McCoy Cells Research Articles

Chlamydial inclusions: Accumulation of fibers bearing an intermediate filament epitope

Chlamydia trachomatis is an obligate intracellular bacterium that replicates in a phagosomal inclusion within a eukaryotic host. Light and transmission electron microscopy studies with monoclonal antibody TIB. 131 detected an intermediate filament (IF) epitope, demonstrating that it is present and co-accumulates in the inclusion along with chlamydia-specific antigens. Light microscope studies established that the material constituted a distinctive mass at the inclusion site and remained associated with elementary bodies when cells lysed to release the infectious particles. Transmission electron microscopy demonstrated that material binding the IF-specific monoclonal antibody lies within the inclusion; it is filamentous and entwines maturing reticulate bodies. An enzyme immunoassay, with mature chlamydial elementary bodies isolated and purified from infected embryonated eggs, examined the reactivity of the anti-IF monoclonal antibody; its binding to elementary bodies was readily titrated, indicating that the intermediate filament-positive epitope, or one cross-reactive with it, was present. Thus, the epitope is retained during purification, and its presence is not a function of growth in McCoy cells.

Comparison of Buffalo green monkey kidney cells and McCoy cells for the isolation of Chlamydia trachomatis in shell vial centrifugation culture

A total of 745 clinical specimens from patients attending hospitals and clinics in an area with a low prevalence of Chlamydia trachomatis were inoculated in parallel into shell vial cultures containing Buffalo green monkey kidney (BGM) and McCoy cells. The shell vial cultures were prepared in-house and were <5 days after seeding when inoculated. A total of 38 specimens (5%) were positive for C. trachomatis. In 36 of the cases, C. trachomatis was detected in both the BGM and McCoy vials. In two cases, C. trachomatis was detected in only the BGM vial. The BGM cells were more resistant to cytotoxicity and seemed to show more and larger inclusions than the McCoy cells. Furthermore, because the BGM cells displayed contact inhibition without the rounding and piling of cells that was encountered with the McCoy cells, they could be stored, ready to use, with an optical monolayer of cells, at 35°C for at least 10 days.

In vitro activity of β-lactam drugs and sulbactam against Chlamydia trachomatis

We tested the in vitro activity of ampicillin, ampicillin-sulbactam, cefoperazone, cefoperazone-sulbactam, and sulbactam against 18 recent clinical isolates of Chlamydia trachomatis and two ATCC strains. Ampicillin (MIC 50, 256 μg/ml) and sulbactam (MIC 50, 128 μg/ml) demonstrated some activity against C. trachomatis it, but cefoperazone had little to no activity. At 2–3 dilutions below the MIC, C. trachomatis treated with ampicillin or sulbactam, but not cefoperazone, formed small inclusions that remained small on passage onto antibiotic-free McCoy cells. It appears that ampicillin and sulbactam suppress rather than kill C. trachomatis.

Mobilization of F-actin and clathrin during redistribution of Chlamydia trachomatis to an intracellular site in eucaryotic cells

Immunofluorescence was used to examine the distribution of Chlamydia trachomatis serovars L2 and E, F-actin, and clathrin in infected McCoy and HeLa cells. After incubation at 4 degrees C, C. trachomatis serovar L2 was randomly distributed on the McCoy cell surface. After a temperature shift to 37 degrees C, chlamydiae redistributed, within 30 min, to one local aggregate in the central or perinuclear region of individual cells. About 90% of these aggregated chlamydiae were intracellularly localized, but some remained randomly distributed on the cell surface. Similar results were obtained with HeLa cells and C. trachomatis serovar E, except that the redistribution was slower in HeLa cells than in McCoy cells and fewer cells infected with serovar E exhibited a local aggregate than those infected with serovar L2. Cytochalasin D inhibited more than 90% of this local aggregation. Instead, in cytochalasin D-treated cells, the entry of chlamydiae was inhibited and the organisms became localized on the cell surface in a peripheral local aggregate that distributed in a manner similar to that of phalloidin-stained actin. In a double immunofluorescence assay, F-actin and clathrin aggregated correspondingly in time and position with central or perinuclear aggregation of chlamydiae. These results indicate that polymerized actin and clathrin participate in a rapid redistribution of chlamydiae to an intracellular aggregate.

