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PubMedDate not supplied

Monoclonal antibodies specific to Mycoplasma iowae.

Monoclonal antibodies (MAbs) against Mycoplasma iowae (MI) were produced to identify common immunogenic determinants shared between antigenically heterogenous MI. Twenty-four MAbs were produced against MI. With western immunoblotting, all 24 MAbs recognized a 45,000-MW protein (p45) of MI strain I-695. One of the MAbs characterized, MAb 2C, identified p45 antigen in western immunoblots with six laboratory strains and 24 field isolates of MI. MAb 2C did not cross-react with Mycoplasma gallisepticum, Mycoplasma synoviae, Mycoplasma meleagridis, or nonpathogenic avian mycoplasmas. Triton X-114 phase separation of MI proteins showed that p45 is an integral membrane protein. In immunofluorescent staining and immunoelectron microscopy of MI, MAb 2C reacted with surface antigen(s). These MAbs specific to MI may be used in detection and diagnosis of MI infections.

Animals↗

PubMed1996

[The detection of pediatric mycoplasma pneumonia cases by polymerase chain reaction and it's clinical significance].

Mycoplasma DNA in nasopharynx secretion from seventy pediatric mycoplasma cases was detected, using rapid feit of PCR DNA sample preparation method and simple PCR processes. The result of PCR showed that: fifty-three positive cases with a positive rate 75.7%, comparing with only 53.8% positive rate using mycoplasma culture method (P < 0.05). Thirteen PCR positive patients were treated by Venoclysis Erythromycin for 10-14 days but mycoplasma DNA was identified by PCR again. Among them, eight cases turned negative, but five cases still remained positive. The resalt showed that this method was not only more sensitive and reliable than conventional culture techniques, but also could simplify the PCR processes. It could be used for routine mycoplasma pneumonia test and for early diagnosis, which leads to early treatment and evaluation of therapeufic effect.

Child↗

PubMedDate not supplied

[Mycoplasmas and AIDS].

AIDS is a complex illness due to HIV type 1 and 2 infection. It is characterized by an important immunodeficiency mainly caused by depletion of CD4+ T lymphocytes. The reasons for this depletion have not been sufficiently clarified yet. In 1986, Shy Ching Lo astonished the scientific community with reported evidence concerning the direct role played by mycoplasma in the etiopathology of AIDS. Since then, different theories have pointed to mycoplasma as cofactors, commensals or opportunistic agents. Although in vivo and in vitro experiments are controversial they suggest a possible mechanism that would explain the synergism between both agents: the mycoplasma belonging to normal intestinal flora could move to urethra, oropharynx or blood due to high risk sexual practice. There it would proliferate favoured by early immunological disorders related to HIV. It has been speculated that several microorganisms including mycoplasma, acting as superantigens, could induce a chronic CD4+ and CD8+ T lymphocytes activation resulting in apoptosis of the infected lymphocytes. The release of cytokines induced by mycoplasma could influence the progression of the disease.

Acquired Immunodeficiency Syndrome↗

PubMed1976

[Investigations on the occurrence of mycoplasma in the amniotic fluid (author's transl)].

In order to elucidate the pathogenetic importance of mycoplasma in amnionitis and in infectious syndrome of the amnion, the amniotic fluid of 185 healthy pregnant women was investigated for mycoplasma, bacteria and fungi. In no case mycoplasma were detected. This means that amniotic fluid is normally free of mycoplasma. If mycoplasma occurs in the amniotic fluid it has to be considered as a possible cause of amnionitis and infectious syndrome of the amnion.

Amnion↗

PubMedDate not supplied

Attachment by mycoplasmas and its role in disease.

Mycoplasmas are the smallest free-living organisms that will grow on lifeless media. It has been demonstrated that these organisms are extracellular parasites in both animal and human diseases and that they attach to mucosal surfaces. Some of the mycoplasma species have differentiated, terminal attachment organelles. A definitive characterization of host-cell receptors and the binding sites on mycoplasmas should aid in the understanding of disease mechanisms. This information may then be used to develop strategies for prevention of diseases due to mycoplasmas.

Adhesiveness↗

PubMed1990

Mycoplasma contamination alters 2'-deoxyadenosine metabolism in deoxycoformycin-treated mouse leukemia cells.

