McCoy Cells Research Articles

Molecular and Cytogenetic Criteria for Identification of Serum-Free Cell Cultures

ABSTRACTThe information about the authenticity of newly or already established animal cell cultures is essential. By using DNA analysis six cell lines has been investigated: 3T3, HeLa, McCoy, HEp-2, McCoy-Plovdiv and HEp-2-Plovdiv E. For the last four was made cytogenetic analysis confirmed that serum-free cell strains McCoy-Plovdiv and HEp-2-Plovdiv E are originally derived from McCoy and HEp-2 cell lines. PCR—analysis demonstrated that 3T3 cells are mouse originally, HeLa, HEp-2 and HEp-2- Plovdiv E are with the human origin and McCoy and McCoy-Plovdiv are hybrid cells carrying mouse and human genes in their genome.

Chemical composition, allelopatic and cytotoxic effects of essential oils of flowering tops and leaves of Crambe orientalis L. from Iran

Crambe orientalis L. (Brassicaceae), a perennial herb, is indigenous to Iran. The essential oils of flowering tops and leaves of the plant were evaluated by GC-MS. Our results showed that while 2-methyl-5-hexenitrile (19.5%) and benzyl cyanide (16.9%) were the major components of flowering tops oil, the oil of the leaves was dominated by octyl-acetate (54.3%) and butenyl-4-isothiocyanates (22.6%). The oils exhibited modest allelopatic effects on lettuce. The leaf oil showed high-cytotoxic effects against Mc-Coy cell lines, with an RC50 value of 16 µg mL−1.

A furanocoumarin from Prangos uloptera roots, biological effects

A known furanocoumarin, 8-geranyloxy psoralen, was isolated from n-hexane extract of Prangos uloptera roots by the TLC method. Its structure was determined by comparison of the spectral data with the literature. Cytotoxic effects of the isolated compound were determined by MTT and Tripan blue assays. The antioxidant and antimicrobial potential of the compound were evaluated by DPPH assay and agar dilution method, respectively. The MTT assay results showed that 8-geranyloxy psoralen reduced cell viability of Hela and Mc-Coy cell lines with IC50 values of 0.792 and 0.835 mM, respectively. Based on the Tripan blue asay, the compound has cytotoxic effects, with an IC50 value of 1.26 mM for Mc-Coy cell line. The compound exhibited weak antioxidant potential, with an RC50 value of 0.262 mg mL−1 and high antimicrobial effects against Staphylococcus epidermidis and Candida kefyr.

Kinematics of Intracellular Chlamydiae Provide Evidence for Contact-Dependent Development

A crucial process of chlamydial development involves differentiation of the replicative reticulate body (RB) into the infectious elementary body (EB). We present experimental evidence to provide support for a contact-dependent hypothesis for explaining the trigger involved in differentiation. We recorded live-imaging of Chlamydia trachomatis-infected McCoy cells at key times during development and tracked the temporospatial trajectories of individual chlamydial particles. We found that movement of the particles is related to development. Early to mid-developmental stages involved slight wobbling of RBs. The average speed of particles increased sharply at 24 h postinfection (after the estimated onset of RB to EB differentiation). We also investigated a penicillin-supplemented culture containing EBs, RBs, and aberrantly enlarged, stressed chlamydiae. Near-immobile enlarged particles are consistent with their continued tethering to the chlamydial inclusion membrane (CIM). We found a significantly negative, nonlinear association between speed and size/type of particles, providing further support for the hypothesis that particles become untethered near the onset of RB to EB differentiation. This study establishes the relationship between the motion properties of the chlamydiae and developmental stages, whereby wobbling RBs gradually lose contact with the CIM, and RB detachment from the CIM is coincidental with the onset of late differentiation.

Prevalence of Infection with Neisseria Gonorrhoeae or Chlamydia Trachomatis in Acute Mucopurulent Cervicitis