A cytopathic effect of Trichomonas vaginalis probably mediated by a mannose/ N-acetyl-glucosamine binding lectin

The pathogenic effect of a highly pathogenic strain of Trichomonas vaginalis on McCoy cell monolayers was investigated. Specific inhibition of the cytopathic effect by monosaccharides, such as N-acetyl-glucosamine (GlcNAc) and mannose (Man), was observed. Our preliminary results suggest that the pathogenicity of T. vaginalis depends on a lectin specifically sensitive to GlcNAc and to a lesser extent to Man. Although N-acetyl-mannosamine was found to be the most efficient inhibitor, this effect seems to be unrelated to the natural biological behaviour of the infested host.

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.

Endocytic mechanisms utilized by chlamydiae and their influence on induction of productive infection

The microfilament-disrupting drug cytochalasin D and, initially, inoculation at 20 degrees C were used to differentiate between phagocytosis (sensitive to both treatments) and pinocytosis (resistant to both treatments) to assess whether chlamydial uptake into McCoy cells occurred by one or both mechanisms and whether each could contribute to productive infection. Both treatments suppressed the infectivity of Chlamydia trachomatis L2/434/Bu and C. psittaci GPIC (the guinea pig inclusion conjunctivitis strain) following static inoculation by only 50%, indicating that there was simultaneous operation of both phagocytosis and pinocytosis during uptake that led to productive infection. Measurement of the entry of organisms by two separate assays established that both strains predominantly used a cytochalasin D-resistant (pinocytic) mechanism, implying that phagocytic uptake was coupled to a higher frequency of productive infection. Integration of the data on infectivity and entry allowed the potential for an organism to infect a host cell to be quantified. This synthesis revealed that for both strains the infectivity potential following phagocytic entry was ca. 10-fold greater than that following pinocytic entry. However, both entry mechanisms were exploited more efficiently by strain L2/434/Bu than by strain GPIC (unless the latter was inoculated with centrifugation), indicating that intrinsic strain properties are more important for infectivity potential than the endocytic mechanism utilized.

Effect of beta-estradiol on production of the cell-detaching factor of Trichomonas vaginalis

Despite over 40 years of study, the pathogenetic mechanisms of Trichomonas vaginalis are just starting to be elucidated. We have recently reported that T. vaginalis produces a virulence factor, cell-detaching factor (CDF), that likely causes the cell sloughing seen in clinical disease. This 200-kDa glycoprotein is acid and heat labile and correlates with clinical symptoms. We applied a McCoy cell culture system to study the effects of various concentrations of beta-estradiol (10(-6) to 10(-10) M) on T. vaginalis growth and CDF production. T. vaginalis growth was unaffected by the different concentrations of beta-estradiol studied, in comparison with the growth of control cultures without beta-estradiol. However, beta-estradiol significantly diminished the activity of CDF at all concentrations and did so most profoundly at 10(-7) and 10(-8) M (P less than 0.0001). This suggests that the symptoms of T. vaginalis infection may be influenced by the vaginal concentration of estrogens, and further studies of the interactions between T. vaginalis and estrogens are warranted.

Immunoelectron microscopic localization of chlamydial lipopolysaccharide (LPS) in McCoy cells inoculated with Chlamydia trachomatis.

