Transient inhibition of cell proliferation in rat glioma monolayer cultures by cortisol.

The effect of 3 muM cortisol on cell proliferation in rat glioma (strain C6) monolayer cultures was investigated. Cell density measurements showed that cortisol-treated C6 cells continued to proliferate at maximum log phase rates for 1 to 2 days. Then cell proliferation ceased as growth in control cultures continued into stationary phase. A 2-day period of growth inhibition followed during which cell densities were 30 to 50% lower relative to controls. Growth resumed subsequently, and final cell densities were similar to those of controls. The presence of epicortisol (the biologically inactive isomer of cortisol) in the culture medium did not alter the rate of log phase growth relative to controls. During the initial period of continued growth after exposure to cortisol, the pH of the medium decreased at the same rate in control and treated cultures. During the growth-inhibitory period, erythrosin B dye was excluded equally well (greater than 94%) by control and treated cells, and no morphological differences were detected by phase contrast microscopy. When the culture medium was replaced daily, the control cells at elevated densities continued to proliferate at a reduced rate. In cortisol-treated cultures, the period of growth inhibition commenced 3 days after the cells were exposed initially to cortisol. A 2-day period of growth inhibition followed during which the pH of the 1-day-old media from both control and treated cultures decreased from 7.4 to 6.9. Growth resumed subsequently in the treated cultures to produce elevated cell densities similar to those of controls. These results demonstrate that cortisol at concentrations considered chemotherapeutic in vivo exerts a transient inhibitory effect on C6 glioma cell proliferation.

Combined growth-inhibitory responses and ultrastructural alterations produced by 1,3-bis(2-chloroethyl)-1-nitrosourea and dexamethasone in rat glioma cell cultures.

The effect of 0.0001 to 10 muM 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and 1 muM dexamethasone on cell proliferation was studied by measuring cell densities in control and drug-treated rat glioma (strain C6) monolayer cultures. When C6 cultures were exposed to 0.01 to 10 muM BCNU, the growth rates decreased for 2 days as control cell populations continued to proliferate at log phase rates. These growth-inhibitory responses were dose dependent and ranged from 20 to 80%, relative to control growth. Subsequently, the growth rates increased and the inhibitory responses ranged from 0 to 12% 4 days later. Cell densities in C6 cultures exposed to 1 muM dexamethasone for 1 day did not differ significantly from controls. Then cell proliferation ceased and the inhibitory response remained at 50% relative to controls in stationary phase. When 0.03 muM BCNU and 1 muM dexamethasone were supplied simultaneously to C6 cultures, a 35% inhibitory response occurred after 1 day. This response did not differ significantly from that observed with 0.03 muM BCNU alone. After 4 days, the inhibitory response did not decrease in cultures containing both drugs, but did decrease to 13% in the 0.03 muM BCNU-treated cultures. In 1 muM BCNU-treated cultures, the response was 66% after 1 day, which decreased to 21% 5 days later. When 1 muM BCNU was supplied to C6 cultures that were pretreated for 1 day with 1 muM dexamethasone, the response was 91% the following day, and this decreased to only 54% 5 days later. Dose-response curves showed that the inhibitory responses after 1 day in these pretreated cultures exposed to 0.001 to 10 muM BCNU increased up to 22% relative to the responses produced by either drug alone. After 5 days, the responses in the pretreated cultures exposed to 0.001 to 1 muM BCNU was 50%, which was similar to the response produced by 1 muM dexamethasone alone. Ultrastructural studies revealed that control and 1 muM BCNU-treated C6 cells contained 18 mitochondria, but the treated cells were 10% smaller after 1 day. Cells exposed to 1 muM dexamethasone for 1 day conount of granular endoplasmic reticulum increased greater than 80% in cells treated with BCNU for 1 day or dexamethasone for 2 days. C6 cells pretreated with dexamethasone and exposed to BCNU for an additional day (a) contained 23 mitochondria, (b) did not decrease in size, and (c) exhibited a greater than 250% increase in the amount of granular endoplasmic reticulum. These results demonstrate that combined growth-inhibitory responses and ultrastructural alterations occur when C6 cells are treated sequentially with 1 muM dexamethasone and BCNU.

Transfer of human membrane surface components by incorporating human cells into intact animal tissue by cell-tissue electrofusion in vivo.

