Production of a T-Cell Clone Which Reacts with Membrane Proteins of Acholeplasma laidlawii

The role of cellular immunity in mycoplasma infection is not completely understood. In this study, we established mycoplasma-specific T-cell clones to evaluate cellular immunity in mycoplasma infection. We developed a T-cell clone (G-10) which was stimulated with Acholeplasma laidlawii. The T-cell clone G-10, CD4⁺ and T-cell receptor (TCR) αβ⁺ recognized the 42- and 65-kilodalton (kDa) membrane proteins of A. laidlawii and responded to A. hippikon. Hence, the application of mycoplasma-specific T cells such as G-10 in animal models may allow the assessment of cellular immune response to mycoplasma infection.     

Changes in superoxide dismutase activity in various larval organs of greater wax moth (Galleria mellonella L., Lepidoptera: Pyralidae) induced by infection with Bacillus thuringiensis ssp. galleriae

We studied the changes in superoxide dismutase activity in organs of Galleria mellonella larvae infected with two strains of Bacillus thuringiensis. A considerable increase in superoxide dismutase activity was observed at the initial stages of infection, later the enzyme activity decreased and this decrease was timed to cessation of feeding and development of sepsis in the infected larvae. Changes in the enzyme activity in the organs of larvae infected with a highly virulent strain of B. thuringiensis correlated with the stages of infection. Involvement of superoxide dismutase in prevention of oxidative stress in the infected larvae is discussed.Translated from Izvestiya Akademii Nauk, Seriya Biologicheskaya, No. 1, 2005, pp. 63–68.Original Russian Text Copyright © 2005 by Khvoshchevskaya, Dubovskii, Glupov.     

PCR-based detection and eradication of mycoplasmal infections from various mammalian cell lines: a local experience

A total of 200 cell lines including different human, monkey, mice, hamster and rat cell types were examined for mycoplasma infection status. PCR assay using generic-specific universal primers showed that 40 (20%) of the cell lines are contaminated with mycoplasma. Employment of species-specific primers within these infected cell lines revealed infection with M. hyorhinis (42.5%), M. fermentas (37.5%), M. arginini (37.5%), M. orale (12.5%) and A. laidlawii (7.5%). A number of the cultures were coinfected with 2 or 3 different species. Contaminated samples were treated with BM-Cyclin, Ciprofloxacin and mycoplasma removal agent (MRA). Mycoplasma eradication was subsequently checked by PCR following 2 weeks continuous culture of treated cells in antibiotic free culture medium. Mycoplasmal infections were eradicated in 100, 70 and 42% of infected cell lines when the samples were treated with BM-Cyclin, MRA and Ciprofloxacin, respectively. However, 12% (BM-Cyclin), 62.5% (MRA) and 82.5% (Ciprofloxacin) of mycoplasma regrowth was observed 4 months after the treatment. Notably, the risk of spontaneous culture death was 17.5, 12.5 and 0% for BM-Cyclin, MRA and Ciprofloxacin, respectively.     

Mycoplasma orale infection affects K+ and Cl− currents in the HSG salivary gland cell line

Summary The relations between K⁺ channel and Cl⁻ channel currents and mycoplasma infection status were studied longitudinally in HSG cells, a human submandibular gland cell line. The K⁺ channel currents were disrupted by the occurrence of mycoplasma infection: muscarinic activation of K⁺ channels and K⁺ channel expression as estimated by ionomycin- or hypotonically induced K⁺ current responses were all decreased. Similar decreases in ionomycin- and hypotonically induced responses were observed for Cl⁻ channels, but only the latter decrease was statistically significant. Also, Cl⁻ currents could be elicited more frequently than K⁺ currents (63% of cases versus 0%) in infected cells when tested by exposure to hypotonic media, indicating that mycoplasma infection affects K⁺ channels relatively more than Cl⁻ channels. These changes occurred in the originally infected cells, were ameliorated when the infection was cleared with sparfloxacin, and recurred when the cells were reinfected. Such changes would be expected to result in hyposecretion of salivary fluid if they occurredin vivo.     

Change in Lectin Specificity of Winter Wheat Seedlings in the Course of Infection with Mycoplasms

The activity of soluble lectins in leaves and roots of seedlings of winter wheat (Triticum aestivum L.) cultivar Mironovskaya 808 increased 1 day and 2 days, respectively, after infection with the mycoplasma Acholeplasma laidlawii 118. Analysis of acid-soluble proteins of wheat leaves by PAGE revealed the appearance of 22- and 20-kDa polypeptides, the disappearance of a 14-kDa polypeptide, and an increase in the content of polypeptides with molecular weights of 76, 48, 25, and 18 kDa. The 18-kDa polypeptide is a subunit of wheat germ agglutinin. A change in the activity of lectins may be a nonspecific response of plants to infection with the pathogen.     

