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Biomedical subjects

M G Gabridge

Publications and source records attributed to M G Gabridge.

At least 19 recordsLinked to original sources
PubMed1998

Cell culture contamination: sources, consequences, prevention, and elimination.

The subject of the chapter is cell culture contamination. Contamination may enter the cell culture system as a physical, chemical, and/or biological component of the environment. The potential sources and consequences of cell culture contamination are unique to the cell culture system and the contaminant. A basic understanding of cell culture contamination is necessary to appreciate the need to develop and practice standardized cell culture procedures. General sources, consequences, and preventative measures are discussed for physical and chemical contamination based on current technology. Mycoplasmal contamination is the focus of the discussion on biological contamination and its impact on cell cultures. The introduction of other biological contaminants should be controlled by the institution of cell culture management procedures needed to minimize the incidence of mycoplasmal contamination. The need to eliminate the routine use of antibiotics in cell culture systems and institute routine testing to detect contamination is emphasized. More rapid detection of contamination should reduce the incidence of cross-contamination and minimize the consequences of any contamination event.

Animals↗
PubMed1988

Species-specific biotinylated DNA probe for the detection of Mycoplasma gallisepticum.

A 5.5 kilobase DNA fragment from an Eco RI digest of the Mycoplasma gallisepticum genome was specific for the detection of M. gallisepticum. This 5.5 kb fragment was initially cloned into bacteriophage lambda gt11 followed by subcloning into the plasmid vector pGEM-3Z. The incorporation of a biotin label was accomplished by utilizing biotin-11-dUTP in a nick translation reaction. This probe, designated pMg6, reacts specifically with M. gallisepticum when tested against various mycoplasma DNAs in Southern blot hybridization analysis. Spot-blot hybridization data indicate the pMg6 is capable of detecting 800 pg of M. gallisepticum DNA.

Biotin↗
PubMed1987

Characterization of a human lung fibroblast receptor site for Mycoplasma pneumoniae.

Mycoplasma pneumoniae, a respiratory pathogen of humans, is the etiologic agent of primary atypical pneumonia. The mycoplasma attaches to the host cell by means of a specialized terminal structure. This structure binds the organism to the corresponding receptor site on the host cell. A glycoprotein receptor site for M. pneumoniae was isolated from MRC-5 human lung fibroblasts. It was isolated from a Triton X-100 extract by column affinity chromatography, utilizing wheat germ agglutinin bound sepharose 6MB. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed it to have an MW of approximately 100 kilodaltons (kDa) and to be composed of subunits of approximately 21 kDa. At a concentration of 56 micrograms/ml, the receptor inhibited the binding of 14C-labeled M. pneumoniae to MRC-5 fibroblasts by 77%. Chemical analysis determined that it does not contain any detectable sialic acid. Knowledge of the biochemical and immunological characteristics of the receptor site are essential to understand the attachment phase of the pathogenic process and to further the development of effective prophylaxis.

Cell Line↗
PubMed1987

Metabolic consequences of Mycoplasma pneumoniae infection.

Mycoplasma pneumoniae is a human respiratory pathogen with the ability to infect a wide variety of cell types. Most studies on the nature of metabolic consequences of infection have used cell culture and explant models. Lung fibroblast monolayer cell cultures and rodent tracheal explant cultures have both served as host cells for M. pneumoniae and display metabolic and morphological alterations post infection. Tracheal explants exhibit a decrease in ciliary motion and desquamation, along with a generalized shutdown of major metabolic pathways. Oxygen uptake, dehydrogenase activity, and ATP content all decrease significantly, though 24 to 96 h may be required for detection. Metabolism is affected more rapidly in homogeneous fibroblast monolayers. In those cells, a significant decrease in de novo purine synthesis occurs within 4 h, whereas other pathways, such as salvage DNA synthesis and RNA synthesis had an increased activity. The molecular signals that mediate these changes remain to be delineated, although the inventory of metabolic changes provides a critical step in this process.

Adenosine Triphosphate↗
PubMed1986

Detection and speciation of common cell culture mycoplasmas by an enzyme-linked immunosorbent assay with biotin-avidin amplification and microporous membrane solid phase.