The aim of this study was to determine the incidence of N. gonorrhoae (NG) and/or C. trachomatis (CT) in acute mucopurulent cervicitis (MPC). The study included 617 non-pregnant women with MPC, who had not been receiving any antimicrobial treatment. The average age of patients was 22.2 years. There were no statistically significant differences according to place of residence, education, and marital status. Samples for laboratory analysis were collected using a routine procedure; NG was identified using the cytochrome oxidase test and Gram staining. CT was isolated on McCoy cell culture and stained with Lugol solution. NG was isolated in three women (0.8%) and CT in 58 women (9.4%). Fifty-six of the CT-positive patients were nullipara and only two were unipara. All NG-positive patients were also nullipara. The mean number of sexual partners was 2.2 in all study subjects, 2.4 in CT-positive subjects, and 2.9 in NG-positive subjects. Vaginal discharge purity according to Schröder was significantly deteriorated in CT-positive patients (p=0.011). When asked about the use of contraceptives, as many as 32.7% patients answered that they did not use any protection, 39% women used the rhythm method and coitus interruptus, 20% were taking oral contraceptives, 6.1% used mechanical devices, and 1.9% used chemical protection. Previous acute and chronic pelvic inflammatory diseases correlated with MPC (p>0.01). Our statistical analysis suggests that chlamydial infection significantly reduces the purity of vaginal discharge, which is more pronounced in nulliparae. Pap smear was not specific enough to demonstrate chlamydial infection. In view of the MPC findings, the prevalence of CT and NG infection is low.

Transcriptional analysis of in vitro expression patterns ofChlamydophila abortuspolymorphic outer membrane proteins during the chlamydial developmental cycle

Chlamydophila abortus is the aetiological agent of ovine enzootic abortion. Sequencing, annotation and comparative analysis of the genome of C. abortus strain S26/3 has revealed variation in the loci encoding the polymorphic membrane proteins (Pmps). These Pmps resemble autotransporter proteins of the type V secretion system, suggesting an important role in chlamydial pathogenesis. The purpose of this study was to characterise the transcriptional expression patterns of this family during the developmental cycle of C. abortus. McCoy cells were infected with C. abortus and analysed for pmp mRNA expression over a 72 h period. Few pmp transcripts were detected in the early stages of the developmental cycle. Peak expression occurred at 48 h post-infection (p.i.) other than for pmp5E, where it was observed at 24 h p.i. Overall, expression of pmps 5E, 18D and 10G were found to be 40 to 100-fold higher than the lowest expressing pmps (6H, 13G and 15G) at 24 h p.i., while pmps 18D and 17G were 14 to 16-fold higher than the lowest (11G, 14G and 15G) at 48 h. Levels of expression for all the other pmp genes were below one copy per genome at any time point. The expression of all the pmps reduced to near base-line levels by 60 h p.i. These results demonstrate that pmp expression in C. abortus is mid to late cycle, consistent with conversion of the reticulate body to the elementary body. The low level of pmp transcription may be indicative of heterogeneity in expression, suggesting a possible role for some of the Pmps in antigenic variation and chlamydial pathogenesis.

Chlamydia trachomatisinfection as a problem among male partners of infertile couples

Chlamydia trachomatis infection is the most common bacterial sexually transmitted disease supposed to cause urethritis, epididymitis, prostatitis and infertility in men. The objective of this study was to assess the frequency of C. trachomatis infection in male partners of infertile couples at childbearing age. Sixty infertile couples and a control group of 40 healthy volunteers were included in the study. Urethral swabs were taken from all the male participants and cervical swabs from the female partners of the infertile couples. Culturing on McCoy cell line and PCR were the methods used for detection of the infection. C. trachomatis was found in five out of the 60 male urethral samples. Three of the female partners of these five positive males were diagnosed with C. trachomatis infection, too. We registered a woman with C. trachomatis infection whose partner's samples were negative for the bacterium. The control group showed one specimen positive for C. trachomatis. The frequency of C. trachomatis infection was 8.3% in the male partners of infertile couples at childbearing age when compared with 2.5% in the control group. It is most likely that infertility in the couples with chlamydial infection was due to the pathogen studied.

Induction of programmed cell death by Prangos uloptera, a medicinal plant

Inhibition of the cell growth or induction of cell death is the most promising area in cancer therapy. The induction of apoptosis by dichloromethane extract of Prangos uloptera was evaluated on the McCoy cell line. This plant's roots, aerial parts and fruit have medicinal value. Cell growth inhibitory and cell cytotoxicity effects of the extract were assayed by MTT and Trypan-blue tests, respectively. Morphological changes and DNA fragmentation were also evaluated. The viability tests showed 0.49 and 0.3 mg/ml as 50% inhibition concentration and 50% cytotoxicity concentration after 24 hours of treatment, respectively. Fluorescent microscopy analysis revealed chromatin fragmentation and scanning electron microscopy showed cell shrinkage and cytoplasmic blebbing. These findings were confirmed by DNA fragmentation analysis. The results demonstrated efficient induction of apoptosis by the plant extract in moderate concentrations, but administration of higher concentrations showed that the primary manner of cell death was necrosis.

