation.     

Interleukin-1 is the initiator of Fallopian tube destruction during Chlamydia trachomatis infection

Chlamydia trachomatis infection is associated with severe Fallopian tube tissue damage leading to tubal infertility and ectopic pregnancy. To explore the molecular mechanisms behind infection an ex vivo model was established from human Fallopian tubes and examined by scanning electron microscopy and immunohistochemistry. Extensive tissue destruction affecting especially ciliated cells was observed in C. trachomatis infected human Fallopian tube organ culture. Interleukin-1 (IL-1) produced by epithelial cells was detected after infection. Addition of IL-1 receptor antagonist (IL-1RA) completely eliminated tissue destruction induced by C. trachomatis. The anti-inflammatory cytokine IL-10 reduced the damaging effect of C. trachomatis infection, however, to a lesser extent than IL-1RA. Furthermore, IL-1 was found to induce IL-8, a neutrophil attractant, using a signal transduction pathway involving p38 MAP kinase. Consequently, IL-1 has the potential to generate a cellular infiltrate at the site of infection in vivo. Blocking the IL-1 receptors by IL-1RA eliminated tissue destruction and cytokine production. Hence, these studies show the importance of IL-1 in initiating the tissue destruction observed in the Fallopian tube following C. trachomatis infection. Because leukocytes are absent in the ex vivo model, this study strongly indicates that IL-1 is the initial proinflammatory cytokine activated by C. trachomatis infection.     

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-1α or tumour necrosis factor (TNF)-α 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.     

Cell death, BAX activation, and HMGB1 release during infection with Chlamydia

Infection by a number of Chlamydia species leads to resistance of the host cell to apoptosis, followed by induction of host-cell death. In a population of infected cells that displays protection against staurosporine-induced apoptosis among the adherent cells, we find that cells that had been recovered from the supernatant share characteristics of both apoptosis and necrosis, as assayed by the propidium iodide (PI)-annexin V double-labeling technique. Cell death was observed in both an epithelial cell line and primary fibroblasts, although the primary cells had a higher propensity to die through apoptosis than the immortalized cell line. Staurosporine-mediated activation of the pro-apoptotic BCL-2 family member, BAX, was inhibited in the epithelial cell line infected for 32 h with the lymphogranuloma venereum (LGV/L2) but not the murine pneumonitis (MoPn) strain of C. trachomatis, but inhibition of staurosporine-mediated BAX activation disappeared after 48 h of infection with the LGV/L2 strain. Conversely, infection with MoPn (C. muridarum) but not LGV/L2 led to BAX activation after 72 h, as previously reported for shorter (48 h) infection with the guinea pig inclusion conjunctivitis (GPIC) serovar of C. psittaci (C. caviae). These results suggest that the ability to inhibit staurosporine-mediated BAX activation or to activate BAX due to the infection itself may vary as a function of the chlamydial strain. Interestingly, both the epithelial cells and the fibroblasts also released high mobility group box 1 protein (HMGB1) during infection, although much less HMGB1 was released from fibroblasts, consistent with the higher level of apoptosis observed in the primary cells. HMGB1 is released preferentially by necrotic or permeabilized viable cells, but not apoptotic cells. In the extracellular space, HMGB1 promotes inflammation through interaction with specific cell-surface receptors. Higher levels of HMGB1 were also measured in the genital-tract secretions of mice infected vaginally with C. trachomatis, compared to uninfected controls. These results suggest that cells infected with Chlamydia release intracellular factors that may contribute to the inflammatory response observed in vivo.     