An enzyme-linked immunosorbent assay (ELISA) was developed in order to serve in detecting and speciating mycoplasmas isolated from cell cultures. Its main features included a biotin-streptavidin amplification step and a solid phase consisting of a microporous membrane. Cell samples in the form of suspensions were applied to nitrocellulose or ion exchange membranes immobilized in commercially-available microtiter, multiwell manifolds. The blocking buffer contained 1% purified alpha-casein. The primary antibodies were monoclonal and the polyclonal secondary antibody was biotinylated. The enzyme utilized was streptavidin-horseradish peroxidase. The substrate-dye complex consisted of either 4-chloro-1-naphthol and hydrogen peroxide or ortho phenylene diamine (OPD) and hydrogen peroxide. The presence of homologous antiserum in the reaction sequence gave clearly visible, colored reactions on the membrane when 50 ul with approximately 10(5) or more cfu/ml were present. This new biotin-avidin microporous membrane (BAMM-ELISA) test can be used both to detect mycoplasmas and to speciate them. The BAMM-ELISA is simple, rapid, sensitive, specific and economical. As such, it has potential for aiding in the control of mycoplasma contamination in cell culture, and could prove useful in clinical diagnostic applications as well.

Animals↗
PubMed1984

Gaseous oxide toxicity evaluated with cell monolayers on collagen-coated, gas-permeable teflon membranes.

A system was developed to evaluate the cytotoxic potential of gaseous oxides in vitro. Target cells were MRC-5 human lung fibroblasts cultivated as monolayers on gas-permeable , FEP-Teflon membranes. Membranes were secured in Chamber/Dishes with a 25 mm diameter well. To promote attachment of fibroblasts to the membranes, the latter were incubated in collagen ( Vitrogen ) solutions for 10 min prior to plating the cells. The collagen pretreatment was significantly more effective than poly-L-lysine, fetal calf serum, polybrene and bovine serum albumin. Several types (mouse and calf) of acid-soluble and alcohol-soluble collagen fractions were evaluated, and all of them promoted cell attachment with equivalent efficiency. Cells on membranes were exposed to gases in a Plexiglass chamber with a gas flow of 2L/min. Sulfur dioxide caused a marked loss in cell viability (as indicated by ATP content of the monolayer) after 30 min exposure to 0.01% and 0.005%. A level of 0.001% did not affect viability, and none of the levels tested caused a sloughing of the monolayer after 90 min. Nitrogen dioxide induced a more modest drop in cell viability after 30 min exposure to 0.1%, while 0.005% and 0.05% were nontoxic. No cell sloughing occurred with NO2 exposures, and exposures to CO2 at levels of 20% for 90 min were nontoxic. This system, with cell culture monolayers on gas-permeable Teflon membranes, is simple and convenient. As such, it has potential application to cytotoxicity evaluations with numerous gases.

Adenosine Triphosphate↗
PubMed1984

In vitro models for host-parasite interactions involving mycoplasmas.

Model systems for the study of mycoplasma infections are available with various degrees of complexity. They offer the advantages of simplicity and control of environmental variables. More significantly, they have a homogeneity of cell type, which is necessary when analyzing the biochemical factors in pathogenicity. Cell cultures of fibroblasts are especially useful in studies of receptor sites. Human lung fibroblasts in monolayer format contain a sialoglycoprotein to which Mycoplasma pneumoniae attaches. Fibroblast membranes are extracted with lithium diiodosalicylate or Triton X-100 to provide a fraction rich in glycoprotein. Polyacrylamide gel electrophoresis revealed less than eight proteins, with the majority of the carbohydrate in a single zone. Autoradiography of gels from fibroblasts grown in radiolabeled medium revealed that this zone also had high concentrations of glucosamine. Detergent extracts competitively inhibited the attachment of M. pneumoniae to fibroblasts. Cultures of fibroblasts also have been used to show that M. pneumoniae disrupts de novo purine synthesis within hours after infection. Monolayers of ciliated respiratory epithelial cells displayed an even distribution of receptor sites over the cell membrane. Intact trachea perfusion cultures were used to establish the pattern of pathogen distribution in upper airways. These in vitro approaches provide valuable insight into the process of mycoplasma attachment and the subsequent metabolic alterations that cause cytotoxicity.