Interactions between Chlamydia trachomatis, host cells, and the immune system are believed to involve lipopolysaccharide (LPS). We used immunogold techniques to study the distribution of chlamydial LPS in cultured cells infected with C. trachomatis LGV-L1. McCoy cells inoculated with C. trachomatis were cultured and then fixed and embedded in situ with acrylic resins. Sections were immunolabeled with a protein A-gold method using antisera to the genus-specific, periodate-sensitive epitope on chlamydial LPS. Pre-embedding immunogold labeling on permeabilized cells was also done. By post-embedding methods, labeling for LPS was equally abundant over the outer membranes of elementary (EB) and reticulate bodies (RB). By post-embedding labeling, the sub-surface side of the EB outer membrane was more heavily labeled than the surface side. By pre-embedding labeling, LPS was found to be less abundant on the surface of EBs than RBs. Labeling for LPS was found over apparent lysosomes in McCoy cells and over electron-dense blebs on or near the surface of the plasma membranes of McCoy cells. These results indicate that the concentration of LPS in chlamydial membranes is constant during development but that with development its location changes from being mostly cell-surface to sub-surface. These results show that the post-embedding immunogold technique can be a useful approach for the cell culture-based study of chlamydial LPS.

Host passage-dependent wheat germ agglutinin-binding proteins of Chlamydia trachomatis

Wheat germ agglutinin (WGA)-binding proteins in the highly purified elementary bodies (EB) of Chlamydia trachomatis were detected by an ELISA-like assay of immobilized EB. Trypsin-sensitivity of the WGA-binding moieties was detected only in the chlamydiae grown in HeLa cells. A nonionic detergent, beta-octyl-D-glucopyranoside (OG), was used to extract proteins from the purified EB. Proteins in the extract were resolved by SDS-PAGE and probed with 125I-labelled WGA. Several proteins reacted with WGA in a specific manner. Some of these WGA-binding proteins in the EB of serovars L1 and E exhibited altered molecular mass after adaptation in the two alternate hosts, HeLa and McCoy cells. These results suggest that WGA-binding proteins exist in chlamydiae.

Host passage-dependent wheat germ agglutinin-binding proteins ofChlamydia trachomatis

Wheat germ agglutinin (WGA)-binding proteins in the highly purified elementary bodies (EB) of Chlamydia trachomatis were detected by an ELISA-like assay of immobilized EB. Trypsin-sensitivity of the WGA-binding moieties was detected only in the chlamydiae grown in HeLa cells. A nonionic detergent, beta-octyl-D-glucopyranoside (OG), was used to extract proteins from the purified EB. Proteins in the extract were resolved by SDS-PAGE and probed with 125I-labelled WGA. Several proteins reacted with WGA in a specific manner. Some of these WGA-binding proteins in the EB of serovars L1 and E exhibited altered molecular mass after adaptation in the two alternate hosts, HeLa and McCoy cells. These results suggest that WGA-binding proteins exist in chlamydiae.

The Use of Fluorescein-conjugated Monoclonal Antibodies, Cell Culture and Transmission Electron Microscopy to Detect Chlamydia psittaci and Associated Lesions in Experimentally Infected Mice

An immunofluorescence test based on a monoclonal antibody (mAb) was used to demonstrate chlamydiae in formalin-fixed and paraffin wax-embedded tissues from 10 adult mice experimentally infected by the oral route with Chlamydia psittaci isolated from the fetal membranes of an aborted ovine fetus. Samples of lung, jejunum and spleen were examined by bright-field microscopy, immunofluorescence and transmission electron microscopy, and were cultured for chlamydia in McCoy cells. These tissues were compared with those of two control mice. All infected mice had splenic hyperplasia and two had pneumonia. The lung appeared to be the target organ for C. psittaci administered by the oral route. Chlamydiae were identified in the lungs of five mice by immunofluorescence, bright-field and transmission electron microscopy. Chlamydiae were cultured from the jejunum of two mice and the spleen of one, but could not be identified at these sites by other methods. Immunofluorescence with an anti-chlamydia mAb was useful for detecting chlamydial antigen in formalin-fixed paraffin wax-embedded samples.