Current animal models employed for the study of the obligate human pathogen Neisseria gonorrhoeae fail to utilize specific human gonococcal attachment receptors required to initiate pathogenesis in a clinically meaningful way. This communication presents evidence that suggests that cell-tissue electrofusion may be employed to create an animal model for this human specific pathogen. This new biotechnology was used to incorporate human membrane gonococcal receptors directly into epithelium of laboratory animals and subsequently infecting the histologically modified tissue with N. gonorrhoeae strain Pgh 3-2.

Electrofusion of individual animal cells directly to intact corneal epithelial tissue.

An electromechanical process was developed to electrofuse human and nonhuman cultured cells directly to rabbit corneal epithelial tissue in vitro and in situ. This new process was utilized successfully to incorporate functional gonococcal membrane attachment receptors from human lymphoma cells into superficial rabbit corneal epithelium. Thus, cell-tissue electrofusion biotechnology may be employed to establish unique and novel animal models for investigating receptor-mediated processes in vivo.

Inhibition of yeast phagocytosis in macrophage cultures treated with slime polysaccharide purified from Pseudomonas aeruginosa.

This study was initiated to determine whether purified slime polysaccharide(PSP) from P aeruginosa inhibits the ingestion of heat killed Saccharomyces cerevisiae particles in macrophage cultures. Relative to controls, direct phagocytosis assays revealed that the percentages of phagocytes and the numbers of ingested yeast particles per phagocyte decreased in a dose-dependent manner in PSP-treated cultures. Thus, PSP may act as a virulence factor in vivo by impairing the phagocytic capacity of macrophages.

An improved spectrofluorometric assay for quantitating yeast phagocytosis in cultures of murine peritoneal macrophages.

An accurate means by which to quantitate phagocytosis in murine resident peritoneal macrophages was developed by improving upon existing methods for measuring this important cellular function. Heat-killed Saccharomyces cerevisiae were conjugated to fluorescein isothiocyanate (FITC) and added to macrophage cultures. Following removal of uningested yeast, the macrophages were lysed and the fluorescence associated with the lysates was quantitated. The SEM were rarely +/- 10%. The improved assay was utilized to demonstrate the suppression of yeast phagocytosis by dexamethasone as measured by our radiometric assay. The spectrofluorometric assay produced results similar to those observed when the radiometric assay was employed to determine steroid induced suppression of yeast phagocytosis. However, the improved spectrofluorometric assay is more accurate, reliable, easier to perform, cost and time efficient, and a much safer method for quantitating yeast phagocytosis.

A rapid radiometric assay for measuring phagocytosis of Saccharomyces cerevisiae in macrophage cultures.

A new assay was developed to measure yeast phagocytosis in cultures of murine resident peritoneal macrophages. Saccharomyces cerevisiae was radiolabeled during exponential growth in nutrient broth supplemented with [3H]glucose. Following ingestion of the radiolabeled heat-killed yeast particles for 15 min, phagocytic capacities were measured in harvested macrophage lysates by liquid scintillation spectrometry. The new procedure compares favorably with light microscopic techniques and appears to be a more sensitive method for quantitating phagocytic function. Dose-response studies indicate, that over a wide range of dexamethasone concentrations, the radiometric procedure consistently measures greater inhibitory effects for the steroid induced suppression of phagocytosis.

Ameliorating effects of hyperbaric oxygenation on experimental allergic encephalomyelitis.

The effect of hyperbaric oxygenation (OHP) on survival and quality of survival of guinea pigs afflicted with experimental allergic encephalomyelitis (EAE) was investigated. EAE was induced in Hartley and Strain 13 animals by intradermal injections of whole guinea pig spinal cord in complete Freund's adjuvant. The inoculated animals were divided into control and treatment groups; the treated animals received OHP in a variety of treatment schedules. Clinical signs of EAE were quantitated and mean survival times were measured. When Hartley animals were exposed to 100% O2 at 2.5 atmospheres absolute (ATA) for 2 hr/day from 5--19 days postinoculation, the mean survival time (+/- SE) was 19.1 +/- 1.6 days relative to 15.7 +/- 0.7 days in the control (p less than 0.050). When Strain 13 guinea pigs were treated with 100% O2 at 2ATA for 4 hr/day on 5--16 days, the mean survival time was 21.6 +/- 0.6 days compared to 16.0 +/- 0.4 days for the control (p less than 0.001). Clinical sign measurements demonstrated that the onset of EAE in the treated animals of both strains occurred between 4--6 days after these signs became detectable in control animals. These results suggest that OHP therapy can ameliorate EAE in afflicted guinea pigs.