Adhesiveness↗
PubMed1983

Differential cytopathogenicity accompanying Mycoplasma pneumoniae infection of human lung fibroblasts maintained in newborn bovine serum or fetal bovine serum.

MRC-5 human lung fibroblasts maintained in Eagle's basal medium (BME) with either 10% fetal bovine serum (FBS) or 10% newborn bovine serum (NBS) did not respond identically to infection by Mycoplasma pneumoniae. Fibroblasts grown in NBS did not develop any cytopathic effect (CPE) when infected with M. pneumoniae, whereas those maintained in FBS developed a pronounced CPE. There was also a difference in sensitivity to infection for fibroblasts maintained in the two sera before the infection. Fibroblasts maintained in NBS, then transferred to FBS 48 h before infection, were still less sensitive to M. pneumoniae infection than cells maintained constantly in FBS. Mycoplasma pneumoniae attached comparably to the fibroblasts grown in the two sera, so the differences in CPE development could not be attributed to differences in mycoplasma attachment. Measurements of DNA, RNA, and protein syntheses of the fibroblasts grown in NBS and FBS indicate that the cells in NBS were growing more rapidly than those in FBS. A determination of the doubling times shows that the doubling time of cells in NBS was 44 h, whereas that of cells in FBS was 51 h. Polyacrylamide gel electrophoresis of samples of NBS and FBS showed significant differences in serum protein composition. The NBS had several protein bands that were lacking in the FBS. This study demonstrates the importance of serum effects in the study of M. pneumoniae infection.

Animals↗
PubMed1983

Improved method for transmission electron microscopy of ciliated cell monolayers maintained on gas-permeable membranes.

A new method is described for preparing thin sections for transmission electron microscopy of ciliated respiratory epithelial cells cultivated in vitro on Teflon membranes. Hamster tracheal explant cultures were treated with collagenase to promote epithelial migration. Patches of monolayer growth occurred in the vicinity of the explants when the latter were incubated on FEP-etched Teflon membranes in Chamber/Dishes. Cells in the monolayer maintained a high degree of differentiation as evidenced by active ciliary motion. The monolayer was fixed and processed in situ. During the embedding process the chamber was supported on a solid Teflon cylinder to keep the membrane taut as it was heated to polymerize the resin. The membrane was removed and the monolayered cells were re-embedded and sectioned. Electron microscopic examination of cultures revealed clear cell profiles on a flat, even basal plane. This method provides for a normal spatial configuration of cells in a monolayer format, and should prove useful for electron microscopy studies of any cells cultured on flexible films.

Animals↗
PubMed1983

De novo purine synthesis, purine salvage, and DNA synthesis in normal and Lesch-Nyhan fibroblasts infected with Mycoplasma pneumoniae.

The effects of Mycoplasma pneumoniae on host cell metabolism were studied by using two types of host cells, MRC-5 human lung fibroblasts, a normal cell line, and Lesch-Nyhan fibroblasts, a cell line deficient in hypoxanthine-guanine phosphoribosyl transferase (EC 2.4.2.8). The susceptibilities of the two cell types were determined by infecting the cells with M. pneumoniae at different multiplicities of infection (MOI). Our data indicate that the Lesch-Nyhan cells were four times more susceptible to damage by M. pneumoniae than the MRC-5 cells. The effects of different MOIs (10 and 50) on de novo purine synthesis. DNA synthesis, and the development of a cytopathic effect were determined. In both cell types, the higher MOI inhibited de novo purine synthesis to a greater extent than the lower MOI. This correlated closely with the cytopathic effect which developed in the monolayers (i.e., the more the inhibition of de novo purine synthesis, the greater the cytopathic effect which developed). In the Lesch-Nyhan cells, DNA synthesis was completely inhibited by the high MOI, whereas in the MRC-5 cells, DNA synthesis was stimulated by the high MOI. In the MRC-5 cells infected with M. pneumoniae, purine salvage activity increased, as indicated by an increase in adenosine deaminase (EC 3.5.4.4) activity. These data indicate that M. pneumoniae alters host cell metabolism, particularly the nucleic acid metabolic pathways. This may explain in part the mechanism of pathogenesis of M. pneumoniae infection.