Are individuals with lower neutrophil oxidative burst activity more prone to Helicobacter pylori infection?

Helicobacter pylori infection has been reported to cause enhanced reactive oxygen species in the gastric mucosa. We examined the relationship between H. pylori infection and neutrophil function of peripheral blood. The subjects were 904 volunteers who participated in the Iwaki Health Promotion Project in 2005. 158 subjects who were infected with H. pylori in 2005 also participated in this project in 2006 and were categorized into two groups: the eradication group, in which H. pylori was successfully eradicated during the 12 month period, and the non‐eradication group, in which eradication was unsuccessful or the subjects did not receive eradication therapy. The laboratory assays performed were: a titre of H. pylori antibody; neutrophil counts; and oxidative burst activity (OBA) of neutrophils. Logistic regression analysis was executed, with H. pylori infection as the dependent variable and other items as the independent variables. OBA showed an inverse association with H. pylori infection in 2005. Additionally, when comparing the eradication and non‐eradication groups, the change rates of OBA between 2005 and 2006 did not show any significant difference. It was concluded that H. pylori infection does not lower OBA, but those individuals in whom OBA was lower were more prone to H. pylori infection. Copyright © 2008 John Wiley & Sons, Ltd.     

Uracil phosphoribosyl transferase activity of mycoplasma and infected cell cultures

Summary Human H. Ep-2 and mouse 3T6 cells infected byMycoplasma hyorhinis showed an increase in [³H]uracil uptake and a more than 20-fold increase in the activity of uracil phosphoribosyltransferase (UraPRT). Uninfected cell cultures gave background levels of this enzyme activity. A survey of 16 strains of mycoplasma showed 13 to possess UraPRT activity. Rabbit kidney cells (RK13) were infected with eight different strains of four mycoplasma species known to be common cell culture contaminants. Seven of the eight cell cultures showed elevated UraPRT activities four days after infection. This enzyme activity may be of value in monitoring cell cultures for mycoplasma and aid in classification. This work was supported by Contract NO1-CP-53530 with the National Cancer Institute, National Institutes of Health, and Contract FDA 74-41 of the Food and Drug Administration, Bethesda, Maryland 20014.     

Interaction of cationic antimicrobial peptides with Mycoplasma pulmonis

We investigated the mode of action underlying the anti-mycoplasma activity of cationic antimicrobial peptides (AMPs) using four known AMPs and Mycoplasma pulmonis as a model mycoplasma. Scanning electron microscopy revealed that the integrity of the M. pulmonis membrane was significantly damaged within 30 min of AMPs exposure, which was confirmed by measuring the uptake of propidium iodine into the mycoplasma cells. The anti-mycoplasma activity of AMPs was found to depend on the binding affinity for phosphatidylcholine, which was incorporated into the mycoplasma membrane from the growth medium and preferentially distributed in the outer leaflet of the lipid bilayer.     

Serological evidence of an antibody response in farmed southern bluefin tuna naturally infected with the blood fluke Cardicola forsteri

In this study, adaptive immune response was investigated in farmed southern bluefin tuna, Thunnus maccoyii, infected with a sanguinicolid Cardicola forsteri. A cohort (Cohort₂₀₀₅) of southern bluefin tuna was sampled between March 2005 and August 2006. Samples were taken at the transfer of wild caught tuna to sea cages and then at regular intervals. Parasite intensity, abundance and prevalence data were recorded. An ELISA was developed to detect and quantify an antibody response against the blood fluke in southern bluefin tuna serum. Intensity and prevalence of the blood fluke were shown to peak in May 2005 at 10.9 flukes per infected fish (SE = 1.72) and 97.5% prevalence and then decreased to low prevalence (10%) and intensity (1.0). There were no significant changes in prevalence or intensity in 2006. Antibody titres and seroprevalence increased from 1.37 U μl⁻¹ and 10% at transfer in March 2005 to reach a peak in December 2005 of 25.86 U μl⁻¹ (SE = 6.26 U μl⁻¹) and 66.66%. No significant changes were observed in antibody titres for the same cohort of fish during 2006. Parasitological and serological values from Cohort₂₀₀₅ were compared to a 2006 cohort (Cohort₂₀₀₆) in March 2006 and August 2006 to determine if prior infection in Cohort₂₀₀₅ elicited any protection against infection in 2006. Although significant differences were not observed in intensities between cohorts it was shown that Cohort₂₀₀₅ had significantly lower abundances and prevalences of blood fluke infection than Cohort₂₀₀₆. Although there was no significant difference in mean antibody titres between cohorts in March 2006, the mean antibody titre of Cohort₂₀₀₆ was significantly greater than that of Cohort₂₀₀₅ in August 2006. No significant differences were observed in seroprevalence. This is one of the few studies to demonstrate the development of acquired resistance in fish against a parasite in an aquaculture environment under natural infection conditions.     

