McCoy Cells Research Articles

Evaluation of strain-specific primer sequences from an abortifacient strain of ovine Chlamydophila abortus ( Chlamydia psittaci) for the detection of EAE by PCR

Strain-specific primer sequences derived from the helicase gene of an ovine abortifacient strain (S26/3) of Chlamydophila abortus ( Chlamydia psittaci) were evaluated for the diagnosis of enzootic abortion in ewes (EAE) by polymerase chain reaction (PCR). C. abortus DNA was amplified from tissues submitted from ovine abortion cases using genus-specific and strain-specific primers in a standard thermal cycler. Amplification was followed by Southern blotting and hybridisation with a strain-specific probe. Real-time PCR was also evaluated using strain-specific primers in a microvolume fluorimeter-based thermal cycler (LightCycler™). Detection using both PCR methods was compared with other diagnostic methods against the standard of McCoy cell culture isolation. In this paper we report the application of strain-specific PCR as a fast, sensitive, specific method for the detection of EAE.

Uptake and intracellular activity of ofloxacin isomers in human phagocytic and non-phagocytic cells

The penetration and intracellular activity of ofloxacin and its isomers (levofloxacin and d-ofloxacin) into human polymorphonuclear leucocytes (PMN), human peritoneal macrophages (PMφ) and tissue cultured epithelial cells (McCoy) were evaluated. The cellular to extracellular concentration (C/E) values of the three fluoroquinolones were higher than 3.6 and 2.6 in PMN and PMφ, respectively. The C/E ratios in McCoy cells were lower than those in PMN, but still higher than 2.0. The uptake of ofloxacin and its isomers was rapid, non-saturable and reversible. All quinolones (extracellular concentrations: 2, 5 and 10 mg/l) produced a significant reduction of viable intraphagocytic Staphylococcus aureus in phagocytic cells. We concluded that ofloxacin and its isomers reach high intracellular concentrations in phagocytic and non phagocytic cells while remaining active in the former.

Hormesis: a stress response in cells exposed to low levels of heavy metals.

Cytotoxicity studies using a 3-(4,5 dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium bromide (MTT)-based in vitro toxicity assay revealed that McCoy cells exposed to low concentrations of mercuric (0.7 microM), cadmium (1 microM) and cupric chloride (3 microM) exhibited significant increases in cellular activity. This increased activity, previously termed hormesis, coincided with the production of high levels of the stress proteins, heat shock protein 70 (Hsp 70) and metallothionein, while the high constitutive expression of these proteins in cadmium-resistant mutant (CRM) cells corresponded to constitutive hormetic activity. Hormesis was found to obey uniform kinetics allowing for a mathematical description of this increased activity. These results suggest that hormetic activity is a specific cellular response, and most likely, a stress response to low but harmful levels of toxic agents and may therefore provide a rapid test for the presence of toxicants at concentrations associated with chronic toxicity.

Cytotoxicity of alkaline-tolerant dairy-associated Bacillus spp.

The cytotoxicity of five Bacillus spp. isolated from alkaline cleaning solutions in South African dairies was evaluated against McCoy mouse cells using a 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT)-based assay, confocal scanning laser microscopy and scanning electron microscopy. According to the MTT-based assay, two of the Bacillus isolates (Bacillus licheniformis 5 and B. pumilus 122) were cytotoxic to McCoy cells and the cytotoxic components were heat labile. Confocal scanning laser microscopy combined with fluorescent staining using propidium iodide and fluorescein diacetate indicated that cytotoxic effects occurred within 3 h, appeared to be membrane active and resulted in cell necrosis. Scanning electron microscopy showed that McCoy cells exposed to the cytotoxic components exhibited morphological damage.

Chlamydial development is adversely affected by minor changes in amino acid supply, blood plasma amino acid levels, and glucose deprivation.