Chlamydia trachomatis infection in women with secondary infertility

To assess the role of Chlamydia in secondary infertility in a prospective study. Forty women with secondary infertility and 30 healthy term pregnant women of similar age composition were studied for past and present Chlamydia trachomatis infection. Women attending the outpatient Department of Obstetrics and Gynaecology with complaint of secondary infertility were enrolled as patients in the study. Forty women with secondary infertility formed the study group, and 30 healthy women served as the controls. Chlamydia IgG was detected by ELISA; titers of 1:320 or more were considered positive. Endocervical swabs were collected for culture on cycloheximide-treated McCoy cell lines, and ELISA was used to detect Chlamydia antigen. Hysterosalpingography was performed to assess tubal patency. A difference was expected between the prevalence of C. trachomatis infection in the infertile study subjects and fertile control group. Immunoglobulin G antibodies were present in 22 (55%) women with secondary infertility, whereas positivity was seen among 2 (5.5%) controls. Tubal occlusion occurred in 16 (63.6%) cases positive for chlamydial antibody. Sensitivity of chlamydial IgG antibody as a diagnostic marker for infertility was 72.7%, and specificity was 44.4%. The majority of Chlamydia IgG antibody-positive cases, 17 (77.2%), were symptomatic. Unfavorable obstetric history was found in 16 (72.7%) cases. Active infection was found in 12 (30%) cases with one (3.3%) case of current infection occurring in the controls. Prevalence of past chlamydial infection is strongly statistically significant in women with secondary infertility. Current infection was also found statistically significantly in these women. Immunoglobulin G antibody detection is an effective and noninvasive tool for detection of Chlamydia and a more viable option than HSG in a developing country such as India. Screening of women with secondary infertility for C. trachomatis is strongly recommended to allow early therapeutic interventions.

Ascorbic acid potentiates the cytotoxicity of the naphthoquinone 5-methoxy-3,4-dehydroxanthomegnin

The interaction of ascorbic acid with 5-methoxy-3,4-dehydroxanthomegnin, an 1,4-naphthoquinone, was investigated using the cytotoxic index for McCoy cells by neutral red assay. The synergstic effect was observed when such compounds were added simultaneously, most probably due to hydrogen peroxide being generated by ascorbate – driven 5-methoxy-3,4-dehydroxanthomegnin redox cycling. Incubation of cells in the presence of 5-methoxy-3,4-dehydroxanthomegnin/ascorbic acid/catalase, an enzyme that destroys H2O2, resulted in an increase of cell survival, reinforcing the involvement of hydrogen peroxide generated as an important oxidizing agent that kills McCoy cells.

Zoe‐philic Desires: Wet media art and beyond

Zoe‐philic Desires: Wet media art and beyond

Chlamydial infection in rheumatic diseases

Система комплексной поддержки и сопровождения научного журнала Elpub позволяет запустить двуязычный сайт журнала со встроенной системой электронной редакции в соответствии с лучшими издательскими практиками, а так же предусматривает техническую и методическую поддержку со стороны специалистов НЭИКОН.

A comparison of the immune parameters of dogs infected with visceral leishmaniasis using Western blot and neutralization techniques

The Western blot technique was used to demonstrate the presence of antibodies in the blood of dogs that presented canine visceral leishmaniasis. This technique was used against some specific molecules present in the lysate of the promastigote form of Leshmania chagasi. Through the association of the results of the Western blot technique with the morphological alterations seen as a result of the serum neutralization technique performed in McCoy cells (which mimetizes the macrophage) it was possible to observe the role of some molecules of great relevance in determining the disease in symptomatic dogs as well as that of some other molecules associated with asymptomatic infected dogs that may become transmitters as well as differentiating them as asymptomatic resistant dogs. In the sera analyses carried out during the immunobloting a variation of 9 to 27 immunoreacting bands was observed, which were then compared using Dice's similarity coefficient. In the dendrogram constructed on the basis of the coefficient, 50% similarity was observed among the total number of reagent bands with the promastigote lysate, thus creating five groups. The main difference observed related to the clinical condition of the dogs: symptomatic and asymptomatic dogs were found in separate groups. The asymptomatic group of dogs was distributed in two different places in the dendrogram because they presented two different behavior patterns regarding the cellular morphology in the serum neutralization reaction: the presence or absence of cellular lysis. According to this analysis it is possible to evaluate the immune status and associate it with specific markers observed in the reaction found in the Western blot strips.