Effect of cell membrane-active antimicrobial agents on membrane potential and viability of mycoplasmas

The membrane potential ofMycoplasma mycoides subsp.capri has been determined to beE _M=−48 mV±10%, inside negative. In this study we investigated the influence of cell membrane-active antimicrobial agents, viz., valinomycin, gramicidin, polymyxin, and clotrimazole, on membrane potential and viability ofM. mycoides subsp.capri. Valinomycin, an ionophore with extreme potassium selectivity, induced a membrane hyperpolarization,E _M=−110 mV. Valinomycin was not cidal, but static to mycoplasmas. Obviously the potassium drain induced by valinomycin can be compensated for by the organisms. Gramicidin is an antibiotic forming cation conduction channels across membranes. It induced a rapid depolarization,E _M=+23 mV, of mycoplasma membranes. At low concentrations, gramicidin had a static effect, whereas at high concentrations it was cidal to mycoplasmas. The rapid permeation of cations through the stationary ion channels formed by gramicidin obviously exerts an inhibitory or even lethal effect on mycoplasma metabolism and growth. Polymyxin B induced a depolarization,E _M=−35 mV, of mycoplasma membranes only when the organisms had been pretreated and hyperpolarized with valinomycin. After treatment with both valinomycin and polymyxin B, a slight inhibition of mycoplasma growth was observed. Clotrimazole, a synthetic imidazole antimycotic, hyperpolarized mycoplasma membranes (E _M=−80 mV). At high concentrations clotrimazole was cidal, whereas at low concentrations it was static to mycoplasmas.     

Proteinase Activity of Prevotella Species Associated with Oral Purulent Infection

Prevotella intermedia and Prevotella nigrescens are often regarded as principal causes of acute dentoalveolar infection; however, other species within the genus are also known to be associated with such infection. The aim of this study was to determine the in vitro proteolytic activity of these different Prevotella species that have been implicated with dentoalveolar infection. A total of 234 strains were obtained from pus specimens from dentoalveolar infection and from the plaque of healthy volunteers. Prevotella loescheii, Prevotella oralis, Prevotella melaninogenica, Prevotella buccae, and Prevotella denticola were all shown to have a proteolytic activity (8.5–10.5 × 10⁻⁸ A-units) lower than that of P. intermedia and P. nigrescens (21.1–23.5 × 10⁻⁸ A-units). In the case of P. loescheii, P. melaninogenica, and P. intermedia, the level of proteolytic activity for clinical strains was significantly (P < 0.05) higher than that recorded for commensal strains. Proteolytic activity for all species of Prevotella examined was inhibited by N-ethylmaleimide and phenymethylsulfonyl fluoride. This study suggests that Prevotella species associated with oral purulent infection produce cysteine and serine proteinases and that in certain species of Prevotella, the strains involved in infection exhibit higher proteolytic activity when compared with strains from healthy sites.     

Influence of mycoplasma infection on the incorporation of different precursors into RNA components of tissue culture cells

Seven different tissue culture cells have been cultured with and without mycoplasma (M. hyorhinis) in the presence of various precursors of RNA. Total cellular RNA was isolated and analysed by electrophoresis on polyacrylamide gels. The results obtained with mycoplasma-infected cells can be summarized as follows:

1. 1. When cells are labelled with [8-³H]guanosine or [5-³H]uridine there is some incorporation into host cell 28S and 18S rRNA, but it is less than into mycoplasma 23S and 16S rRNA. [8-³H]guanosine or [5-³H]uridine are also incorporated into host cell and mycoplasma tRNA and mycoplasma 4.7S RNA, but the incorporation into host cell 5S rRNA and low molecular weight RNA components (LMW RNA) is reduced.

2. 2. [5-³H]uracil is not incorporated into host cell RNA but into mycoplasma tRNA, 4.7S RNA, a mycoplasma low molecular weight RNA component M₁ and 23S and 16S rRNA.