This study has demonstrated the extreme sensitivity of Chlamydia trachomatis growing in McCoy cells to small changes in external amino acid supply. In the absence of cycloheximide, a decrease in the amino acid concentration of medium to 75% of control values was sufficient to induce the growth of enlarged chlamydial forms of reduced infectivity. Morphology became more distorted and the yield of infectious particles from inclusions declined as medium amino acid levels were further reduced. These events correlated with a general decline in intracellular amino acids, as measured by high-performance liquid chromatography, suggesting that chlamydiae require a minimum concentration of each amino acid for normal development. Cycloheximide enhanced the production of normal organisms and increased infectivity yield in media, suggesting that the drug increased the available pool of amino acids. This was supported by intracellular amino acid analyses. Aberrant forms with reduced infectivity were also induced during supply of infected cell cultures with medium containing blood plasma amino acid concentrations, supporting the proposal that nutrient levels in vivo could promote abnormal chlamydial development. Markedly abnormal forms were also observed during glucose deprivation, providing further evidence that aberrant development is a general stress-related response.

Peripheral blood T cell proliferative response to chlamydial organisms in gonococcal and non-gonococcal urethritis and presumed pelvic inflammatory disease.

OBJECTIVE: To study peripheral blood mononuclear cell (PBMC) proliferative response to Chlamydia trachomatis elementary bodies in (a) controls, (b) various stages of gonococcal (c) and non-gonococcal urethritis, and (d) women...

Tumor Necrosis Factor in Peritoneal Fluid From Asymptomatic Infertile Women

Background Tumor necrosis factor-α (TNF-α) is a cytokine that can be found in the peritoneal fluid (PF) of patients with endometriosis and pelvic inflammatory disease (PID) as a response to inflammatory disorders and infections. The cytotoxic effect of this cytokine could be a factor participating in the pathology of various gynecological diseases, and could also be accountable for the high immunological response and damage to the tubal epithelium. The objective of this study was to establish the presence of TNF-α in asymptomatic infertility and its association with various isolated bacteria. Methods Ten milliliters of PF were collected from each of 73 patients by means of laparoscopy and cultured in synthetic medium and McCoy cells for the isolation of aerobic and anaerobic bacteria, as well as for Chlamydia trachomatis. The activity of TNF-α was determined by means of a bioassay using L-929 cells. Results Forty-three percent of the PFs showed positive TNF-α activity, while the laparoscopic evaluation showed that 32 patients had Fallopian tube occlusion (FTO), 7 had endometriosis, 30 had PID, and 4 had myomas and adhesions. TNF-α activity was found to be high in FTO patients ( p <0.05). Positive cultures were found in 50.7% of patients; of these, 31.5% had PID ( p <0.05), and only 20.5% of positive cultures were TNF-α positive. Chlamydia trachomatis (16%) was the most frequently isolated bacteria in these patients. Conclusions The detection of TNF-α could be useful in the diagnosis of active infectious and inflammatory diseases in asymptomatic infertile patients.

Uptake and intracellular activity of moxifloxacin in human neutrophils and tissue-cultured epithelial cells.

The penetration by moxifloxacin of human neutrophils (polymorphonuclear leukocytes [PMN]) and tissue-cultured epithelial cells (McCoy cells) was evaluated by a fluorometric assay. At extracellular concentrations of 5 mg/liter, the cellular-to-extracellular concentration ratios (C/E) of moxifloxacin in PMN and McCoy cells were 10.9 +/- 1.0 and 8.7 +/- 1.0, respectively (20 min; 37 degrees C). The uptake of moxifloxacin by PMN was rapid, reversible, nonsaturable (at extracellular concentrations ranging from 1 to 50 microg/ml), and not affected by cell viability. The uptake of moxifloxacin was affected by external pH and the environmental temperature. The incubation of PMN in the presence of sodium fluoride, sodium cyanide, and carbonyl cyanide m-chlorophenylhydrazone significantly decreased the C/E of this agent. Neither PMN stimulation nor phagocytosis of opsonized Staphylococcus aureus significantly affected the uptake of moxifloxacin by human PMN. This agent, at concentrations of 0.5, 1, and 5 mg/liter, induced a significant reduction in the survival of intracellular S. aureus in human PMN. In summary, moxifloxacin reaches much higher intracellular concentrations within phagocytic and nonphagocytic cells than extracellular ones, remaining active inside the neutrophils.

Cytostatic Activity of Some Phenolic Acids of Scrophularia frutescens L. var. frutescens

Abstract The cytostatic activity of seven phenolic acids: coumaric, caffeic, ferulic, gentisic, protocatechuic, syringic and isovanillic acid previously isolated from Scrophularia frutescens has been evaluated against Hep -2 cells (de­rived from a human epidermoid carcinoma of the lar­ynx) and McCoy cells (derived from the synovial fluid in the knee joint of a patient suffering from degenerative arthritis). The compounds belonging to the cinnamic group present the highest activity.

