Biomedical subjects
Publications and source records attributed to M G Gabridge.
The effect of fusogenic agents on the interaction of Mycoplasma pneumoniae with human lung fibroblasts was evaluated. A 60-s exposure of the fibroblasts to polyethylene glycol (molecular weight 200; 10%) significantly increased attachment of mycoplasmas to the fibroblasts.
Mycoplasma pneumoniae induces pneumonia-like symptoms in hamsters and causes ciliostasis and cytonecrosis in hamster tracheal explants. 2,4-Dimethylphenol and, to a lesser extent, its 2,3-, 2,5-, and 2,6-dimethylphenol isomers protected tracheal explants from these changes after exposure to virulent M. pneumoniae strain PI 1428. The effect was concentration, time, and isomer dependent. At concentrations of 10(-9) M or greater, 2,4-dimethylphenol completely prevented the morphological (loss of ciliated cells) and biochemical (decreased dehydrogenase activity) changes normally observed after exposure to M. pneumoniae. Apparently, 2,4-dimethylphenol interfered with an early event in the infection process. Complete protection required that it be present during the first 2 h of exposure of the explants to the infecting mycoplasmas. These xylenols may prove to be useful tools for helping to define the mechanisms of pathogenesis in certain respiratory infections.
Exogenous cyclic AMP and dibutyryl cyclic AMP decreased the relative ciliary activity values of tracheal organ cultures. In contrast, theophylline and cholera toxin were not ciliostatic. The use of a radioimmunoassay for cyclic AMP indicated that all of the tested substances increased intracellular cyclic AMP levels to some extent (from 3-fold for cholera toxin to almost 40-fold for dibutyryl cyclic AMP). Physical inactivation of explants by either freeze-thaw or heat destroyed all ciliary activity and greatly decreased intracellular cyclic AMP levels. Cyclic AMP levels of explants remained relatively constant during in vitro cultivation. Three strains of Mycoplasma pneumoniae were found to contain extremely low amounts of cyclic AMP. Infection of tracheal explants produced a significant decrease in relative ciliary activity, but only a slight decline in organ-culture cyclic AMP levels.
The interaction of pathogenic Mycoplasma pneumoniae and host cells was studied in cell cultures of MRC-5 human lung fibroblasts. A comparison of results obtained with fibroblasts in a monolayer format and with hamster tracheal explant cultures indicated that the former can bind significantly larger numbers of mycoplasmas. In addition, the attachment was 96% specific, that is, mediated through a neuraminidase-sensitive receptor on the host cell. Uptake of mycoplasmas was directly related to the number of mycoplasma cells present in the inoculum, and attachment was virtually complete within a 30-min period at 37 degrees C. High doses of M. pneumoniae induced a marked cytopathic effect, whereas doses of less than or equal to 10(6) colony-forming units per ml produced grossly observable cell damage that was moderate and variable. Transmission electron microscopy studies indicated that attachment of M. pneumoniae to the surface of lung fibroblasts occurred with the specialized terminal structure or binding site oriented closest to the epithelial cell surface. The filamentous mycoplasma cells were spatially arranged in several configurations and were not limited to a vertical orientation. The advantages and disadvantages of human lung fibroblast monolayer cultures, in reference to other in vitro models are discussed. A new mycoplasma agar medium (G-200 agar) with a defined tissue culture base and 10% horse serum is also described.
The biochemical nature of the neuraminidase-sensitive Mycoplasma pneumoniae receptor site on human lung fibroblast cells was studied. Purified, mixed sialoglycolipid (ganglioside) preparations from human and bovine tissues did not bind to M. pneumoniae organisms and block their subsequent attachment to fibroblasts. Fibroblasts incubated for 24 h in sialoglycolipid solutions to increase the ganglioside content of their membranes did not show increased pathogen attachment when later incubated with mycoplasmas. HeLa cells grown in the presence of sodium butyrate to increase GM3 ganglioside levels likewise did not have significantly increased uptake of M. pneumoniae organisms. Treatment of fibroblasts with enzymes indicated that the mycoplasma receptor site is trypsin and papain resistant but Pronase sensitive. Pronase digests of fibroblast membranes contained a product(s) which combined with M. pneumoniae cellls and cosedimented with them during centrifugation. Glycoproteins, purified from fibroblast membranes by a lithium diiodosalicylate solubilization technique, similarly bound to M. pneumoniae organisms. Collectively, these data suggest that the major component of the M. pneumoniae receptor site is a sialoglycoprotein with little or no lipid.
Explore the source record for details and available documents.