A novel automated method for enumeration of Chlamydia trachomatis inclusion forming units

Chlamydia trachomatis is an obligate intracellular pathogen that primarily infects epithelial cells. Traditional methods for quantification of inclusion forming units (IFUs) rely upon infection of epithelial cell monolayers in vitro. Following incubation for ∼ 2 days, inclusion bodies that result from infection of cells are detected by immunofluorescent staining with an antibody conjugated to a fluorescent dye. These inclusion bodies are then manually counted by microscopic examination of multiple, randomly selected fields of view. This requires substantial operator time and is subject to investigator bias. We have developed a novel method in which we utilize an automated microplate ImmunoSpot reader to count C. trachomatis IFUs. Following infection of epithelial cells in a 96-well plate and subsequent incubation, IFUs are fixed and detected with an anti- C. trachomatis LPS monoclonal antibody. Immobilized antibody is detected with a biotinylated secondary antibody and visualized enzymatically with streptavidin-alkaline phosphatase and the colorimetric substrate nitro-blue tetrazolium chloride/5-bromo-4-chloro-3-indolyl-phospate (NBT/BCIP). IFUs are then enumerated with the ImmunoSpot system. This method has been used to quantify IFUs from all cell lines traditionally used for chlamydial propagation, including L929, McCoy, HeLa and HaK cells. IFU numbers obtained are comparable to those determined by traditional microscopic counting. In addition, the method can be applied to rapid determination of serum-neutralizing titers for vaccine studies, and we have also applied this approach to quantify Chlamydia recovered from vaginal swabs collected from infected animals. This method provides for rapid enumeration of IFU counts while minimizing investigator bias and has potential applications for both research and diagnostic use.

Effect of the water extracts of propolis on stimulation and inhibition of different cells

The water extracts of propolis (WEP) could inhibit growth of different cell lines namely McCoy, HeLa, SP2/0, HEp-2, and BHK21 and stimulate growth of normal cell named human lymphocyte, rat kidney, rat liver, and rat spleen. In these experiments 1 and 2 mg of WEP were added to 1 ml RPMI media with 5% FCS. Cell counts and cell viability of propolis-treated and propolis-free cells were assessed by Trypan blue dye exclusion test and MTT assay. The results showed that in case of McCoy, HeLa, SP20, HEp-2, and BHK21 cell lines, the water extracts of propolis could inhibit cell growth as well as reduction on size of the cells. In contrast the same amount of WEP could stimulate growth of normal cells up to 60% with the same concentration used for cell lines. Thus our study indicates that although WEP consists only of the soluble part of propolis, it enables to inhibit different cell lines and increase growth of normal cells. This indicates also that WEP contains the specific compounds with bioactivity against cell lines. Although propolis contain different number of compounds it is clear that WEP has enough biological compounds useful for the treatment of some diseases, medical and related applications.

Kinetics of growth of Leishmania (Leishmania) chagasi cycle in McCoy cell culture

The kinetics of growth of Leishmania performed in vitro after internalization of the promastigote form in the cell and the occurrence of the transformation of the parasite into the amastigote form have been described by several authors. They used explants of macrophages in hamster spleen cell culture or in a human macrophage lineage cell, the U937. Using microscopy, the description of morphologic inter-relationship and the analysis of the production of specific molecules, it has been possible to define some of the peculiarities of the biology of the parasite. The present study shows the growth cycle of Leishmania chagasi during the observation of kinetic analysis undertaken with a McCoy cell lineage that lasted for a period of 144 hours. During the process, the morphologic transformation was revealed by indirect immunofluorescence (IF) and the molecules liberated in the extra cellular medium were observed by SDS-PAGE at 24-hour intervals during the whole 144-hour period. It was observed that in the first 72 hours the promastigote form of L. chagasi adhered to the cell membranes and assumed a rounded (amastigote-like) form. At 96 hours the infected cells showed morphologic alterations; at 120 hours the cells had liberated soluble fluorescent antigens into the extra cellular medium. At 144 hours, new elongated forms of the parasites, similar to promastigotes, were observed. In the SDS-PAGE, specific molecular weight proteins were observed at each point of the kinetic analysis showing that the McCoy cell imitates the macrophage and may be considered a useful model for the study of the infection of the Leishmania/cell binomial.