Comparative In Vitro Susceptibility of a Tetracycline-Resistant Chlamydia trachomatis Strain Isolated in Toulouse (France)

We recently reported the first isolation of a tetracycline-resistant Chlamydia trachomatis strain in Toulouse from a woman treated with tetracycline. To characterize this isolate, its in vitro susceptibility was compared with those of 34 other C. trachomatis isolates recovered in Toulouse. The susceptibilities of C. trachomatis strains were determined in terms of minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) using McCoy cells in 96-well microdilution plates, with an inoculum of 5.10(3) to 1.10(4) inclusion-forming units/ml. The antimicrobial agents tested were tetracycline, azithromycin, erythromycin, ofloxacin, and pristinamycin. No difference was observed between the MICs and MBCs except for the tetracycline. Tetracycline-resistant strain MIC and MBC were > 64 micrograms/ml, although < 1% of the bacterial population showed resistance. For the other isolates, the MIC of tetracycline was < or = 0.25 microgram/ml. The antibiotics other than tetracycline were active in vitro against all strains. These results show that the tetracycline resistance observed in Toulouse differs from the "heterotypic resistance" described previously in the United States in multiresistant C. trachomatis isolates. They confirm that the resistance we observed may be a new phenomenon.

Conserved in VivoPhosphorylation of Calnexin at Casein Kinase II Sites as Well as a Protein Kinase C/Proline-directed Kinase Site

Calnexin is a lectin-like chaperone of the endoplasmic reticulum (ER) that couples temporally and spatially N-linked oligosaccharide modifications with the productive folding of newly synthesized glycoproteins. Calnexin was originally identified as a major type I integral membrane protein substrate of kinase(s) associated with the ER. Casein kinase II (CK2) was subsequently identified as an ER-associated kinase responsible for the in vitro phosphorylation of calnexin in microsomes (Ou, W-J., Thomas, D. Y., Bell, A. W., and Bergeron, J. J. M. (1992) J. Biol. Chem. 267, 23789-23796). We now report on the in vivo sites of calnexin phosphorylation. After 32PO4 labeling of HepG2 and Madin-Darby canine kidney cells, immunoprecipitated calnexin was phosphorylated exclusively on serine residues. Using nonradiolabeled cells, we subjected calnexin immunoprecipitates to in gel tryptic digestion followed by nanoelectrospray mass spectrometry employing selective scans specific for detection of phosphorylated fragments. Mass analyses identified three phosphorylated sites in calnexin from either HepG2 or Madin-Darby canine kidney cells. The three sites were localized to the more carboxyl-terminal half of the cytosolic domain: S534DAE (CK2 motif), S544QEE (CK2 motif), and S563PR. We conclude that CK2 is a kinase that phosphorylates calnexin in vivo as well as in microsomes in vitro. Another yet to be identified kinase (protein kinase C and/or proline-directed kinase) is directed toward the most COOH-terminal serine residue. Elucidation of the signaling cascade responsible for calnexin phosphorylation at these sites in vivo may define a novel regulatory function for calnexin in cargo folding and transport to the ER exit sites.

Susceptibility of Chlamydia trachomatis to chlorhexidine gluconate gel.

To identify topical antimicrobial preparations which may be effective in preventing the transmission of sexually transmitted diseases, we examined the activity of chlorhexidine gluconate (CHG) against Chlamydia trachomatis. Chlamydial elementary bodies were incubated with dilutions of CHG gel for various times from 0 to 120 min. An aliquot of each dilution was further diluted and was inoculated onto McCoy cell monolayers in individual wells in a 96-well microtiter plate. The cultures were incubated for 48 h, and the chlamydial inclusions were stained and counted. CHG gel diluted fourfold (0.0625% CHG) killed C. trachomatis serovar D, and CHG gel diluted eightfold (0.0313% CHG) killed serovar F immediately upon exposure. CHG gel diluted 16-fold (0.0156% CHG) killed serovar D, and CHG gel diluted 32-fold (0.0078% CHG) killed serovar F after 120 min of exposure. Alteration of the pH over the range of from 4 to 8 did not significantly affect its activity. The addition of 10% whole human blood decreased the CHG gel activity at 0 min but had no significant effect after 120 min of exposure. We conclude that CHG gel may be effective topically against C. trachomatis at concentrations that can be used and under conditions that are found in the female genital tract and that further studies of its antimicrobial efficacy and toxicity in vivo are warranted.