A tracheal ring explant system, when used with 25% cystic fibrosis (CF) serum, displayed obvious ciliostasis. Hamster, rabbit, and guinea pig explants all had measurable decreases in ciliary activity after 24 hr of incubation in the serum. The differential response to CF serum (relative to normal serum) was greatly increased by using explants which were maintained 24-72 hr in minimal essential medium (MEM) with 10% horse serum and which were selected on the basis of optimal ciliary activity and vigor. With such a bioassay system of guinea pig tracheal explants, incubation with 25% normal serum would produce essentially no change in relative ciliary activity (score of 242 of a possible 300), whereas CF serum resulted in an 86% decrease (score of 33). Scanning electron microscopic observation indicated that the explants displaying the CF-ciliostatic effect had significant accumulations of mucous over the ciliated epithelial surface. A biochemical viability assay (dehydrogenase activity) showed no cytonecrosis when CF serum-treated tissues were compared to standard explants (10% horse serum in MEM) or control explants (25% normal human serum).
The effect of environmental conditions on the relative pathogenicity of Mycoplasma pneumoniae for hamster tracheal explants was investigated. Organisms from the early stages of the growth cycle (e.g., day 1 to 2) were more effective in the induction of ciliostasis than were older cultures. Both the degree of ciliostasis and the speed of onset were affected. The type of explant culture medium also affected pathogenic potential. M. pneumoniae infection produced significantly greater ciliostasis and cytonecrosis in a "permissive" medium, i.e., one capable of supporting mycoplasma metabolism and replication, than in a "nonpermissive" medium. However, no adenine protection effect could be detected under permissive conditions, though it was quite striking when a nonpermissive medium was used as the post-infection explant medium. This suggests that the cell damage noted under permissive conditions may result from processes distinct from those operative in the actual host-parasite cellular interaction.
Adenine sulfate and several related compounds were evaluated for their ability to retard the cytotoxic effect which normally accompanies M. pneumoniae infections of hamster tracheal explants. Adenine sulfate, at the 0.01 mM level, was found to exert a significant protective effect. Little or no ciliostasis or loss of cell viability was detectable when organ cultures were infected with 10(7) CFU of virulent M. pneumoniae in the presence of the adenine supplement. Mycoplasmas grew in broth and on plastic surfaces in the presence of adenine, and no significant diminution of growth rate or cell yield was detectable. Organisms adhered to the tracheal epithelial surface regardless of the presence or absence of adenine. When explants were incubated in the presence of 14C-(8)-adenine, rinsed, and then infected with M. pneumoniae, the adenine label could be recovered from the mycoplasmas 20 h after the infection. These data are compatible with the known nucleic acid requirement of mycoplasmas and with a model which ascribes a role for purine/pyrimidine competition and/or depletion in the infective process.
Hamster respiratory epithelial cells were cultured in a monolayer format, and 20% of the cells were ciliated. Mycoplasma pneumoniae attached to the epithelial cells in a neuraminidase-specific fashion and induced ciliostasis and cytonecrosis.
The amount of adenosine triphosphate (ATP) in hamster trachea organ cultures was determined with a technique based on light emission from a luciferin/luciferase/ATP reaction. The amount of ATP, expressed as ng per mg dry weight, was consistent in tracheal explants prepared from various animals and changed negligibly when explants were cultivated in vitro for several days. The amount of ATP was related directly to cellular activity and integrity in the epithelium since inactivation by heat or freeze-thaw rapidly depleted measurable ATP, and ciliary activity and ATP content were related directly. When tracheal explants were infected with 10(5) to 10(7) CFU of virulent Mycoplasma pneumoniae cells, both ciliary activity and ATP content in the tissue dropped dramatically after approximately 5 to 8 days (up to 85% and 60% decreases, respectively). Exposure of explants to 50 to 200 microgram per ml of purified M. pneumoniae membranes also caused significant decreases in ciliary activity and ATP. When explants were infected with attenuated or nonvirulent mycoplasmas, ciliary activity was only slightly decreased, while ATP values often rose slightly. The technology associated with the determination of ATP levels in tracheal explants should prove useful as a new, objective, analytical approach to cell viability in organ cultures.
The morphology of the inner aspect of the adult hamster trachea was examined by scanning electron microscopy. Relatively large patches of unciliated cells were observed in the epithelial layer. The patches, which covered several hundreds to thousands of square microns, were most conspicuous on the ventral surface of the trachea, especially in the middle third. The frequency of these areas of unciliated cells, both isolated and in patches, was much greater in hamsters than in mice, rats, or cats. Greatest ciliation in the hamster trachea was observed over the strip of trachealis muscle between the open ends of the cartilaginous rings. Areas with the heaviest ciliation also had the greatest activity of cellular metabolism, as measured by the tetrazolium reduction assay. The attachment of tritium-labeled cells of Mycoplasma pneumoniae was inversely correlated with extensive ciliation, since the greatest numbers of counts were found on the middle third and ventral regions of the tracheal surface. The results of this study suggest that the regional differences in ciliation of respiratory epithelium in hamsters may influence studies of pathogenesis and isolation of M. pneumoniae and that these differences should therefore be considered and controlled in the experimental design.