3. 3. [³H]methyl groups are incorporated into mycoplasma tRNA, 23S and 16S rRNA, but not into host cell 28S, 18S, 5S rRNA nor into mycoplasma 4.7S RNA.

4. 4. With [³²P]orthophosphate or [³H]adenosine as precursors, the labelling is primarily in the host RNA.

Mycoplasma infection influences the labelling of RNA primarily by an effect on the utilization of the exogenously added radioactive RNA precursors, since the generation time of mycoplasma infected cells is about the same as that of uninfected cells. Mycoplasma infection may completely prevent the identification of LMW RNA components.     

Detection of mycoplasma contamination in tissue cultures by fluorescence microscopy

Summary The in situ staining method of Chen (1977) for the detection of mycoplasma contaminants in tissue cultures was tested in cultures of human skin fibroblasts after controlled contamination with Mycoplasma arginini. It is concluded that this method is reliable only at infection rates of 100% or higher, i.e., at one mycoplasma or more per tissue-culture cell.     

Detection ofMycoplasmain Avian Live Virus Vaccines by Polymerase Chain Reaction

The polymerase chain reaction (PCR) was evaluated to detect mycoplasma contamination of avian live virus vaccines. The specificity of the primers showed that 34 strains belonging to nine species of avian mycoplasma DNA could be detected. The sensitivity of PCR to detect mycoplasma DNA was 10^0·2colony forming units (cfu) ofMycoplasma synoviaeand 10^0·7cfu ofMycoplasma gallisepticum. WhenM. synoviaeandM. gallisepticumwere spiked into several avian live virus vaccines, PCR gave a positive reaction except for the avian pox and the avian encephalomyelitis vaccines which were prepared from organ homogenates. Short-term incubation of avian encephalomyelitis vaccine improved the sensitivity of PCR to detect bothM. synoviaeandM. gallisepticum. Therefore, PCR, combined with the short-term incubation, were shown to be most effective in detecting mycoplasma contamination in all of avian live virus vaccines.     

Biological Enrichment of Mycoplasma Agents by Cocultivation with Permissive Cell Cultures

In this study, we describe our results on the evaluation of the ability of different permissive mammalian cell lines to support the biological enrichment of mycoplasma species known to be bacterial contaminants of cell substrates. The study showed that this approach is able to significantly improve the efficiency of mycoplasma detection based on nucleic acid testing or biochemical technologies (e.g., MycoAlert mycoplasma detection). Of 10 different cell lines (Vero, MDBK, HEK-293, Hep-G2, CV-1, EBTr, WI-38, R9ab, MDCK, and High Five) used in the study, only MDCK cell culture was found to support the efficient growth of all the tested mycoplasmas (Mycoplasma arginini, M. bovis, M. fermentans, M. gallinaceum, M. gallisepticum, M. synoviae, M. hominis, M. hyorhinis, M. orale, M. salivarium, and Acholeplasma laidlawii) known to be most frequently associated with contamination of cell substrates and cell lines in research laboratories or manufacturing facilities. The infection of MDCK cells with serial dilutions of each mycoplasma species demonstrated that these common cell line contaminants can be detected reliably after 7-day enrichment in MDCK cell culture at contamination levels of 0.05 to 0.25 CFU/ml. The High Five insect cell line was also found to be able to support the efficient growth of most mycoplasma species tested, except for M. hyorhinis strain DBS1050. However, mycoplasma growth in insect cell culture was demonstrated to be temperature dependent, and the most efficient growth was observed when the incubation temperature was increased from 28°C to between 35 and 37°C. We believe that this type of mycoplasma enrichment is one of the most promising approaches for improving the purity and safety testing of cell substrates and other cell-derived biologics and pharmaceuticals.     

Purification and Properties of a β-1,3-glucanase from Chaetomium sp. that is Involved in Mycoparasitism

A β-1,3-glucanase was detected, using laminarin as substrate, in the culture broth of Chaetomium sp. Major activity was associated with a 70 kDa protein band visualized on a polyacrylamide gel. β-1,3-Glucanase was purified by a one-step, native gel purification procedure. Optimal activity was observed at pH 6.0 and 30 °C (over 30 min). It could degrade cell walls of plant pathogens including Rhizoctonia solani, Gibberella zeae, Fusarium sp., Colletotrichum gloeosporioides and Phoma sp. The N-terminal amino acid residues of the purified β-1,3-glucanase are PYQLQTP, which do not exhibit homology to other fungal β-1,3-glucanases suggesting it may be a novel enzyme. Received 20 July 2005; Revisions requested 2 August 2005 and 27 September 2005; Revisions received 16 September 2005 and 3 November 2005; Accepted 6 November 2005     