Deoxycoformycin-treated P388 and L1210 mouse leukemia cells salvage 2'-deoxyadenosine from the medium only inefficiently, because deoxyadenosine deamination is blocked and its phosphorylation is limited by feedback controls. Mycoplasma contamination at a level that had no significant effect on the growth of the cells increased the salvage of deoxyadenosine greater than 10 fold over a 90 min period of incubation at 37 degrees C, but in this case deoxyadenosine was mainly incorporated into ribonucleotides and RNA via adenine formed from deoxyadenosine by mycoplasma adenosine phosphorylase. Deoxyadenosine was an efficient substrate for this enzyme, in contrast to 2',3'-dideoxyadenosine which was not phosphorolyzed. Mycoplasma infection was confirmed by the presence of uracil phosphoribosyltransferase activity and by culture isolation. The contaminant has been identified as Mycoplasma orale. Mycoplasma infection had no effect on the deamination and phosphorylation of deoxyadenosine and adenosine, on the salvage of hypoxanthine and adenine, or on the degradation of dAMP and dATP by the cells or on their acid and alkaline phosphatase activities.

Animals↗

PubMed2000

Induction of leukemia cell differentiation and apoptosis by recombinant P48, a modulin derived from Mycoplasma fermentans.

P48 is a 48-kDa monocytic differentiation/activation factor which was originally identified in the conditioned medium of the Reh and other leukemia cell lines and has recently been shown to be a Mycoplasma fermentans gene product. Previously, conditioned medium P48 has been shown to induce differentiation of HL-60 (human promyelocytic leukemia) cells. Recently our laboratory isolated cDNA clones for P48 from Reh cells and genomic clones from Mycoplasma fermentans and expressed the recombinant protein as a maltose binding protein (MBP) fusion protein in E. coli. In this report we present the initial characterization of this recombinant P48 fusion protein (rP48-MBP). We show that rP48-MBP induces differentiation of HL-60, U937 (human histiocytic lymphoma), and M1 (mouse myeloid leukemia) cell lines. Interestingly, rP48-MBP also induces apoptosis of U937 and HL-60 cells as assessed by terminal transferase (TUNEL) assays. This is the first report of induction of apoptosis by a Mycoplasma gene product. P48 is a Mycoplasma-derived immunomodulatory molecule which has differentiation and apoptosis-inducing activities and may be important in the pathophysiology of Mycoplasma infections. The recombinant protein may be useful in studying the mechanisms of differentiation, cytokine production, and apoptosis in malignant and nonmalignant hematopoietic cells.

Animals↗

PubMed1998

The ftsZ gene as a tool for detection of Mycoplasma fermentans.

Mycoplasma fermentans was reported as a common contaminant of cell cultures, and was shown to either induce or suppress several immunological functions. A strain of M. fermentans was recently isolated from a mouse T-lymphoma cell line, which differs from other M. fermentans strains by its growth characteristics and was designated (in the authors' records) as strain 609. Using the differential display technique (DD), a differentially expressed gene that was identified as the M. fermentans 609 ftsZ gene was isolated. Comparison of the nucleotide sequence of the M. fermentans 609 ftsZ gene to other ftsZ genes showed a 98% homology with Mycoplasma fermentans strain K7 and approximately 50% homology with Mycoplasma pulmonis and Mycoplasma genitalium. Comparison of the putative amino acid sequences of the FtsZ proteins showed similar homology. A polymerase chain reaction (PCR) assay to detect the presence of this ftsZ gene was established; it is a fast and convenient assay to detect infection of cells by the M. fermentans species. This work demonstrates that: (i) DD can be used as a useful technique to identify and isolate mycoplasmal genes from infected cells; and (ii) the ftsZ gene can be a useful marker to distinguish between different species of mycoplasma.

Amino Acid Sequence↗

PubMed1983

Some properties of mycoplasma virus Br 1.

Morphologically mycoplasma virus Br 1 is a typical contractile-tailed bacteriophage with a head 77 nm in diameter and a tail 104 nm long. Its type of nucleic acid was not determined. Br 1 was closely associated with its host cell and assays reflected infectious centres. During growth of Br 1 in mycoplasma cultures at multiplicities of infection (MOI) greater than 0.001, there was a lag period: this was up to 23 hours at an MOI of 35. The mean generation time of a mycoplasma culture infected at MOI up to 235 was 2 hours, compared with 1 hour for an uninfected culture. However in these infected cultures there were viable mycoplasmas all of which appeared to be fully susceptible to Br 1 infection and did not seem to be carrying the virus. Br 1 formed plaques on M. bovirhinis but failed to produce plaques on strains of 8 Mycoplasma, 2 Acholeplasma and 4 bacterial species.