An organ culture system for hamster trachea was developed for maintenance of the ciliated respiratory epithelium during periods of extended cultivation (i.e., greater than 20 days). Evaluation of five serum types showed that horse serum and fetal calf serum were best for the maintenance of epithelial ciliary activity and morphology. Rings that were opened on one side ("split rings") had the best maintenance of the ciliated epithelium as judged by the retention of ciliary activity and normal histological appearance after 3 to 4 weeks in culture. The in vitro induction of squamous metaplasia was achieved by cultivating explants in Waymouth MAB 87/3 (vitamin A-free) medium, without serum. This system allowed a direct comparison of the effects of Mycoplasma pneumoniae infection in two epithelial types, ciliated pseudostratified columnar and keratinizing squamous. Attachment of 14C-labeled mycoplasmas was more than twofold greater in the normal epithelium. Pretreatment of explants with neuraminidase decreased attachment for both squamous and pseudostratified epithelial surfaces to a similar basal level. Recovery of viable organisms from infected tissue of both epithelial types indicated that the organism titer remained essentially constant during the infection period, but was significantly higher for the pseudostratified ciliated epithelium. These results suggest that specific receptor sites for M. pneumoniae are markedly reduced by the induction of squamous metaplasia and, hence, appear to be specific for the normal respiratory surface containing goblet cells and pseudostratified, ciliated epithelial cells.
Hamster trachea organ cultures were exposed to isolated membranes of Mycoplasma pneumoniae, PI 1428. Attachment, monitored by the uptake of tritiated membranes, was relatively insensitive to neuraminidase pretreatment, unlike the attachment of viable cells. Membrane attachment was optimal when explants were incubated with 50 to 100 micrograms of membrane protein per ml in minimal essential medium broth while gently being rotated (1 rpm) in a roller apparatus for 90 to 120 min at 37 degrees C. Saturation of the receptor sites with viable cells failed to inhibit subsequent membrane attachment. Induction of squamous metaplasia by extended cultivation of tracheal explants in a vitamin A-free medium reduced the content of ciliated cells without significantly affecting total cell viability, but did not alter the attachment of M. pneumoniae membranes. Collectively, the data indicate that the mechanism of attachment of M. pneumoniae membranes to respiratory epithelium is distinct from the receptor site-mediated attachment of M. pneumoniae cells.
Explore the source record for details and available documents.
A new medium, which contained a chemically defined tissue culture base ("medium 199"), was developed for the cultivation of mycoplasmas. When supplemented with albumin, glucose, serum, and yeast extract, the new medium adequately supported the growth of Mycoplasma and Acholeplasma species.
The ability of Mycoplasma pneumoniae cells and membranes to affect tetrazolium reduction by hamster trachea organ cultures was evaluated. Uninfected trachea explants reduced 2,3,5-triphenyl tetrazolium chloride (TTC) and nitro-blue tetrazolium when incubated at 37 C in the absence of air. Reduced tetrazolium salts (formazans) were extractable with acetone or ethylene glycol and could be quantitated spectrophotometrically. The optimal assay system involved the use of three or more tracheal rings incubated for 2 h in 0.12% TTC in Tyrode balanced salts supplemented with 1.2% sodium succinate. Formazan was extracted for 5 min with acetone, and the optical density (490 nm) was determined. Trachea explants with metabolic activity reduced or obliterated by freeze-thaw lysis, heat (56 C X 30 min), or cyanide (0.1 M NaCN X 30 min) had negligible ciliary activity and tetrazolium reduction activity (optical density at 490 nm [dry weight]). Tracheas exposed to mycoplasma cells or membranes also showed significantly decreased ciliary activity and tetrazolium reduction; e.g., only 5pc of the ciliary activity and reduction capacity remained after 5 days in culture when infected with M. pneumonia PI 1428 cells. The data indicate that the exposure of ciliated respiratory epithelium to mycoplasma cells or membranes results in diminished oxidative metabolism, and that the ability to reduce TTC to its formazan is correlated with relative ciliary activity.
The effects of chemical, physical, and infectious cytotoxic agents on primary and cultured cells were evaluated by measurements of oxygen uptake for various time periods. A newly developed respirometer used a Clark oxygen electrode in a 1.0-ml chamber, with provisions for constant mixing and for temperature control of both the sample and electrode chambers. The device was unique because the electrode and instrumentation were provided by a clinical blood-gas analyzer. Oxygen uptake by blank controls was negligible, whereas cells and tissue consumed oxygen at rates of approximately 1 to 5 mul/h in a dose- and temperature-dependent fashion. Cyanide, heat, and freeze-thaw lysis reduced the oxygen uptake to less than 0.6 mul/mg per h. Infection of trachea organ cultures with Mycoplasma pneumoniae significantly reduced relative ciliary activity, tetrazolium reduction capacity, and oxygen consumption in a coordinated fashion.