Adenosine phosphorylase activity in a mutant Hep-2 cell line contaminated withmycoplasma hyorhinis

Summary Metabolic studies in HEp-2/MP,MIR cells (an adenosine kinase, hypoxanthine phosphoribosyltransferase negative mutant) indicated the presence of adenosine phosphorylase activity. This activity, unknown in established mammalian cell lines, resulted in the glycosidic cleavage of both adenosine and the antiviral drug arabinosyladenine. The activity was observed readily in the presence or absence of the adenosine deaminase inhibitor coformycin. Isopycnic separation of [³H]thymidine-labeled DNA species in CsCl density gradients resulted in the appearance of two distinct peaks. The heavier peak coincided with [¹⁴C]thymidine-labeled marker DNA of human origin, whereas the lighter peak was within the range associated with mycoplasmal DNA. Testing by commercial laboratories confirmed the presence of mycoplasma in HEp-2/MP,MIR cells. The contaminant was identified asMycoplasma hyorhinis, a porcine mycoplasma. Following γ-irradiation (3000 rads) to block cellular mitosis, the mycoplasma-contaminated HEp-2/MP,MIR cells were cocultivated with mycoplasma-free wild-type HEp-2 cells which did not exhibit adenosine phosphorylase activity. Following serial cocultivation in a medium designed to favor the survival of the wild-type cells, adenosine phosphorylase activity was found in the previously uninfected cells. Studies of this nature emphasize the need for investigators to carefully monitor their cell lines for mycoplasma. Presented at the 25th Annual Meeting of the Tissue Culture Association, Philadelphia, Pa., June 1976. This work was supported by Public Health Service Grants DE 02731 from the National Institute of Dental Research and CA 16219 from the National Cancer Institute.     

Production of laccases by Pleurotus ostreatus in submerged fermentation in co‐culture with Trichoderma viride

Aims: To evaluate the production and stability of laccases by Pleurotus ostreatus in liquid co‐cultures with Trichoderma viride as a function of infection time and agitation rate. Methods and Results: Pleurotus ostreatus cultures were infected with T. viride spores at 30 and 48 h. Maximal laccase volumetric activity was seen after 48 h (control cultures) or 72 h (co‐cultures) of cultivation time. Only the cultures infected at 30 h showed an increased laccase volumetric activity compared to control cultures. After maximal laccase volumetric activity value was reached, a sharp decrease in it was observed in control cultures. Co‐cultures exhibited a comparatively lower loss of activity. The influence of P. ostreatus and/or T. viride on the stability of laccase volumetric activity and isoenzyme pattern was evaluated. Trichoderma viride induced changes in the laccase isoenzyme pattern. Agitated cultures increased biomass growth and specific productivity threefold and sevenfold, respectively, to the static cultures. Conclusions: The laccase volumetric activity is very likely the result of the balance between biosynthesis and degradation/biotransformation rates occurring during the cultures. The individual presence of P. ostreatus or T. viride in the culture negatively affected the volumetric laccase activity. Significance and Impact of the Study: The evaluation of culture parameters that could influence Trichoderma–basidomycetes interaction and laccase production during submerged fermentation has not been reported. This study showed how laccase production in co‐cultures of P. ostreatus and T. viride was influenced by the infection time and agitation/oxygenation conditions.     

Mycoplasma Suppression of THP-1 Cell TLR Responses Is Corrected with Antibiotics

Mycoplasma contamination of cultured cell lines is a serious problem in research, altering cellular response to different stimuli thus compromising experimental results. We found that chronic mycoplasma contamination of THP-1 cells suppresses responses of THP-1 cells to TLR stimuli. For example, E. coli LPS induced IL-1 β was suppressed by 6 fold and IL-8 by 10 fold in mycoplasma positive THP-1 cells. Responses to live F. novicida challenge were suppressed by 50-fold and 40-fold respectively for IL-1β and IL-8. Basal TLR4 expression level in THP-1 cells was decreased by mycoplasma by 2.4-fold (p = 0.0003). Importantly, cell responses to pathogen associated molecular patterns are completely restored by mycoplasma clearance with Plasmocin. Thus, routine screening of cell lines for mycoplasma is important for the maintenance of reliable experimental data and contaminated cell lines can be restored to their baseline function with antibiotic clearance of mycoplasma.