Anti-Bacterial Agents↗

PubMed1994

Characterization and purification of a mycoplasma membrane-derived macrophage-activating factor.

A highly hydrophobic component derived from the membrane of Mycoplasma capricolum has been characterized, purified and assessed for its ability to activate macrophages to tumor cytotoxicity. Initially, crude membranes were evaluated for their solubility in a wide range of solvents. Despite differential solubility in the various solvents, the mycoplasma membranes retained their ability to potentiate macrophage tumor cytotoxicity. Mycoplasma membranes were further characterized by appraising their macrophage-activating ability subsequent to various chemical treatments: cleavage of ester and thioester bonds, oxidation of vicinal hydroxyl groups, and exposure to a broad range of pH. Only strong alkaline treatment (pH > 12) caused a reduction in mycoplasma membrane activity; all other chemical treatments were inconsequential. With potential therapeutic applications in mind, mycoplasma membranes were subjected to various physical treatments including heating, freezing/thawing, sonication, lyophilization and storage. The ability of the membranes to induce macrophage activation was stably maintained following all these treatments. Purification of membranes was initiated by a chloroform/methanol lipid extraction. Macrophage-activating ability was found predominantly in the interphase. Proteolytic cleavage with trypsin increased specific activity at least sixfold. Trypsinized fractions were solubilized in 2-chloroethanol and gel filtration was performed on a hydroxylated Sephadex LH-60 column. The active fraction from this column had a further tenfold increase in specific activity. Subsequent rounds of reverse-phase HPLC on this fraction yielded three to four peaks absorbing at 280 nm, of which only one had macrophage-activating ability.

Animals↗

PubMed1976

Pathogenicity factors of mycoplasmas.

The pathogenicity of mycoplasmas is caused by several factors, e.g. exotoxin, toxic properties of membrane components, exoenzymes, peroxide, and immunological factors. The absence of a rigid cell wall and the small genome tend to influence the interactions between mycoplasmas and host tissue. Mycoplasmas do not have a cell wass and are therefore resistant to the action of the host's lysozymes. They appear in some patients to be immunologically inconspicuous and in other patients they have been reported to have an immuno-suppressive effect. Recently there have been reports of central nervous system disorders due to mycoplasma. The pathogenic factors involved in these reactions have not been elucidated. Other aspects of Mycoplasma pneumoniae pathogenicity are also discussed.

Animals↗

PubMed1995

Microparticle agglutination versus antibody-capture enzyme immunoassay for diagnosis of community-acquired Mycoplasma pneumoniae pneumonia.

Community-acquired Mycoplasma pneumoniae pneumonia is a common disease which is usually diagnosed by serological methods. The objective of the present study was to understand the diagnostic significance and test characteristics of two different serological tests used to identify current Mycoplasma pneumoniae infection. Three hundred sixty-six patients who suffered from community-acquired pneumonia served as the study population. Six hundred ninety-four (328 paired and 38 unpaired) sera were examined for the presence of antibodies to Mycoplasma pneumoniae with commercial kits based on two serological methods, microparticle agglutination and antibody-capture EIA. Agreement between the two kits was 85.2% when individual sera were compared (kappa = 0.62) and 88.5% when patients were compared (Kappa = 0.69). The positive predictive value and the specificity for the identification of current Mycoplasma pneumoniae infection using a single acute-phase serum were 49.3% and 86.9%, respectively, for the microparticle agglutination method, compared to 91.3% and 97.7% for the antibody-capture EIA method (p < 0.001). The negative predictive value and the sensitivity were 86.3% and 48.1% for the microparticle agglutination, not significantly different from the corresponding values of 86.5% and 61.2% for the antibody-capture EIA. It is concluded that the overall agreement between the two methods tested is good, but not perfect. The methods complement each other in the identification of Mycoplasma pneumoniae as the causative agent in patients with community-acquired pneumonia.