A feasibility study to determine if microbicidal activity can be measured in dexamethasone-treated macrophage cultures.

Dexamethasone action severely limits the ingestion of Saccharomyces cerevisiae in cultures of murine peritoneal macrophages. In spite of this inhibitory response, numbers of viable yeasts sufficient for microbicidal studies can be recovered shortly after their limited ingestion from lysed steroid-treated phagocytes.

Loss of host-controlled restriction of lambda bacteriophage in Escherichia coli following methionine deprivation.

lambda Bacteriophages produced in Escherichia coli C (designated as lambda . C) are restricted in their ability to grow in E. coli K-12. The rare successful infections that arise in the K-12 population occur in "special" cells which have lost their capacity to restrict lambda . C. These infections yield modified progeny phage (designated as lambda . K) which, unlike lambda . C, plate equally well on E. coli C and E. coli K-12. When methionine, but no other amino acid, was removed from the growth medium of a mutant strain of E. coli K-12, the number of special cells rapidly increased 500- to 3,000-fold. These new special cells retain their capacity to produce modified lambda . K progeny. This conversion of restricting cells into special cells does not require the synthesis of new protein. The special cells formed when methionine was removed from the culture did not revert into restricting cells when methionine was restored. Such cells have also lost the ability to divide for at least 4 hr after methionine supplementation. When methionine was restored, the remaining restricting cells, but not the special cells, immediately resumed growth. Removing methionine from cultures of E. coli B caused a similar increase in the number of special cells able to support the growth of lambda . C and lambda . K. However, when E. coli K-12 (P1) cultures were deprived of methionine, the number of special cells increased for lambda . C but not for lambda . K. Thus, retention of the P1-restriction system, unlike the B- and the K-12-systems, does not require the presence of methionine.

Suppression of phagocytosis by dexamethasone in macrophage cultures: inability of arachidonic acid, indomethacin, and nordihydroguaiaretic acid to reverse the inhibitory response mediated by a steroid-inducible factor.

Glucocorticoid steroids inhibit phagocytosis and cell spreading in cultures of murine resident peritoneal macrophages. It is postulated that these suppressive responses are mediated by a steroid-induced substance elaborated from dexamethasone-treated macrophages. Accordingly, dialyzed medium from dexamethasone-treated cultures was analyzed for the presence of a factor that inhibits phagocytosis and cell spreading in macrophage cultures not exposed to the steroid. When previously untreated macrophages were supplied dialysates from steroid-treated cultures, cell spreading and the phagocytic capacity (i.e. percentages of phagocytes in macrophage populations and the ability of phagocytes to ingest heat-killed Saccharomyces cerevisiae particles) decreased dramatically between 2 and 24 h after exposure. A lesser transient inhibitory response was observed when dialysates from untreated cultures were used. Relative to these controls after 48 h, phagocytic capacity and cell spreading remained suppressed in cultures treated with dialysates from dexamethasone-treated cultures. The addition of arachidonate, in the absence and presence of cyclooxygenase and lipoxygenase inhibitors, did not affect the phagocytic capacity of control macrophages. Furthermore, the addition of these compounds, either alone or in combination, to dexamethasone-treated cultures did not modulate the steroid-induced suppression of phagocytosis. These results support the hypothesis that the inhibition of phagocytosis and cell spreading may be mediated by a dexamethasone-induced non-dialyzable factor. In addition, the inability of arachidonic acid and inhibitors of prostaglandin and leukotriene biosynthesis to reverse the steroid-induced suppression of phagocytosis implies that the inhibition of this important macrophage function is not associated with the failure of dexamethasone-treated macrophages to release these mediators.

Suppression of yeast ingestion by dexamethasone in macrophage cultures: evidence for a steroid-induced phagocytosis inhibitory protein.

