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1.
Mycoplasmal infections prevent apoptosis and induce malignant transformation of interleukin-3-dependent 32D hematopoietic cells 总被引:17,自引:0,他引:17
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32D cells, a murine myeloid cell line, rapidly undergo apoptosis upon withdrawal of interleukin-3 (IL-3) supplement in culture. We found that 32D cells, if infected by several species of human mycoplasmas that rapidly activated NF-kappaB, would live and continue to grow in IL-3-depleted culture. Mycoplasma-infected cells showed no evidence of autocrine production of IL-3. Pyrrolidine dithiocarbamate (PDTC) blocked activation of NF-kappaB and led to prominent cell death. Heat-killed mycoplasmas or mycoplasmal membrane preparations alone could support continued growth of 32D cells in culture without IL-3 supplement for a substantial period of time. However, upon removal of heat-inactivated mycoplasmas, 32D cells quickly became apoptotic. In comparison, live Mycoplasma fermentans or M. penetrans infection for 4 to 5 weeks induced malignant transformation of 32D cells. Transformed 32D cells grew autonomously and no longer required support of growth-stimulating factors including IL-3 and mycoplasmas. The transformed 32D cells quickly formed tumors when injected into nude mice. Karyotyping showed that development of chromosomal changes and trisomy 19 was often associated with malignant transformation and tumorigenicity of 32D cells. Mycoplasmal infections apparently affected the fidelity of genomic transmission in cell division as well as checkpoints coordinating the progression of cell cycle events. 相似文献
2.
M Kita Y Ohmoto Y Hirai N Yamaguchi J Imanishi 《Comptes rendus des séances de la Société de biologie et de ses filiales》1990,184(5-6):380-384
Mycoplasma shows a variety of effects on immune system, including the activation of macrophage, the increase in T cell cytotoxicity, and the enhancement of the proliferation and maturation of B cells, etc. As it is well known that many cytokines regulate the immune system, it is interesting to examine whether or not human peripheral blood mononuclear cells (PBMC) produce interleukin (IL) in response to mycoplasmas. In the present study, human PMBC were incubated with 7 species of mycoplasmas for 48 hours, and IL-1 beta, IL-2 and IL-6 activities in the supernatants were determined by ELISA. All the species of mycoplasmas were able to induce IL-1 beta and IL-6, although IL-2 was induced only by M. pneumoniae. These results suggest that the influence of mycoplasma infection on immune system may be partly due to the interleukins induced by mycoplasmas. 相似文献
3.
Mycoplasma contamination of cell cultures: Incidence, sources, effects, detection, elimination, prevention 总被引:11,自引:0,他引:11
The contamination of cell cultures by mycoplasmas remains a major problem in cell culture. Mycoplasmas can produce a virtually
unlimited variety of effects in the cultures they infect. These organisms are resistant to most antibiotics commonly employed
in cell cultures. Here we provide a concise overview of the current knowledge on: (1) the incidence and sources of mycoplasma
contamination in cell cultures, the mycoplasma species most commonly detected in cell cultures, and the effects of mycoplasmas
on the function and activities of infected cell cultures; (2) the various techniques available for the detection of mycoplasmas
with particular emphasis on the most reliable detection methods; (3) the various methods available for the elimination of
mycoplasmas highlighting antibiotic treatment; and (4) the recommended procedures and working protocols for the detection,
elimination and prevention of mycoplasma contamination. The availability of accurate, sensitive and reliable detection methods
and the application of robust and successful elimination methods provide powerful means for overcoming the problem of mycoplasma
contamination in cell cultures.
This revised version was published online in August 2006 with corrections to the Cover Date. 相似文献
4.
Induction of cytokines in human peripheral blood mononuclear cells by mycoplasmas. 总被引:12,自引:0,他引:12
Various species of mycoplasmas were tested for their ability to induce cytokine production in human peripheral blood mononuclear cells (PBMC). Human PBMC were incubated with Mycoplasma pneumoniae, M. hyorhinis, M. arginini, M. salivarium, M. orale, M. gallisepticum or A. laidlawii for 48 hr, and the activities of interleukin-1 beta (IL-1 beta), IL-2, IL-4, IL-6, tumor necrosis factor-alpha (TNF-alpha) and interferon (IFN) in the supernatants were determined by ELISA or bioassay. All mycoplasma species induced IL-1 beta, IL-6 and TNF-alpha, although IL-2 was induced only by M. pneumoniae. IFN was induced by 5 of the 7 species, and the IFN produced was antigenically confirmed to be mainly IFN-alpha. On the other hand, mycoplasma-stimulated cultures did not contain detectable amounts of IFN-beta and IL-4 activities. Furthermore, the cytokines were induced by mycoplasmal contaminating cells in human PBMC as well as by mycoplasma alone. These results suggest that many kinds of cytokines induced by mycoplasma contamination in cell culture affect immunological experiments in vitro. 相似文献
5.