Toxic effects of sulfur mustard on respiratory epithelial cells in culture

Sulfur mustard (SM) is known to induce cutaneous injury and to cause acute damage to the respiratory tract. Although skin vesication has been demonstrated on human epidermal keratinocytes in culture, no study has been carried out to analyze the effects of SM on the ultrastructural and functional activity of surface respiratory epithelial cells. To evaluate this SM toxicity, we developed an in vitro model of respiratory epithelial cells in primary culture. The study was performed on surface epithelial cells from rabbit trachea cultured according to the explant-outgrowth technique. The functional activity of the cultures was evaluated by measuring the ciliary beating frequency (CBF) of the ciliated cells with a videomicroscopic method. The morphological aspects of the cells were analyzed by light and electron microscopy. Addition of 0.1 mM SM directly into the culture medium produced a sudden and irreversible CBF inhibition, first observed after 2 hr on the ciliated cells of the outgrowth periphery. The arrest of the ciliary beating progressively reached the whole surface of the outgrowth and was simultaneously observed with a detachment of the outgrowth cells. It began at the outgrowth border, leading to the exfoliation of cell sheets, and then to the whole culture after 48 hr. Morphological damage was expressed by intense vacuolisation and disorganization of cytoplasmic and nuclear structures. These findings suggest that the detachment of the respiratory epithelial cells from the matrix represents a major toxic effect of 0.1 mM SM. SM dramatically affects the viability of respiratory epithelial cells in culture. Moreover, the sudden CBF inhibition is more likely due to the death of the ciliated cells than to a specific ciliotoxic effect of SM.Abbreviations CBF ciliary beating frequency - HEPES N2-hydroxyethylpiperazine-N'2ethanesulfonic acid - PBS phosphate buffer saline - SM sulfur mustard - TEM transmission electron microscopy     

Enzyme-ultracytochemical study of adenylate and guanylate cyclases in normal and pathologic human nasal mucosa

The ultracytochemical localization of adenylate cyclase (AC) and guanylate cyclase B (GC-B) and C (GC-C) activity was studied after stimulation with pituitary adenylate cyclase activating peptide, C-type natriuretic peptide and guanylin, respectively, in normal human respiratory nasal mucosa and mucosa of nasal polyps. To demonstrate these enzymatic activities, we employed enzyme-ultracytochemical methods for electron microscopy. Both normal and pathologic nasal mucosa contained AC, GC-B and GC-C activity. In the upper portion of respiratory epithelium, the enzymes were detected on ciliary and microvillar membranes. In ciliary membranes, GC-B was the predominant form expressed. In goblet cells and in glands of the lamina propria, enzymatic activities were localized mainly on plasma membranes and on membranes lining secretory granules. The results did not reveal any evident differences between the enzymatic activities in normal and pathological nasal mucosa and suggest complementary activities for these enzymes and their stimulators in the regulation of mucociliary transport and glandular secretion.     

Mucociliary Clearance Defects in a Murine In Vitro Model of Pneumococcal Airway Infection

Mucociliary airway clearance is an innate defense mechanism that protects the lung from harmful effects of inhaled pathogens. In order to escape mechanical clearance, airway pathogens including Streptococcus pneumoniae (pneumococcus) are thought to inactivate mucociliary clearance by mechanisms such as slowing of ciliary beating and lytic damage of epithelial cells. Pore-forming toxins like pneumolysin, may be instrumental in these processes. In a murine in vitro airway infection model using tracheal epithelial cells grown in air-liquid interface cultures, we investigated the functional consequences on the ciliated respiratory epithelium when the first contact with pneumococci is established. High-speed video microscopy and live-cell imaging showed that the apical infection with both wildtype and pneumolysin-deficient pneumococci caused insufficient fluid flow along the epithelial surface and loss of efficient clearance, whereas ciliary beat frequency remained within the normal range. Three-dimensional confocal microscopy demonstrated that pneumococci caused specific morphologic aberrations of two key elements in the F-actin cytoskeleton: the junctional F-actin at the apical cortex of the lateral cell borders and the apical F-actin, localized within the planes of the apical cell sides at the ciliary bases. The lesions affected the columnar shape of the polarized respiratory epithelial cells. In addition, the planar architecture of the entire ciliated respiratory epithelium was irregularly distorted. Our observations indicate that the mechanical supports essential for both effective cilia strokes and stability of the epithelial barrier were weakened. We provide a new model, where - in pneumococcal infection - persistent ciliary beating generates turbulent fluid flow at non-planar distorted epithelial surface areas, which enables pneumococci to resist mechanical cilia-mediated clearance.     