Sexually active adolescents and young adults: a high-risk group for Chlamydia trachomatis infection.

The importance of travel as a risk factor for Chlamydia trachomatis infection was evaluated among a series of young people consecutively tested. We studied 130 sexually active young subjects, aged 14-25 years, all living in the Rome, Italy, urban area. Ninety-eight females and 32 males attended hospital-based clinics or were the partners of an infected female. About half of these subjects had traveled abroad either for pleasure or for work, mostly to Europe, but also to North America or to Asia, where they admitted to having had casual sex. We used two "gold standard" methods to diagnose infection with C. trachomatis: culture on McCoy cells grown in shell vial, and direct immunofluorescence with monoclonal antibodies. Subjects were considered infected when at least one test was positive. Thirty-nine of 130 (30%) subjects were asymptomatic, and 27/130 (20.8%) subjects were infected with Chlamydia trachomatis, of whom 6/25 (24%) asymptomatic females and 3/14 (21.4%) asymptomatic males were infected. Among teen-aged (ages 14-19) youngsters with more than one sex partner, international travel was an additional significant risk factor for C. trachomatis infection (p<.02; OR 20; 95% CI 1.47-40%). Urethritis/cystitis and vaginal pathology/discharge were the prevalent manifestations of illness among the females, while urethritis was the only clinical condition found in the males. In a series of young subjects, travel abroad, sex with more than one partner, and teen age, combined together, were significant risk factors for the acquisition of Chlamydia trachomatis genitourinary infection.

Cytostatic activity of some compounds from the unsaponifiable fraction obtained from virgin olive oil

Oleuropein, tyrosol, squalene and the fraction of sterols and triterpenoid diacohols from the unsaponifiable fraction obtained from virgin olive oil have been tested for its possible cytostatic activity McCoy cells, using 6-mercaptoropurine as a positive control. The samples of sterols and triterpenic dialcohols showed a strong activity.

Killing of Chlamydia trachomatis by novel antimicrobial lipids adapted from compounds in human breast milk.

The development of new methods for prevention of sexually transmitted Chlamydia trachomatis infection is a top public health priority. Topical self-administered vaginal microbicides represent one such approach in which the organism is eradicated at the time of initial exposure. To this end, we examined the activity of five synthetic lipids adapted from naturally occurring compounds found in human breast milk. C. trachomatis serovar D or F elementary bodies were added to serial dilutions of the lipids and incubated for various times. Aliquots were then cultured in monolayers of McCoy cells, and inclusions were counted. A 7.5 mM concentration of 2-O-octyl-sn-glycerol completely prevented growth of C. trachomatis after 120 min of contact with the organism. The remaining lipids, 1-O-octyl-, 1-O-heptyl-, 2-O-hexyl-, and 1-O-hexyl-sn-glycerol, showed less activity. On electron microscopic examination, the lipids were shown to have disrupted the chlamydial inner membrane, allowing leakage of the cytoplasmic contents from the cell. Lipid activity was unaffected by the presence of 10% human blood or alterations in pH from 4.0 to 8.0, conditions reflecting those sometimes found in the vagina. Our results suggest that these lipids, especially 2-O-octyl-sn-glycerol, may be effective as topical microbicides in preventing the transmission of C. trachomatis. Further efficacy and toxicity studies with these lipids and assessment of their activity against other sexually transmitted disease pathogens are in progress.