In situ detection of mycoplasma contamination in cell cultures by fluorescent Hoechst 33258 stain 总被引:97,自引:0,他引:97
T. R. Chen 《Experimental cell research》1977,104(2):255-262
A simple, fast, and easily reproducible routine laboratory technique for detecting mycoplasma contamination in cell cultures is reported. Cells grown on a coverslip are fixed directly with Carnoy's, air-dried, stained with DNA-specific fluorescent Hoechst 33258, and examined microscopically. All cultures that were infected with mycoplasmas had readily discernible, small, morphologically uniform, bright fluorescent bodies in the extranuclear and intercellular space in contrast to the non-contaminated control cultures in which the extra-nuclear background appeared uniformly dark. To probe the degree of sensitivity to detect mycoplasmas, control cultures were infected with aliquots from serially diluted cells or media collected from Mycoplasma hyorhinus infected cultures. The lowest infection rate (0.40% by sampling 1 000 cells in average per culture 4–24 h after infection) scored presently, however, can easily be lowered by increasing sample size since a cell infected with even one mycoplasma can be discerned. These mycoplasmas resisted centrifugation at 2 500 rpm for 30 min and easily filtered through 0.22 μm pore-size filter membrane. Amazingly infection rate of 0.63% scored from 24 h post-infection incubation attained 100% contamination with several hundreds of mycoplasmas per host cell within 120 h. 相似文献
6.
The T cell-secreted lymphokine interleukin 13 (IL-13) exerts pleiotropic effects on monocytes (Mphi) and B cells. Since accessory cells, like Mphi and B cells, also act in antigen-presenting and lymphokine augmentation of T cells, Mphi and B cells may be able to effect T cell IL-13 production. Purified T cells produced slightly less IL-13 than the lower T cell numbers contained in peripheral blood mononuclear cell population, further suggesting accessory cell augmentation. Addition of 10% B cells [either unstimulated or pokeweed mitogen (PWM)-stimulated] to autologous T cells only moderately augmented T cell IL-13 levels. PWM-stimulated B cell culture supernates had even less augmenting effect on T cell IL-13 levels and unstimulated B cell culture supernates did not augment T cell IL-13 production. In contrast to the moderately augmenting effect of B cells or their stimulated culture supernates, addition of 10% Mphi, either unstimulated or muramyl dipeptide (MDP)+IFN-gamma stimulated, to autologous T cells produced a highly significant increase in T cell IL-13 production. Mphi culture supernates were equally effective in augmenting T cell IL-13 levels, suggesting both that cell-to-cell contact is not critical for Mphi augmentation of T cell IL-13 levels, and that Mphi secreted factors are pivotal. CD64(+) Mphi (or their culture supernates), which are known as poor antigen-presenting cells, also effectively augmented T cell IL-13 production, further supporting the involvement of Mphi secreted factors. Finally, experiments with exogenous addition of recombinant monokines, as well as neutralization experiments with different cytokine antibodies, suggested IL-1beta as a primary cytokine involved in the augmentation of T cell IL-13 levels by accessory cells. However, these experiments also indicated other unidentified Mphi factors as playing a significant role in producing maximal T cell IL-13 production. 相似文献
7.
8.
Normal human embryonic lung fibroblasts (WI-38) were infected with Ureaplasma urealyticum, a urea hydrolysing mycoplasma. It was possible to observe reduced rates of multiplication of infected cells and reduced plating efficiency as well as the morphological changes usually associated with mycoplasma infection of animal cells in vitro. The cytotoxic effect on multiplication was sensitive to aureomycin but not penicillin. It was not related to depletion of amino acids or nucleic acid precursors from the cell culture medium but appeared to require that the host cells be growing. Ureaplasmas could not be recovered from cell culture medium after 4 days post infection and their characteristic urease activity could not be demonstrated either in cell culture medium or associated with the cells after the first cell subcultivation. [3H]TdR was incorporated into the nuclei of infected cells and the percent labelled nuclei was reduced compared with uninfected cells. Nuclear labelling indices of infected cells increased as the cells were subcultivated by trypsinization suggesting that the ureaplasmas were removed from the host cell surface by this treatment. In general, the effects of ureaplasmas on WI-38 cells do not appear to be as pronounced as effects of other mycoplasmas on animal cells in culture. It is clear, nonetheless, that the urea hydrolysing mycoplasmas can infect cells in culture and cause discernible effects on the growth and metabolism of these cells. 相似文献
9.