Hematopoietic cells are required to initiate a Chlamydia trachomatis-specific CD8+ T cell response

Chlamydia trachomatis is a global human pathogen causing diseases ranging from blinding trachoma to pelvic inflammatory disease. To explore how innate and adaptive immune responses cooperate to protect against systemic infection with C. trachomatis L2, we investigated the role of macrophages (Mphi) and dendritic cells (DCs) in the stimulation of C. trachomatis-specific CD8(+) T cells. We found that C. trachomatis infection of Mphi and DCs is far less productive than infection of nonprofessional APCs, the typical targets of infection. However, despite the limited replication of C. trachomatis within Mphi and DCs, infected Mphi and DCs process and present C. trachomatis CD8(+) T cell Ag in a proteasome-dependent manner. These findings suggest that although C. trachomatis is a vacuolar pathogen, some Ags expressed in infected Mphi and DCs are processed in the host cell cytosol for presentation to CD8(+) T cells. We also show that even though C. trachomatis replicates efficiently within nonprofessional APCs both in vitro and in vivo, Ag presentation by hematopoietic cells is essential for initial stimulation of C. trachomatis-specific CD8(+) T cells. However, when DCs infected with C. trachomatis ex vivo were adoptively transferred into naive mice, they failed to prime C. trachomatis-specific CD8(+) T cells. We propose a model for priming C. trachomatis-specific CD8(+) T cells whereby DCs acquire C. trachomatis Ag by engulfing productively infected nonprofessional APCs and then present the Ag to T cells via a mechanism of cross-presentation.     

Isolation, in silico characterization and chromosomal localization of a group of cDNAs from ciliated epithelial cells after in vitro ciliogenesis

Background

Immotile cilia syndrome (ICS) or primary ciliary dyskinesia (PCD) is an autosomal recessive disorder in humans in which the beating of cilia and sperm flagella is impaired. Ciliated epithelial cell linings are present in many tissues. To understand ciliary assembly and motility, it is important to isolate those genes involved in the process.

Results

Total RNA was isolated from cultured ciliated nasal epithelial cells after in vitro ciliogenesis and expressed sequenced tags (ESTs) were generated. The functions and locations of 63 of these ESTs were derived by BLAST from two public databases. These ESTs are grouped into various classes. One group has high homology not only with the mitochondrial genome but also with one or more chromosomal DNAs, suggesting that very similar genes, or genes with very similar domains, are expressed from both mitochondrial and nuclear DNA. A second class comprises genes with complete homology with part of a known gene, suggesting that they are the same genes. A third group has partial homology with domains of known genes. A fourth group, constituting 33% of the ESTs characterized, has no significant homology with any gene or EST in the database.

Conclusions

We have shown that sufficient information about the location of ESTs could be derived electronically from the recently completed human genome sequences. This strategy of EST localization should be significantly useful for mapping and identification of new genes in the forthcoming human genome sequences with the vast number of ESTs in the dbEST database.     

Effect of Pseudomonas aeruginosa rhamnolipids on mucociliary transport and ciliary beating.