Adenosine Deaminase↗
PubMed1982

Effects of heavy metals on structure, function, and metabolism of ciliated respiratory epithelium in vitro.

Tracheal explants were used to evaluate the relative ciliostatic and cytotoxic potential of heavy metal salts (cadmium chloride, chromium chloride, nickel chloride, and copper sulfate). Explants from hamster, rat, and guinea pig were all sensitive to the metals, though guinea pig explants showed the greatest difference between the untreated and metal treated tissues. Dosage levels were 50, 100, and 500 microM, for 24 to 148 h. Cadmium caused the greatest degree of ciliostasis and cell necrosis. Copper was less toxic, and nickel and chromium caused marginal damage when tested at 100 microM or lower. In each instance, damage became detectable at approximately 24 to 48 h and was nearly stabilized by 72 h. A significant loss of ciliary motion was always accompanied by a decrease in metabolic activity (dehydrogenase activity and ATP content). Transmission and scanning electron microscopy revealed a severely necrotic epithelium after exposure to cadmium, with only subtle morphological alterations after exposure to other metals. With all of the treatments there was no overt structural damage to cilia and little alteration in membranes of cells remaining in the epithelium. Some coagulation or vacuolization was noted in cadmium and copper treated explants but most cellular organelles did not display obvious damage. The most significant changes in the tracheal epithelium exposed to heavy metal salts in vitro were a loss of ciliary motion and a decrease in total ATP content.

Animals↗
PubMed1982

Scanning electron microscopy of Mycoplasma pneumoniae on the membrane of individual ciliated tracheal cells.

Cell monolayer cultures were prepared from hamster tracheal explants by a collagenase exposure and subsequent incubation in Waymouth's MAB 87/3 medium. The epithelial outgrowth occurred on glass cover slips. Cilia on the monolayers continued to beat normally after the "parent" explant was removed. Monolayer cultures infected with Mycoplasma pneumoniae had significant amounts of attachment. A morphological analysis of the attachment was conducted with scanning electron microscopy. Clusters, cocci, and filaments of M. pneumoniae all attached to the epithelial cells, but the filaments were especially common. Mycoplasmas were seen in association with both ciliated and nonciliated cell membranes. On ciliated cells, mycoplasmas were on the ciliary strands and on the cell membrane. When located immediately adjacent to or in between cilia, mycoplasmas were oriented vertically with the constricted attachment tip oriented down toward the host cell membrane. When located more than a micron away from the ciliary fibers, mycoplasmas lay horizontally along the epithelial cell membrane. The photographic data suggest that clusters or "sperules" of mycoplasmas may liberate individual mycoplasmas that attach to the cell membrane. It appears that the receptor sites for M. pneumoniae are rather uniformly distributed along the ciliated cell membrane, and are not restricted to the interciliary areas.

Cell Membrane↗
PubMed1982

Alterations in nucleotide content of human lung fibroblasts infected with Mycoplasma pneumoniae.

The nucleotide content of normal MRC-5 human lung fibroblasts and fibroblasts infected with Mycoplasma pneumoniae PI 1428 was determined. Nucleotides from control and infected fibroblasts were extracted with 5% trichloracetic acid. After neutralization of the extracts, the nucleotides in the extracts were separated by anion-exchange chromatography. Significant differences were found between the nucleotide content of the control and infected cells. Nucleotide triphosphate levels were twofold higher in the control fibroblasts than in the infected fibroblasts 4 h after the initiation of infection. At the same time, nucleotide diphosphate and monophosphate levels were higher in the infected fibroblasts than in the control fibroblasts. Determination of the energy charge ratio for each set of nucleotides (adenosine, guanosine, cytidine, and uridine) demonstrated a shift of nucleotide content in the infected fibroblasts. Immediately after infection, the energy charge for each set of nucleotides was higher for the control fibroblasts than it was for the infected fibroblasts. This pattern continued throughout the infection period with only minor exceptions. The work presented here indicates a loss of energy charge in fibroblasts infected with M. pneumoniae and may help to explain some of the metabolic changes and cell damage which accompany infection.

Adenine Nucleotides↗
PubMed1982

Cytotoxicity and ciliostasis in tracheal explants exposed to cadmium salts.