In VitroModels for Biochemical Investigations on Programmed Cell Death

Isolated rat thymocytes treated with methylprednisolone (MPS), cultured human synovial (McCoy's) cells exposed to cold shock and human mammary adenocarcinoma (BT-20) cells treated with Tumour Necrosis Factor (TNF), all showed DNA fragmentation and the morphological and biochemical features of cell death characteristic of a process known as apoptosis.DNA fragmentation in MPS-treated thymocytes and in cold shock-exposed McCoy's cells was preceded by a marked increase in cytosolic free Ca2+concentration resulting either from a sustained influx of Ca2+from the extracellular medium or from the release of Ca2+from intracellular pools. On the other hand, in TNF-treated BT-20 cells, DNA fragmentation was not associated with any early, marked increase in the cytosolic free Ca2+level. In all three models, however, DNA fragmentation was efficiently prevented by intracellular Ca2+chelators, calmodulin inhibitors and activators of protein kinase C (PKC). These results suggest that the activation of common mechanisms involving Ca2+and PKC may be essential for the development of apoptosis.

The hemolytic and cytolytic activity of group B streptococcal hemolysin and its possible role in early onset group B streptococcal disease

The in vitro and cytolytic properties of the hemolysin of group B streptococcus (GBS) were investigated using sheep erythrocytes and McCoy cells adapted for growth in a serum-deficient medium. The relationship between the hemolysin, various carrier molecules and phospholipids was examined. Starch-based carriers interfered with the inhibitory activity of phospholipids and solvents for the phospholipids reduced the activity of the hemolysin. These technical problems were resolved by use of an albumin-based carrier, a strain producing large amounts of hemolysin and sonication of the phospholipid. The hemolysin was cytolytic for McCoy cells and this activity and its hemolytic action on sheep erythrocytes were inhibited by a number of phospholipid components of surfactant. It is possible that GBS hemolysin has a direct or indirect role in the pathogenesis of the pneumonitis of early onset GBS infection.

Comparative activities of amoxycillin, amoxycillin/clavulanic acid and tetracycline against Chlamydia trachomatis in cell culture and in an experimental mouse pneumonitis

The activity of amoxycillin, amoxycillin/clavulanic acid and two tetracycline antibiotics was investigated against three strains of Chlamydia trachomatis in vitro. McCoy cells were infected and single doses of antibiotic administered 24 h after infection. The percentage of infected cells was calculated at intervals up to 72 h after infection. Amoxycillin and clavulanic acid, alone and in combination, reduced the incidence of inclusion formation of all three strains. Particularly good activity was observed against the laboratory-adapted strain C. trachomatis Sa2f and a clinical isolate C. trachomatis LB1, where a progressive reduction in numbers of inclusions was observed with time. Minocycline and oxytetracycline were the most active agents tested. In an experimental animal model, mice were inoculated intranasally with C. trachomatis MoPn (ATCC VR123) which caused a fatal pneumonia within 16 days, and treated orally for four days commencing at 24 h after infection. At doses producing clinically achievable serum concentrations, amoxycillin (10 mg/kg), amoxycillin/clavulanic acid (10 + 5 mg/kg) and minocycline (5 mg/kg) all protected the mice over a 21-day period. The majority of the animals treated with clavulanic acid alone (20 mg/kg) survived the infection. Treatment with oxytetracycline was less effective, a dose of 160 mg/kg being required to protect 70% of the mice. The results indicate that amoxycillin and amoxycillin/clavulanic acid were more effective against C. trachomatis MoPn in vivo than might be predicted from in-vitro data, suggesting that amoxycillin/clavulanic acid may have potential for the treatment of polymicrobial infections involving C. trachomatis.

Use of ultrasound to increase infection of McCoy cell monolayers by Chlamydia trachomatis strain

An ultrasound cell disrupter with a cooled cup tip was used to increase rapidly Chlamydia trachomatis infection in vitro. After three growth cycles of the NI-1 strain (serovar E), the pulsed ultrasound use enhanced the number of infected McCoy cells by approximately 12-times, as compared with control; and 8·8-times over the shaking with glass beads and centrifugation technique. After three growth cycles of the fast-growing LB-1 strain (serovar L2), the enhancement was by 15 and 10·8 respectively. Consequently, ultrasound treatment with a cooled cup tip can offer a working standard procedure to increase rapidly the number of cells infected with Chlamydia.