Pseudomonas aeruginosa rhamnolipid causes ciliostasis and cell membrane damage to rabbit tissue, is a secretagogue in cats, and inhibits epithelial ion transport in sheep tissue. It could therefore perturb mucociliary clearance. We have investigated the effect of rhamnolipid on mucociliary transport in the anesthetized guinea pig and guinea pig and human respiratory epithelium in vitro. Application of rhamnolipid to the guinea pig tracheal mucosa reduced tracheal mucus velocity (TMV) in vivo in a dose-dependent manner: a 10-microgram bolus caused cessation of TMV without recovery; a 5-micrograms bolus reduced TMV over a period of 2 h by 22.6% (P = 0.037); a 2.5-microgram bolus caused no overall changes in TMV. The ultrastructure of guinea pig tracheal epithelium exposed to 10 micrograms of rhamnolipid in vivo was normal. Application of 1,000 micrograms/ml rhamnolipid had no effect on the ciliary beat frequency (CBF) of guinea pig tracheal rings in vitro after 30 min, but 250 micrograms/ml stopped ciliary beating after 3 h. Treatment with 100 micrograms/ml rhamnolipid caused immediate slowing of the CBF (P less than 0.01) of human nasal brushings (n = 7), which was maintained for 4 h. Mono- and dirhamnolipid had equivalent effects. The CBF of human nasal turbinate organ culture was also slowed by 100 micrograms/ml rhamnolipid, but only after 4 h (CBF test, 9.87 +/- 0.41 Hz; control, 11.48 +/- 0.27 Hz; P less than 0.05, n = 6), and there was subsequent recovery by 14 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temperature effect on the ciliary beat frequency of human nasal and tracheal ciliated cells.

Even though all human respiratory cilia are similar in structure, they experience a wide range of temperatures between the initial part of the nasal fossae which behave as heat exchangers and the inferior part of the trachea, particularly when we inhale exceedingly cold or hot air. The ciliary beat frequency of ciliated cells from human nasal mucosa and from bronchial mucosa averages 8 Hz when measured at room temperature. In the present study we compared the ciliary beat frequency of human cells from nasal and tracheal mucosa brushings at different temperatures from 5 degrees C to 50 degrees C using two different techniques, ex vivo and in vitro: ex vivo in culture medium less than 24 h after sampling and in vitro after demembranation and reactivation according to a standard procedure developed in our laboratory. Measuring the ATP-reactivated ciliary beat frequency allowed us to check the thermal parameters of the dynein ATPase and all the axonemal machinery. No significant difference in frequency was observed between nasal fossae cilia and tracheal cilia when comparing extreme temperatures in both experimental procedures.     

TGF-beta 1 downregulates CFTR expression and function in nasal polyps of non-CF patients

Nasal polyposis is a chronic inflammatory disease of the upper airways. It has been suggested that ion transports and CFTR expression could be modified in epithelial cells from nasal polyps of non-cystic fibrosis patients. We compared human nasal epithelial cells from nasal polyps (NP) with control nasal mucosa (CM). The level of CFTR mRNA was studied by Northern blot analysis and protein expression was studied by immunoprecipitation both ex vivo and in vitro in primary cultures of human nasal epithelial cells at the air-liquid interface. Ion transports were evaluated by short-circuit measurements in vitro. CFTR gene and protein expressions were significantly decreased in NP native tissues and in culture on day 4, when a global defect of ion transports was observed in NP cultures, but not in CM. We evaluated the effect of transforming growth factor (TGF)-beta 1 on CFTR expression and function in NP cultures on day 14 and showed, for the first time, that TGF-beta 1 was able to significantly downregulate the level of CFTR mRNA and cAMP-dependent current in NP cultures. Finally, we showed that the effects of TGF-beta 1 on ion transports could be reversed after 48-h removal of TGF-beta1 in NP cultures. In conclusion, our data strongly suggest that chronic inflammation in nasal polyposis downregulates CFTR gene and protein expression.     

An in vitro model of Chlamydia trachomatis infection in the regenerative phase of the human endometrial cycle

An in vitro model of the regenerative phase of the human endometrial cycle was developed in order to study the growth of Chlamydia trachomatis during the period following menses. Glandular epithelial fragments were prepared from curettings of endometria and explanted onto coated substrata. Epithelial cells migrated rapidly from the explant in a fashion which closely mimicked the regeneration of the surface epithelium after menses. The cultures were then experimentally infected with C. trachomatis serotype E at various times during formation of the outgrowth. Chlamydial inclusions developed both within the explants and in the outgrowing epithelial sheets. They were also found in isolated epithelial and non-epithelial cells. However, the most striking feature of chlamydial inclusion development within these cultures was the tendency for inclusions to be located in cells at the periphery of the epithelial sheets. This was partly due to the failure of the cells within the sheets to bind chlamydiae after centrifugation of the organisms onto the culture and partly due to a phenomenon similar to phagokinesis. During this process infectious chlamydial particles were cleared from the substratum by migrating cells with free motile edges, which occasionally led to internalization and inclusion development within these cells.     