S. J. D. Vitkus S. A. Hanifin D. W. McGee 《In vitro cellular & developmental biology. Animal》1998,34(8):660-664
Summary Intestinal epithelial cells (IEC) have previously been shown to produce several cytokines including interleukin-6 (IL-6).
However, many factors which may regulate IL-6 secretion by human IEC still remain a mystery due in part to the lack of appropriate
model cell lines and the difficulty of culturing human IEC over long periods of time. We have determined that the human colonic
carcinoma cell line Caco-2 is capable of secreting IL-6 when stimulated by the inflammatory cytokines IL-1β or tumor necrosis
factor-α (TNF-α), and stimulation of these cells with IL-1β plus TNF-α induced a synergistic enhancement of IL-6 secretion.
The inflammatory cytokine-induced enhancement in IL-6 secretion was greatest when the cells were cultured in a 10% CO2 atmosphere as compared to cells grown in 5% CO2, suggesting that environmental CO2 levels may affect IEC cytokine secretion. Finally, long-term culture of the Caco-2 cells to induce cellular differentiation
had no effect on the capacity of these cells to produce IL-6, indicating that the regulation of IL-6 secretion was not affected
by differentiation. Taken together, these studies provide important information on the factors which regulate IL-6 secretion
by human IEC as they may contribute to the cytokine network during a mucosal inflammation. The results also suggest that the
Caco-2 cell line is an appropriate model for further studies on the regulation of cytokine secretion by human IEC. 相似文献
10.
Mycoplasmas cause numerous human diseases and are common opportunistic pathogens in cancer patients and immunocompromised individuals. Mycoplasma infection elicits various host immune responses. Here we demonstrate that mycoplasma-infected tumor cells release exosomes (myco+ exosomes) that specifically activate splenic B cells and induce splenocytes cytokine production. Induction of cytokines, including the proinflammatory IFN-γ and the anti-inflammatory IL-10, was largely dependent on the presence of B cells. B cells were the major IL-10 producers. In splenocytes from B cell deficient μMT mice, induction of IFN-γ+ T cells by myco+ exosomes was greatly increased compared with wild type splenocytes. In addition, anti-CD3-stimulated T cell proliferation was greatly inhibited in the presence of myco+ exosome-treated B cells. Also, anti-CD3-stimulated T cell signaling was impaired by myco+ exosome treatment. Proteomic analysis identified mycoplasma proteins in exosomes that potentially contribute to the effects. Our results demonstrate that mycoplasma-infected tumor cells release exosomes carrying mycoplasma components that preferentially activate B cells, which in turn, are able to inhibit T cell activity. These results suggest that mycoplasmas infecting tumor cells can exploit the exosome pathway to disseminate their own components and modulate the activity of immune cells, in particular, activate B cells with inhibitory activity. 相似文献
11.
12.
Portnoy J Pan T Dinarello CA Shannon JM Westcott JY Zhang L Mason RJ 《American journal of physiology. Lung cellular and molecular physiology》2006,290(2):L307-L316
Alveolar type II (ATII) cells inhibit fibroblast proliferation in coculture by releasing or secreting a factor(s) that stimulates fibroblast production of prostaglandin E2 (PGE2). In the present study, we sought to determine the factors released from ATII cells that stimulate PGE2 production in fibroblasts. Exogenous addition of rat IL-1alpha to cultured lung fibroblasts induced PGE2 secretion in a dose-response manner. When fibroblasts were cocultured with rat ATII cells, IL-1alpha protein was detectable in ATII cells and in the coculture medium between days 8 and 12 of culture, correlating with the highest levels of PGE2. Furthermore, under coculture conditions, IL-1alpha gene expression increased in ATII cells (but not fibroblasts) compared with either cell cultured alone. In both mixed species (human fibroblasts-rat ATII cells) and same species cocultures (rat fibroblasts and ATII cells), PGE2 secretion was inhibited by the presence of IL-1 receptor antagonist (IL-1Ra) or selective neutralizing antibody directed against rat IL-1alpha (but not IL-1beta). Conditioned media from cocultures inhibited fibroblast proliferation, and this effect was abrogated by the addition of IL-1Ra. Addition of keratinocyte growth factor (KGF) resulted in an earlier increase in PGE2 secretion and fibroblast inhibition (day 8 of coculture). This effect was inhibited by indomethacin but was not altered by IL-1Ra. We conclude that in this coculture system, IL-1alpha secretion by ATII cells is one factor that stimulates PGE2 production by lung fibroblasts, thereby inhibiting fibroblast proliferation. In addition, these studies demonstrate that KGF enhances ATII cell PGE2 production through an IL-1alpha-independent pathway. 相似文献
13.