マクロライド系抗生剤のクラミジア培養細胞内への移行性の検討

Minimum inhibitory concentration (MIC) of Macrolide antibiotic (ML) against C. trachomatis was found to greatly vary with the cell culture system used for the assay. We then investigated the ability of various cell cultures to uptake MLs in the relation to MIC determination. Penetration of the 14C-labeled MLs, Erythromycin, Jasamycin and Rokitamycin into cells was quantitatively studied by measuring the radioactivity incorporated into McCoy, HeLa229W and HeLa229F cells. It was found that HeLa229W cell showed the lowest MIC for the drugs, followed by HeLa229F cell and then by McCoy cell. Reversely, McCoy cell showed the lowest intracellular concentration of an ML, followed by HeLa229F cell and then by HeLa229W cell. These results indicate that MIC of an ML significantly varies depending on the ability of the test cell to uptake the drug.

The Effect of Filtration of Amniotic Fluid on the Growth ofChlamydia TrachomatisandEscherichia Coli

There is increasing concern about Chlamydia trachomatis infection during pregnancy, because of reports of increased maternal, fetal, and neonatal risks. Amniotic fluid is known to possess antibacterial activity and has recently been shown to inhibit formation of chlamydial inclusions in McCoy cell culture. To further characterize the anti-chlamydial factor, we investigated the effect of filtering the fluid (0.45 microns pores) prior to incubation. Amniotic fluid was obtained from 12 women at term gestation, either by amniocentesis, or at cesarean section, Chlamydial inclusion formation was studied in McCoy cell cultures, and Escherichia coli growth was studied by a plate-count method. Filtered amniotic fluid had significantly less inhibitory activity against chlamydial inclusion formation than nonfiltered fluid did. Both filtered and nonfiltered amniotic fluid were equally effective in inhibiting E. coli colony growth. These data suggest that the chlamydial inhibitor in amniotic fluid does not pass through 0.45 microns pores and is larger than the bacterial inhibitor that was reported to be a peptide of low molecular weight.

Comparison of Cotton Wool Swab and Cytobrush for Detection of Chlamydial Infection in Women Attending a Genitourinary Medicine Clinic

Endocervical specimens obtained by cytobrush and conventional cotton wool swabs from 90 women attending a genitourinary medicine clinic were compared for their efficiency in detecting chlamydial infection. Isolation of Chlamydia trachomatis and detection of the chlamydial lipopolysaccharide antigen were attempted on each specimen. Antigen was detected in 18% of cytobrush and 17% of swab specimens. The cytobrush proved less suitable than swabs for isolation because 8 cytobrush specimens (9%) were toxic to the McCoy cells. Toxicity was significantly associated with an infected endocervix (2P = 0.004). Cytobrush therefore appeared to have little advantage over the much cheaper alternative, the cotton wool swab.

Growth of Trichomonas vaginalis in a Serum-Free McCoy Cell Culture System

Axenic cultures of Trichomonas vaginalis normally require serum for proliferation, yet serum-containing medium may interfere with the detection of T. vaginalis-secreted virulence factors. Trichomonas vaginalis can, however, grow in coculture with a McCoy cell monolayer in both the presence and absence of serum. For 6 T. vaginalis isolates examined, growth in this serum-free system shows lower peak concentrations of T. vaginalis and longer doubling times than those apparent in a serum-containing McCoy cell system. McCoy cells employed in the system did not appear to secrete soluble growth factors for T. vaginalis. The presence of McCoy cells was required for serum-free proliferation of T. vaginalis possibly indicating that eukaryotic cell membrane components may be important in supporting serum-free growth in this system.