Induction of HLA class I-restricted CD8+ CTLs specific for the major outer membrane protein of Chlamydia trachomatis in human genital tract infections.

HLA class I-restricted CD8+ CTLs specific for the major outer membrane protein (MOMP) of Chlamydia trachomatis are present in the peripheral blood of humans who acquired genital tract infections with the organism. Three HLA-A2-restricted epitopes and two HLA-B51-restricted epitopes were identified in serovar E-MOMP. One of the five epitopes spans a variable segment of MOMP and is likely a serovar E-specific epitope. The other four epitopes are localized in constant segments and are C. trachomatis species specific. CTL populations specific for one or more of the four constant segment epitopes were isolated from all 10 infected subjects tested, regardless of infecting serovars, but from only one of seven uninfected subjects tested. The CTLs failed to recognize corresponding peptides derived from Chlamydia pneumoniae MOMP, further suggesting that they indeed resulted from genital tract infections with C. trachomatis. Significantly, ME180 human cervical epithelial cells productively infected with C. trachomatis were killed by the MOMP peptide-specific CTLs. Further investigations of the ability of such CTLs to lyse normal infected epithelial cells and their presence at inflamed sites in the genital tract will help understand the protective or pathological role of CTLs in chlamydial infections. The MOMP CTL epitopes may be explored as potential components of a subunit vaccine against sexually transmitted diseases caused by C. trachomatis. Moreover, the knowledge provided here will facilitate studies of HLA class I pathways of chlamydial Ag processing and presentation in physiologically relevant human APCs.     

A proposed mouse model for acute epididymitis provoked by genital serovar E, Chlamydia trachomatis

This study was conducted to determine the efficacy of the male mouse as a model for epididymitis caused by human genital serovar E, Chlamydia trachomatis. C. trachomatis was reisolated from all tissues removed on Days 3, 5, and 7 post inoculation (pi). Although some infected epididymides removed on Days 10, 14, and 21 pi were positive, control tissues remained negative. Histopathology of tissues showed a heavy, mixed inflammatory infiltrate consisting of polymorphonuclear cells and lymphocytes. Serum antibody to C. trachomatis was detected in the infected mice only (titer greater than or equal to 1:32). Chlamydial inclusions and individual elementary bodies were confirmed by immunofluorescent and immunoperoxidase staining up to Day 7 pi. These data show that the male mouse is susceptible to C. trachomatis infection and is appropriate for studies dealing with the effect of C. trachomatis on male fertility.     

The effect of cortisol on glycogen and fructose-1,6-bisphosphatase in baby hamster kidney cells infected with Chlamydia trachomatis

This study was designed to assay changes in glycogen synthesis which may occur as a result of cortisol treatment of chlamydial-infected cells. Monolayers of baby hamster kidney (BHK) cells, unlabeled and prelabeled with [6-14C]glucose, were treated with various concentrations of cortisol before (pretreated) or during (post-treated) infection with Chlamydia trachomatis. At designated times after absorption, the cells were harvested and assayed for total glycogen and 14C accumulation in glycogen. The total amount of glycogen accumulated in cells during the period of greatest chlamydial glycogen synthesis (36 h) was not affected by cortisol treatment. Cortisol treatment appeared to have retarded the accumulation of glycogen in treated infected cells until 30 h after infection. Treated infected cells prelabeled with [6-14C]glucose accumulated a greater amount of 14C in glycogen than untreated infected cells. All cortisol-treated, infected cells exhibited elevated levels of fructose-1,6-bisphosphatase activity, whereas untreated infected cells did not. The hypothesis that cortisol affects chlamydia multiplication by altering the intracellular environment of the host cell is compatible with the results obtained.