Efficacy of cytology for the diagnosis of Chlamydia trachomatis in pregnant women

This study evaluated the effectiveness of Papanicolaou staining for the initial diagnosis of Chlamydial infection in pregnant women. A hundred thirteen patients were examined with a Papanicolaou test, independent of gestational age, parity or maternal age. Three endocervical samples were collected; the first two were collected with a brush (Cytobrush plus, Mediscand, Sweden) and the third with Ayre's spatula. The first specimen was used for McCoy cell culture and the other two were examined cytologically. Chlamydial infection was detected in 9 (7.9%) patients. Only one (0.8%) was diagnosed by cytological exam. The sensitivity and specificity of the cytological examination were 10 and 98%, respectively. The estimated positive predictive value was 33.3% and the negative predictive value was 92.7%. When Papanicolaou stain diagnosis suggests Chlamydia, a more specific complementary exam should be added to confirm infection; subsequently adequate treatment can be implemented, thereby preventing the frequent complications of untreated subclinical infections.

Effect of subinhibitory concentration of chlorhexidine on Streptococcus agalactiae virulence factor expression

The aim of the present study was to investigate the effect of chlorhexidine at subinhibitory concentration (50% minimal inhibitory concentration (MIC)) on the growth, cytolysin expression and phagocytosis of Streptococcus agalactiae ATCC 13813. Bacterial growth with and without chlorhexidine treatment was monitored by turbidity measurements, and exocytolysins were estimated by neutral red uptake assay by the McCoy cell line. The phagocytic process was evaluated using luminol-enhanced chemiluminescence to follow the respiratory burst of polymorphonuclear neutrophils exposed to bacteria. Chlorhexidine-treated culture did not exhibit a detectable decrease in cell growth, and no statistically significant reduction in the respiratory burst of polymorphonuclear neutrophils was observed. However, growth in the presence of chlorhexidine resulted in a significant reduction of S. agalactiae exocytolysins. Although 50% MIC of chlorhexidine did not interfere with S. agalactiae growth and phagocytosis, the knowledge that this concentration was still able to alter some bacterial virulence parameters may be useful in its therapeutic applications.

Reproduction of Leishmania (Leishmania ) infantum chagasi in conditioned cell culture growth medium

Leishmanias can be produced by inoculation in conditioned McCoy cell culture growth medium (CGM). Leishmania (Leishmania) infantum chagasi (100 parasites) grown in NNN medium was inoculated in 2.5 mL CGM, kept in plates (24 wells) and its multiplication was observed for five days (120 hours). After day 5, the medium was saturated with the flagellate forms of the parasite (promastigotes). The reproduction of the leishmanias was observed every 24 hours and the number of parasites was calculated by counting the parasites in a drop of 10 microL and photomicrographed. So the number of Leishmanias was adjusted to 1 mL volume. The advantage of the technique by isolation of Leishmania in CGM demonstrated in this study is its low cost and high efficacy even with a small quantity of parasites (10(2) promastigotes) used as inoculum. Additionally, isolation of the leishmania can be obtained together with an increase in their density (180 times) as observed by growth kinetics, within a shorter time. These results justify the use of this low-cost technique for the isolation and investigation of the behavior and multiplication of Leishmania both in vertebrates and invertebrates, besides offering means of obtaining antigens, whether whole antigens (leishmanias) or the soluble antigens produced by the parasites which may be useful for the production of new diagnostic kits.

Reliability of Detecting rRNA Sequences of Chlamydia trachomatis With Fluorescence in Situ Hybridization Without Amplification

To use fluorescence in situ hybridization (FISH) using ribosomal RNA (rRNA) oligonucleotide probes as the target nucleic acid for the detection of Chlamydia trachomatis.Suitable sequences selected from the rRNA sequence of C trachomatis were labeled with a fluorescent dye and used in FISH for detecting chlamydial inclusion bodies and/ or elementary bodies in paraformaldehyde-fixed urogenital swab samples. The sensitivity and specificity of the FISH assay were compared with those of the polymerase chain reaction (PCR) using plasmid primers. Positive known C trachomatis-infected McCoy cells were used as positive controls. Urogenital swab specimens that were C trachomatis negative on culture and PCR were used as negative controls.Among the 128 samples included in the study, FISH was positive in 28 (21.8%) and PCR in 33 (25.7%). A significant correlation was found between the 2 detection methods. Results of PCR and FISH were consistent in 115 of the 128 samples (R = 0.89). Thirteen samples showed discordant results. Of these, 9 FISH negative samples were PCR positive and 4 FISH positive samples were PCR negative.FISH was a highly specific and fairly sensitive technique for detecting C trachomatis. Signal amplification techniques and use of different fluorophores may further increase the sensitivity of this technique.