Gabriele Mohr Alexandra Preininger Michèle Himmelspach Barbara Plaimauer Christine Arbesser Heinz York Friedrich Dorner Uwe Schlokat 《Biotechnology and Bioprocess Engineering》2000,5(2):84-91
Mycoplasma contamination of tissue culture cells easily evades detection and, thus, represents a continous threat to cell
biologists. In cases where infected cell can not simply be replaced, attempts have to be made to eradicate mycoplasma from
the tissue culture cells. A variety of anti-microbial agents have been shown to be toxic to mycoplasma strains; however, cell
associated mycoplasmas are often protected from antibiotics at concentrations shown to be effectivein vitro. Antibiotic concentrations high enough to be lethal to cell asso|ciated mycoplasmas frequently are also detrimental to the
host cells, while moderately increased antibiotic levels tolerated by the host cells often lead to only temporary growth suppression
and/or to the emergence of mycoplasma strains resistant even to high concentrations of the antibiotic applied. Here, a genetic
approach for the elimination of mycoplasma from tissue culture cells that overcomes these limitations is described. By expression
of a selection marker conferring resistance to an otherwise toxic agent,Acholeplasma laidlawii infected BHK-21 cells used as the model system were enabled to temporarily tolerate antibiotic concentrations high enough
to be lethal to cell associated mycoplasma while leaving the host cells unharmed. Upon successful mycoplasma eradication,
cultivation of the cured host cells in the absence of the selective agent yielded revertant cell clones that had regained
susceptibility to the toxic agent. Cessation of the selection marker expression was shown to result from the loss of the selection
marker DNA, which is a consequence of the fact that the stable and permanent integration of foreign DNA in eucaryotic cell
chromosomes is highly inefficient. Thus, the cells were cured from mycoplasma yet remained biochemically unaltered. 相似文献
14.
Rajbabu Pakala Reuven Laskov Shlomo Rottem Uriel Bachrach 《FEMS microbiology letters》1988,49(3):357-361
Abstract Infecting NIH 3T3 cells with different species of mycoplasmas resulted only in a slight decrease in ornithine decarboxylase (ODC) activity and in the appearance of cadaverine in the infected cells. Similarly, the presence of mycoplasma in NIH 3T3 cells infected with a temperature-sensitive mutant of Rous Sarcoma virus did not bring about any significant changes either in the pattern of ODC activity or in putrescine levels, when transferred to the permissive temperature. This indicates that mycoplasmal contamination of cultures may not significantly change the putrescine metabolism in host cells. On the other hand, the presence of cadaverine in cultured cells may be attributed to contamination by mycoplasma. 相似文献
15.
The contamination of cell lines with mycoplasmas is certainly one of the major problems occurring in cultured cells. Analyzing more than 460 human leukemia-lymphoma (LL) cell lines, we found that 28% of the cultures were mycoplasma-positive. Mycoplasmas can produce extensive changes, growth arrest and cell death in the infected cultures. While mycoplasma-infected cell lines can be truly cleansed from the contaminants, all the efforts would be in vain when the cells return to a mycoplasma-infested environment or are handled with unsuitable culture practices. Hence, the main focus of mycoplasma control should be on preventing cell culture contamination. Mycoplasmas can be introduced through several routes including culture reagents and laboratory personnel. Cross-contamination from infected cell cultures within one laboratory continues to be the major source for the spread of mycoplasma. Specific technical protocols and cell culturing guidelines may be followed in order to minimize the risk of mycoplasma contamination of cell lines. This "good culture practice" is of utmost importance as faulty cell culture techniques appear to be also the main reason for the high incidence of cross-contaminated LL cell lines which according to our experience using DNA fingerprinting of some 500 LL cell lines is about 15%. 相似文献
16.