Antibiotic susceptibility testing forChlamydia trachomatis using flow cytometry

The aim of this study was to compare the effectiveness of two methods of antibiotic susceptibility testing performed on Chlamydia trachomatis-infected cells: a flow cytometric detection method and the standard method, which consists of a microscopic reading of minimal inhibitory concentration (MIC). The L2 reference strain and 13 clinical strains isolated from six patients presenting recurrent infections were tested. McCoy cells infected with an inoculum of 10(5) inclusion forming units (IFU)/ml were incubated with serial dilutions of doxycycline, ofloxacin, and erythromycin. Mean fluorescence intensity (MFI) of cells was determined by flow cytometry after staining of chlamydial inclusions with an anti-Chlamydia fluorescent monoclonal antibody. The end-point values determined by flow cytometry and microscopic reading were equivalent but presented the same imprecision. Calculation of the inhibitory concentration 50 (IC50) by flow cytometry, defined as the antibiotic concentration required to reduce the drug-free control MFI by 50%, allowed a more objective and precise evaluation of antibiotic activity than MIC. Moreover, IC50 values were reproducible, independent of the antibiotic dilution series tested, and could be used to compare the in vitro efficiency of various drugs on C. trachomatis. No resistant strain was found among the 13 clinical isolates of C. trachomatis tested.

Antibiotic susceptibility testing for Chlamydia trachomatis using flow cytometry

The aim of this study was to compare the effectiveness of two methods of antibiotic susceptibility testing performed on Chlamydia trachomatis-infected cells: a flow cytometric detection method and the standard method, which consists of a microscopic reading of minimal inhibitory concentration (MIC). The L2 reference strain and 13 clinical strains isolated from six patients presenting recurrent infections were tested. McCoy cells infected with an inoculum of 105 inclusion forming units (IFU)/ml were incubated with serial dilutions of doxycycline, ofloxacin, and erythromycin. Mean fluorescence intensity (MFI) of cells was determined by flow cytometry after staining of chlamydial inclusions with an anti-Chlamydia fluorescent monoclonal antibody. The end-point values determined by flow cytometry and microscopic reading were equivalent but presented the same imprecision. Calculation of the inhibitory concentration 50 (IC50) by flow cytometry, defined as the antibiotic concentration required to reduce the drug-free control MFI by 50%, allowed a more objective and precise evaluation of antibiotic activity than MIC. Moreover, IC50 values were reproducible, independent of the antibiotic dilution series tested, and could be used to compare the in vitro efficiency of various drugs on C. trachomatis. No resistant strain was found among the 13 clinical isolates of C. trachomatis tested. Cytometry 31:37–44, 1998. © 1998 Wiley-Liss, Inc.

Evaluation of the microparticle enzyme immunoassay Abbott IMx Select Chlamydia and the importance of urethral site sampling to detect Chlamydia trachomatis in women.

OBJECTIVE: To evaluate the commercial microparticle enzyme immunoassay (MEIA), Abbott IMx Select Chlamydia, for the detection of Chlamydia trachomatis in women and to compare its performance with endocervical cell culture....

Evaluation of 2-SP transport medium for detection of Chlamydia trachomatis and Neisseria gonorrhoeae by two automated amplification systems and culture for chlamydia.

AIMS: To assess the performance of 2-sucrose-phosphate based transport medium (2-SP) for the detection of Chlamydia trachomatis and Neisseria gonorrhoeae by an automated commercial polymerase chain reaction (PCR) and ligase...

Cloning and expression of the 75 kDa DnaK-like protein of Chlamydia psittaci and the evaluation of the recombinant protein by immunoblotting and indirect ELISA

The 75 kDa dnaK-like gene of Chlamydia psittaci ovine abortion strain S26 3 was isolated from an EMBL 3 chlamydial DNA library. A 7 kb DNA fragment containing the gene was subcloned into Bluescribe (M13 +) plasmid and used to transform competent E. coli. These cells were found to express a cytoplasmic protein of 75 kDa. Monospecific antibodies against the protein prepared by antibody elution reacted with the native 75 kDa protein. Recombinant clones did not adhere to McCoy cell monolayers in cell adhesion studies. The 75 kDa protein purified by ion-exchange chromatography was used in immunoblotting and indirect enzyme-linked immunosorbant assay (ELISA) studies using sera previously screened for chlamydial antibodies by an indirect ELISA incorporating solubilised chlamydial elementary bodies and by micro-immunofluorescence. Immunoblotting identified 6 11 sera from infected ewes that had either typical placental lesions or had been found positive on examination of stained placental smears and 1 11 sera from ewes that had no typical placental lesions. The ELISA gave positive reactions with 29 of 65 known positive sera and 15 of the 76 negative sera. It is concluded that the 75 kDa DnaK-like protein is unsuitable as an antigen for antibody detection but its potential as a component for a sub-unit vaccine against ovine enzootic abortion warrants further study.