Identification of Mycoplasma binding proteins utilizing a 100 kilodalton lung fibroblast receptor.

A 100 kilodalton glycoprotein receptor for Mycoplasma pneumoniae has been isolated from MRC-5 human lung fibroblasts. This receptor, as well as anti-receptor serum, were both capable of inhibiting the attachment of 14C-labelled M. pneumoniae to MRC-5 fibroblasts. The receptor was also capable of inhibiting the attachment of C-labelled M. gallisepticum and M. genitalium, but not M. pulmonis, to MRC-5 fibroblasts. This indicates that a common sequence may exist in these binding proteins of M. pneumoniae, M. genitalium, and M. gallisepticum. This receptor and anti-receptor serum were utilized to probe M. pneumoniae, M. genitalium, and M. gallisepticum for their corresponding binding proteins. A 32 kilodalton protein in M. pneumoniae, a 90 kilodalton protein in M. genitalium and a 139 kilodalton protein in M. gallisepticum were recognized.

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.

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.

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.