Adolescent↗

PubMed1994

Treatment of mycoplasma contamination in a large panel of cell cultures.

Mycoplasmal contamination remains a significant impediment to the culture of eukaryotic cells. For certain cultures, attempts to eliminate the infection are feasible alternatives to the normally recommended disposal of the contaminated culture. Here, three antibiotic regimens for mycoplasmal decontamination were compared in a large panel of naturally infected cultures: a 1-wk treatment with the fluoroquinolone mycoplasma removal agent (MRA), a 2-wk treatment with the fluoroquinolone ciprofloxacin, and three rounds of a sequential 1-wk treatment with BM-Cyclin containing tiamulin and minocyclin. These antibiotic treatments had a high efficiency of permanent cure: MRA 69%, ciprofloxacin 75%, BM-Cyclin 87%. Resistance to mycoplasma eradication was observed in some cell cultures: BM-Cyclin 0%, MRA 20%, ciprofloxacin 20%. Nearly all resistant contaminants that could be identified belonged to the species Mycoplasma arginini and M. orale. Detrimental effects of the antibiotics were seen in the form of culture death caused by cytotoxicity (in 5 to 13% of the cultures). Alterations of the cellular phenotypic features or selective clonal outgrowth might represent further untoward side effects of exposure to these antibiotics. Overall, antibiotic decontamination of mycoplasmas is an efficient, inexpensive, reliable, and simple method: 150/200 (75%) chronically and heavily contaminated cultures were cured and 50/200 (25%) cultures could not be cleansed and were either lost or remained infected. It is concluded that eukaryotic cell cultures containing mycoplasmas are amenable to antibiotic treatment and that a cure rate of three-quarters is a reasonable expectation.

Cells, Cultured↗

PubMed1995

Elevation of endothelin biosynthesis in human endothelial cells with mycoplasma infection.

The effects of mycoplasma infection on the biosynthesis of endothelin-1 (ET-1) in cultured human vascular endothelial cell lines were examined to understand regulatory mechanisms of ET-1 biosynthesis and causes of angiopathy due to mycoplasma infection. The growth of normal endothelial cells from the umbilical cord vein and of an immortal endothelial cell line transfected with prepro ET-1 cDNA was decreased, while the secretion of ET-1 into the culture medium was enhanced by mycoplasma infection. However, the rate of ET-1 production in these cell cultures was much higher at the growing phase than at the stationary phase. Immunocytochemical studies with anti-ET-1 antibody and an autoradiographic examination of the labeled nuclei with 3H-TdR revealed that mycoplasma infection induced an elevation of ET-1 production in both S and non-S phase cells. The expression of prepro ET-1 mRNA as examined by in situ hybridization and by RNase protection assay was not altered by mycoplasma infection. Thus, the biosynthesis of ET-1 in vascular endothelial cells may be regulated at the posttranscriptional level.

Cell Division↗

PubMed2005

Mycoplasma-mediated alterations of in vitro generation and functions of human dendritic cells.

While tumor cell-derived factors have been demonstrated to hamper the in vitro differentiation and maturation of dendritic cells (DCs) from hematopoietic stem cells, their effects on DC differentiation from CD14+ plastic-adherent monocytic precursors have been controversial. To address this issue, we examined the effects of the culture supernatants from six tumor cell lines on in vitro DC differentiation and maturation from monocytes. Two tumor cell supernatants, MDA468 and 293T, were found to be able to affect the in vitro differentiation of DCs from monocytic precursors, leading to the generation of a distinct type of DC with markedly reduced expression of DC-SIGN, downregulation of CD11c, HLA-DR and CD1a, and upregulation of CD123, HLA-ABC, CD80, CD40, CD86, CD54, CD83, CD25 and CCR7. Functionally, these DCs exhibited reduced phagocytosis and enhanced allostimulatory capacity. Further investigation demonstrated that the changes in DC phenotype and functions were due to the presence of mycoplasmas in these two cell lines; eradication of mycoplasmas completely abolished the observed effects, and importantly, pure mycoplasmas in the absence of tumor cell supernatants were able to produce the same effects. Since mycoplasmas are common contamination agents in routine tissue culture, our results caution that many reported effects of DCs in culture warrant re-evaluation. The distinct effects of mycoplasmas on DC differentiation described in this report could potentially benefit future development of DC-based vaccination and therapeutic applications.