The mechanism by which glucocorticoid steroids suppress yeast phagocytosis in cultures of resident and thioglycollate-elicited murine peritoneal macrophages was examined. Time course and dose-response studies demonstrated that the phagocytic capacity of resident macrophages was suppressed by dexamethasone to the same extent in both newly established cultures and cultures that were incubated for several days. In contrast, relative to newly established cultures of elicited cells that were treated with the drug, elicited macrophages that were incubated for at least 1 day prior to exposure to dexamethasone, exhibited enhanced sensitivity to the action of the steroid. Steroid-induced phagocytic inhibitory responses were blocked by the metabolic inhibitors cycloheximide and actinomycin D. The suppression of phagocytosis by dexamethasone was mediated by a factor, present in the cellular homogenates of steroid-treated macrophages, that was partially purified by Sephadex G-25 chromatography. Since the phagocytic inhibitory activity in these homogenates was destroyed following exposure to heat and trypsin, the factor has been named phagocytosis inhibitory protein (PIP). The antiphagocytic activity of PIP was neutralized by treatment with RM23, a monoclonal antibody directed against lipocortin. The results support the hypothesis that the suppression of yeast ingestion is mediated by the action of PIP, which is induced in dexamethasone-treated macrophage cultures. Moreover, PIP appears to belong to the lipocortin family of phospholipase inhibitory proteins.

Glucocorticoid receptor properties and glucocorticoid regulation of glutamine synthetase activity in sensitive C6 and resistant C6H glial cells.

The relationship between induction of glutamine synthetase activity by dexamethasone and binding of the steroid to cytosolic glucocorticoid receptors was examined in sensitive C6 and resistant C6H glial cell cultures. Glutamine synthetase activity increased 3-4-fold when C6 cultures were exposed to 7.6 x 10(-6) M dexamethasone. This inductive response was reversible, dose-dependent (ED50 approximately 2 x 10(-8) M), required de novo protein and RNA synthesis, and was elicited only by glucocorticoid steroids. Progesterone, but not epicortisol, antagonized the dexamethasone-induced enzyme increase. In contrast, only a slight inductive effect was observed in dexamethasone-treated C6H cells. Competitive binding assays demonstrated that specific binding of [3H]-dexamethasone to cytosolic receptors was also dose-dependent. The ED50 was approximately 10(-8) M for both C6 and C6H cells. Scatchard analysis revealed that each C6 cell contained approximately 10,800 receptor sites and that the equilibrium dissociation constant (Kd) was 4.5 x 10(-9) M. Each C6H cell possessed approximately 12,200 sites, and the Kd was 6.7 x 10(-9) M. Unlabeled dexamethasone and cortisol (but not epicortisol) competed effectively with [3H]-dexamethasone for binding to cytosolic receptor sites and nuclear sites of both cell types. These results suggest that induction of glutamine synthetase activity in dexamethasone-treated C6 cells is a glucocorticoid-directed response. Since C6H cells are refractory in this regard but contain functional cytosolic receptors which interact with cell nuclei, the basis for their resistance appears to involve some step beyond these cellular processes.

Elevated amounts of protein in C6 glial cells during the growth-inhibitory response elicited by dexamethasone.

Dexamethasone suppresses C6 glial cell proliferation in vitro. This growth-inhibitory response is accompanied by elevated amounts of acid-insoluble protein in the steroid-treated cells relative to controls. These results provide additional evidence that the glucocorticoid acts to arrest C6 cell proliferation in G2.

Inhibition of yeast phagocytosis and cell spreading by glucocorticoids in cultures of resident murine peritoneal macrophages.

This study was initiated to determine whether the inhibition of phagocytosis and cell spreading in cortisol-treated cultures of resident murine peritoneal macrophages are glucocorticoid-directed responses. Phagocytosis of heat-killed Saccharomyces cerevisiae and cell spreading were measured in control and steroid-treated macrophage cultures over 6 days. When the cultures were exposed to testosterone, progesterone, or epicortisol, phagocytosis and cell spreading were similar to controls. In contrast, both macrophage functions were inhibited significantly in cultures treated with cortisol, methylprednisolone, dexamethasone, and triamcinolone acetonide. In addition, the rate of phagocytosis was retarded and phagocytic indices (i.e., yeast particle number/cell) were reduced in glucocorticoid-treated cultures. Dose-response studies with dexamethasone demonstrated that the ED50 for the inhibitory effect on phagocytosis was 20 nM. These results indicate that the inhibition of yeast phagocytosis and cell spreading in the steroid-treated cultures are specific glucocorticoid-directed responses.