Monoclonal antibodies were prepared against two different human tumour cell lines, the melanoma cell line SK-Mel-25 and the acute lymphoblastic leukemia T cell line CCRF-CEM. Presence of antibodies against human tumour cells in the supernatants of hybridoma cultures was tested by binding of 125I-F(ab′)2 anti-mouse IgG. On two occasions a hybridoma culture, initially selected for subsequent cloning as it seemingly produced antibodies against tumour cells, was later found to produce monoclonal antibodies specific for Mycoplasma hyorhinis. In immunofluorescent staining patchy structures were visible which seemed to be attached to the cell surface. By combined staining with FITC-conjugated anti-mouse immunoglobulin for monoclonal antibody, Evans blue for cytoplasm and Hoechst compound no. 33258 for DNA, the reaction against mycoplasma could be recognized. These results demonstrate that if cultured cells are used for preparation of monoclonal antibodies, there is a good chance that the selected hybridomas may produce antibodies against ‘culture artifacts’ such as mycoplasmas, in addition to the target antigens. Thus mycoplasma contamination of cell cultures poses a serious problem in the hybridoma research and the testing system for antibody specificity should be carefully monitored. 相似文献
17.
Effect of mycoplasma infection on pyruvate dehydrogenase complex activity of normal and pyruvate dehydrogenase complex-deficient fibroblasts 总被引:1,自引:0,他引:1
The fermentative mycoplasmas A. laidlawii JS, M. hyorhinis DBS-50, M. hyorhinis GDL and M. pneumoniae FH have very high apparent activities of pyruvate dehydrogenase (PDH) (EC 1.2.4.1) and pyruvate dehydrogenase complex (PDHC). Infection of normal and PDHC-deficient fibroblasts with these mycoplasma species resulted in a marked increase of the specific activity of these two enzymes, and under certain conditions could conceal the enzymatic defect. The non-fermentative mycoplasmas M. salivarium VV and M. arthritidis PG-6 have very low apparent activities of these two enzymes. Normal fibroblasts infected with non-fermentative mycoplasmas could appear as deficient in these two enzymes. The degree of interference depends on the number of mycoplasmas associated with the harvested cells. Besides the mycoplasma species, this depends (1) on the duration of infection which determines mycoplasmal titers and also can have a killing effect on both host cells and/or mycoplasmas; (2) harvest of the cells by scraping or trypsinization; (3) centrifugal force used in the collection of the cells; (4) washing and the inherent mechanical treatment; and (5) other possibilities. 相似文献
18.
19.
Highly purified human T cells from peripheral blood fail to produce interferon (IFN)-gamma in the absence of accessory cells. The ability of T cells to produce IFN-gamma upon stimulation with phytohemagglutinin (PHA) or concanavalin A could be restored by the addition of cultured allogeneic human foreskin fibroblasts. Addition of antibodies specific for HLA-DR, DQ, and DP antigens failed to block this accessory function of the fibroblasts. In contrast, antibodies to HLA-DR and DQ antigens inhibited the accessory cell activity of autologous monocytes. Allogeneic fibroblasts failed to exert accessory activity when exogenous interleukin 2 (IL-2) was used as the stimulus for IFN-gamma production. In contrast, autologous monocytes were active as accessory cells for IL-2-stimulated T cells. Addition of recombinant human interleukin 1 alpha (IL-1 alpha) or IL-1 beta to PHA-stimulated T cells co-cultured with fibroblasts stimulated IFN-gamma production. In contrast, preincubation of fibroblasts with IL-1 alpha or IL-1 beta caused a dose-dependent suppression of the ability of fibroblasts to augment PHA- and concanavalin A-induced IFN-gamma production by T cells. Preincubation of fibroblasts with recombinant human tumor necrosis factor (TNF) also reduced their accessory activity. Incubation of fibroblasts with IFN-gamma produced some reduction in their accessory activity and the inhibitory effect of TNF was further enhanced in the presence of IFN-gamma. A 4- to 10-hr incubation of fibroblasts with IL-1 or TNF was sufficient to produce a maximal suppression of accessory activity. Fixation of fibroblasts with formaldehyde decreased their accessory activity, but fixation did not abolish the suppression of accessory function induced by earlier incubation with IL-1. Supernatants of IL-1-treated fibroblast cultures had less suppressive activity than the IL-1-treated fibroblasts per se, and no suppressive activity at all was detected in the supernatants of TNF-treated fibroblasts. Enhanced prostaglandin synthesis may play a role in the IL-1- and TNF-induced suppression of accessory cell function, but other factors are likely to be involved. Our results show that fibroblasts can have a marked effect on T cell function and that IL-1 and TNF can exert immunoregulatory activities indirectly by altering the interactions of fibroblasts with T cells. 相似文献
20.
Hans G. Drexler Suzanne M. Gignac Zhen-Bo Hu Anne Hopert Elke Fleckenstein Maren Voges Cord C. Uphoff 《In vitro cellular & developmental biology. Animal》1994,30(5):344-347
Summary 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 speciesMycoplasma arginini andM. 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. 相似文献