Cadmium salts were examined for their biological effects on ciliated respiratory epithelium in hamster tracheal explants. Cadmium chloride and cadmium acetate both caused significant decreases in ciliary motion when tested at 100 micrograms M and above. Reductions in relative ciliary activity were dose-dependent and were first demonstrable at 8-32 hr. The decreased ciliary motion was accompanied by decreases in two key metabolic compound (ATP and dehydrogenase) which are normally associated with cell viability. Histopathological examination of cadmium-treated tissues showed an epithelium thinner than normal, with extensive vacuolization and few, if any, intact ciliated cells. The various biological effects exerted by cadmium are presented, along with potential mechanisms of pathogenesis for the observed ciliostasis and cytonecrosis. Decreases in adenosine triphosphate appear to play a critical role in the development of cadmium-related effects on cellular function and metabolism.

Adenosine Triphosphate↗
PubMed1981

The Chamber/Dish: an improved vessel for cell and explant culture.

A vessel consisting of a Teflon well former, metal base plate, rubber gasket, and variable growth surface has been constructed. It is nontoxic, autoclavable, and reusable. It provides for ready access into the growth chamber, plus an optically clear lower surface for improved microscopic resolution. The vessel has been used successfully with several different eucaryotic and procaryotic cell systems. Morphology of cells and yields (as measured by total protein and ATP) are comparable to those noted with other vessels. This Chamber/Dish can accommodate almost any type of growth substrate in the form of films or sheets. It appears to have widespread application for the growth of cells and explants in vitro.

Animals↗
PubMed1981

Mycoplasma pneumoniae infection of intact guinea pig tracheas cultured in a unique matrix-embed/perfusion system.

A new method for the in vitro culture of entire, intact tracheas from adult guinea pigs is described. Matrix-embed/perfusion (MEP) culture is based on an immobilization of the tissue in nutrient agar The tubular piece of agar-embedded organ was contained in a special perfusion block with two wells for liquid medium at either end. When incubated on a rocker platform, liquid medium flows through the trachea and supplies oxygen an nutrients. In this configuration, tracheas maintain near-normal metabolism (ATP content and dehydrogenase activity), structure (as determined by light and electron microscopy), and function (ciliary motion). Tissues could be maintained in vitro in a normal for at least 4 wk, with reduced ciliary motion and cell metabolism detectable for at least 6 wk. Agar-embedded tissues from the MEP cultures were nearly identical to those cultivated with standard tracheal ring explant techniques. Tracheas in the MEP cultures were infected with Mycoplasma pneumoniae. Attachment was neuraminidase-sensitive. Mycoplasma attachment was lowest on the epithelium along the dorsal ridge, but was uniform along the length of the trachea. Ciliostasis and cytonecrosis induced by M. pneumoniae was dose dependent. The matrix-embed/perfuse technique appears to have considerable potential for several types of in vitro studies on trachea or other tubular organs.

Adhesiveness↗
PubMed1981

Role of host cell metabolism in the pathogenesis of Mycoplasma pneumoniae infection.

Human lung fibroblasts develop a cytopathic effect (CPE) when infected with Mycoplasma pneumoniae. This study was designed to determine the relationship between host cell metabolism and the formation of the CPE. Human lung fibroblasts were grown in different serum concentrations, plated at different densities, and grown for different periods of time to alter the metabolic activity of the cells. Deoxyribonucleic acid, ribonucleic acid, and protein syntheses were measured by the ability of the cells to incorporate thymidine, uridine, and leucine, respectively. With each treatment, leucine incorporation remained constant. Thymidine and uridine incorporation was higher when the cells were in high serum, at low cell densities, or grown for 2 or 3 days. The appearance of an observable CPE, which was corroborated with a protein synthesis assay, correlated closely with thymidine and uridine incorporation. A more pronounced CPE was seen when thymidine and uridine incorporation was high. In addition, it was found that the first 2 h after infection by M. pneumoniae were the critical hours in determining whether a CPE would develop. This was accomplished by altering the serum concentration of the culture medium at different times postinfection and thereby altering the metabolism of the fibroblasts. These results demonstrate the importance of the metabolic state of the host cells in studying mycoplasma infections and establish a correlation between the nucleic acid metabolism of the cells and the production of a CPE.

Blood↗