Biomarkers↗

PubMed1982

Effects of energization on membrane organization in mycoplasma.

Fluorescence polarization and ESR experiments using various probes demonstrated that addition of glucose to resting Mycoplasma capricolum and Mycoplasma mycoides subs capri had, if any, a very limited effect on the physical state of their membrane lipids. Under the same conditions the degree of exposure of primary amino groups of membrane proteins to the aqueous surrounding, estimated from fluorescence labeling by fluorescamine and the cycloheptaamylose-fluorescamine complex was significantly increased. This energy dependent increase was blocked by dicyclohexylcarbodiimide (DCCD), an inhibitor of the membrane bound Mg2+ stimulated ATPase of mycoplasma and by carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) which, in mycoplasma, only affects the chemical component of the proton-motive force. Variations in the proton activity gradient across the membrane induced by changing the pH of the labeling medium resulted in parallel variations in the ratio of relative intensities of labeling of energized to resting cells. The values taken by this ratio were up to two for a maximal proton gradient of 0.9 pH unit and tended to unity when the intracellular and extracellular pH tended to equalize. It is concluded that, upon mycoplasma cell energization, membrane proteins undergo a conformational change resulting in the exposure of new free amino groups. This conformational change is primarily dependent on the existence of a delta ph across the membrane and occurs in the absence of important modifications in the physical state of membrane lipids.

Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone↗

PubMed1980

Interferon production in cocultures between mouse spleen cells and tumor cells: possible role of mycoplasmas in interferon induction.

Interferon production was measured in the murine mixed lymphocyte tumor cell interaction (MLTI) using spleen cells of three inbred mouse strains and a number of in vitro grown lymphoma cells. No difference in interferon production was observed when syngeneic or allogeneic combinations were compared. Interferon was not detectable during the first 6 h of culture and reached its maximum after 12--24 h. In contrast, natural killer cell activity of spleen cells against one of these lymphoma lines (YAC-1) was already high after 4h. A cell line that was not susceptible to natural killing (MDAY-D2) induced the same amount of interferon as YAC-1. These findings suggest that natural killer cell activity and interferon induction in the MLTI are not correlated. Several lymphoma cell lines induced interferon in the MLTI whereas some others did not. All lines that were inductive were found to contain mycoplasmas. Furthermore, even the cell-free supernatants of inducing lines contained mycoplasmas and induced interferon. Purified mycoplasmas and membranes thereof were able to induce interferon production in mouse spleen cells. Our data strongly suggest that interferon production in the MLTI is caused by mycoplasmas. This artifact is the relevance for three reasons. First, cocultures between lymphoma cells and lymphocytes are widely studied in immunology. Secondly, contamination with mycoplasmas is extremely common and often goes unnoticed in immunological laboratories. Thirdly, interferon is known to affect profoundly a number of in vitro functions of immunocompetent cells.

Animals↗

PubMed1993

Specificity and sensitivity of polymerase chain reaction (PCR) in comparison with other methods for the detection of mycoplasma contamination in cell lines.

The polymerase chain reaction (PCR) amplification was used for the detection of mycoplasma contamination in 42 continuous cell lines. Using the microbiological cultivation on agar as the reference method, 29 cell lines were regarded as positive and 13 cell lines as negative. The double-step PCR analysis employed nested primers that anneal to gene sequences coding for the evolutionarily conserved 16 S rRNA of some 25 different mycoplasma species (including the ones most commonly found in cell cultures). In terms of the positivity or negativity of mycoplasma infection the results were identical for the agar assay and PCR amplification. All positive cell lines displayed distinct, unequivocal, objectively discernible bands on agarose gels while the non-infected specimens showed no DNA amplification. A simultaneously performed comparison with four other commonly used detection methods (DNA-RNA hybridization in solution, DAPI DNA fluorescence staining, immunostaining with a monoclonal antibody and an ELISA) showed that PCR produced significantly less false-negative or false-positive results than all the other methods. Furthermore, in dilution experiments, PCR correctly detected the infecting mycoplasmas at the lowest level of 1/10(4) whereas the other assays were less sensitive. It is concluded that double-step PCR employing nested primers is superior to other mycoplasma detection methods in many respects: simplicity and speed, high specificity and extreme sensitivity, objectivity and accuracy.

Animals↗