Dexamethasone action inhibits the release of arachidonic acid from phosphatidylcholine during the suppression of yeast phagocytosis in macrophage cultures.

Dexamethasone suppresses phagocytosis of heat killed Saccharomyces cerevisiae in cultures of murine peritoneal macrophages. Recent observations suggest that dexamethasone induces a phagocytic inhibitory protein that suppresses yeast ingestion by inhibiting macrophage phospholipase A2 activity. The present investigation, therefore, examined whether macrophage lipid metabolism is modulated by dexamethasone. Control and steroid treated macrophages were allowed to incorporate radiolabeled arachidonate and were incubated subsequently in the absence and presence of yeast. Following ingestion by control macrophages, arachidonate from phosphatidylcholine was readily cleaved to free fatty acid and transferred to the neutral lipid fraction. In contrast, arachidonate release was inhibited in dexamethasone treated macrophages. These results suggest that the suppression of yeast phagocytosis by dexamethasone action may be associated with the inhibition of phospholipase A2 activity.

Glucocorticoid-mediated establishment of an antiviral state coincident with other glucocorticoid-induced biochemical activities in L929 cells.

Incubation of L929 cells with three different glucocorticoids, hydrocortisone, dexamethasone and triamcinolone acetonide, rendered the cells unable to support plaque formation by several unrelated DNA and RNA viruses. The establishment of this antiviral state by dexamethasone coincided with an inhibition of cell growth and induction of glutamine synthetase activity. These steroid-mediated activities occurred only in cultures of L929 cells and not in cultures of rabbit skin or rat glioma cells.

Inhibition of yeast phagocytosis by dexamethasone in macrophage cultures: reversibility of the effect and enhanced suppression in cultures of stimulated macrophages.

This investigation was initiated to characterize further the ability of 1 microM dexamethasone to suppress the ingestion of heat-killed Saccharomyces cerevisiae particles in cultures of murine resident peritoneal macrophages. Time course studies revealed that the inhibitory response required the continual presence of the steroid in the culture medium. In addition, increased inhibitory responses occurred after dexamethasone was supplied to previously untreated cultures of resident macrophages that became stimulated by differentiating in vitro. These findings indicate that glucocorticoids act directly on macrophages to decrease their phagocytic capacity, which in vivo would reduce host resistance.

Modulatory effects of heat-labile serum components on the inhibition of phagocytosis by dexamethasone in peritoneal macrophage cultures.

This investigation examined the effects of heat-labile serum substances on the suppression of yeast phagocytosis in dexamethasone-treated cultures of murine resident peritoneal macrophages. When 4-6 day old untreated control cultures were supplemented with either heat-inactivated (56 degrees C, 30 min) or intact (non-heat-inactivated) fetal bovine serum, more than 90% of the macrophage population ingested at least 1 yeast particle during 15 min phagocytosis assays. In cultures treated with 10(-6) M dexamethasone, approximately 30% of the macrophages were phagocytic. In contrast, approximately 70% of the steroid-treated population consisted of phagocytes in cultures supplemented with intact serum. Medium shift experiments demonstrated that the type of serum present during the 15 min yeast phagocytosis assays, but not the 4-6 day incubation periods, determined the size of the phagocytic subpopulations in the treated cultures. Whereas the majority of control phagocytes ingested more than 8 yeast particles, most dexamethasone-treated phagocytes ingested far fewer than 8 particles regardless of the size of the phagocytic subpopulations. In contrast to yeast, the ingestion of latex particles was inhibited to the same extent in dexamethasone-treated cultures that contained either heat-inactivated or intact serum. Thus, dexamethasone action impairs the ability of macrophages to accumulate yeast particles even though the phagocytic subpopulation is larger in treated cultures containing intact serum. This larger subpopulation may result from the activation of the alternative complement pathway by yeast during phagocytosis.

Increased sensitivity of a new coagglutination test for rapid identification of Haemophilus influenzae type b.

A newly developed rapid coagglutination test for identifying Haemophilus influenzae type b organisms isolated from clinical specimens correlated 100% with the slide agglutination test but was 100- to 200-fold more sensitive.