Participation of glutathione in the elimination of Porphyromonas gingivalis in vivo.

INTRODUCTION: Glutathione is involved in immune responses such as cell proliferation and bactericidal activity. The aim of this study was to see whether glutathione influences the intraperitoneal elimination of Porphyromonas gingivalis in Fusobacterium nucleatum-immunized mice. METHODS: Mice were immunized with P. gingivalis or F. nucleatum, and then P. gingivalis was inoculated into the peritoneal cavity of the mice. After various lengths of time, the numbers of bacteria were determined by a colony-forming assay and by polymerase chain reaction. The effect of glutathione on the elimination of P. gingivalis was explored by changing the intracellular glutathione level. Furthermore, we examined the effects of glutathione on the peritoneal levels of interferon-gamma, a macrophage activator, and of nitrite, a derivative of nitric oxide that acts as an antimicrobial agent when produced by macrophages and neutrophils. RESULTS: Inoculated P. gingivalis was eliminated more rapidly from F. nucleatum-immunized mice than from P. gingivalis-immunized mice. Interferon-gamma levels in peritoneal lavage fluid and glutathione levels in peritoneal exudate cells were higher in F. nucleatum-immunized mice than in P. gingivalis-immunized mice. When P. gingivalis-immunized mice were given glutathione monoethylester (a derivative of glutathione that is converted to glutathione intracellularly through hydrolysis) into the peritoneal cavity, the elimination of P. gingivalis was accelerated. On the other hand, when F. nucleatum-immunized mice were given L-buthionine-[S,R]-sulfoximine (an inhibitor of glutathione synthesis) into the peritoneal cavity, the elimination of P. gingivalis was suppressed. CONCLUSION: In F. nucleatum-immunized mice, glutathione may have a key role in the defense against P. gingivalis infections.

Oncogene transformation frequency of nonsenescent SFME cells is increased by c-myc.

Immortalized, postcrisis mouse embryo cell cultures derived in serum-containing medium display genomic abnormalities and an altered, preneoplastic phenotype. These lines can be transformed with single oncogenes, such as Ha-ras, while efficient transformation of precrisis, genomically unaltered rodent embryo cultures require cooperating oncogenes, such as Ha-ras and the mouse c-myc gene constitutively expressed. Serum-free mouse embryo (SFME) cells, cultured under conditions in which serum is replaced by growth factors and other supplements, are 'immortalized' in the genomically unaltered state. SFME cells do not exhibit growth crisis or gross chromosomal aberration, and are dependent on epidermal growth factor for survival, growth inhibited by serum, and are nontumorigenic. Transformation of SFME cells can be achieved with ras alone, but the introduction of c-myc increased the transfection frequency upon subsequent transfection with ras by as much as twenty fold. Similar results were obtained with mutationally activated neu oncogene and with genomic human tumor DNA. Constitutive expression of c-myc alone did not alter the properties of the SFME cells. These results demonstrate that c-myc alters cellular responses to oncogenes in a culture system in which oncogene-induced immortalization is not a factor, indicating that the effects of myc may extend beyond an 'immortalization' function in these cells.

Serum inhibition of proliferation of serum-free mouse embryo cells.

Serum-free mouse embryo (SFME) cells, derived in medium supplemented with insulin, transferrin, high density lipoprotein, epidermal growth factor, and fibronectin, do not undergo crisis, maintain a predominantly diploid karyotype with no detectable chromosomal abnormalities for well over 100 population doublings in vitro, and are growth inhibited by concentrations of serum that are growth-stimulatory for most cell lines in culture. Serum inhibition of SFME cell proliferation was reversible and was not prevented by addition of the supplements of the serum-free medium, even when added repeatedly during the culture period. The serum effect on SFME cell proliferation could be detected after incubation in serum-containing medium for as little as 8 h. SFME cells in serum-containing medium were arrested in the G1 phase of the cell cycle with a greatly reduced rate of incorporation of precursors into DNA and thymidine kinase activity, while a reduction in rate of incorporation of amino acids into protein was not observed. SFME cultures maintained for extended periods in serum-containing medium underwent a crisis-like period followed by the appearance of variant cells capable of growing in serum-supplemented medium. These cells exhibited abnormal karyotype and were resistant to several inhibitors of proliferation active on the parent SFME cell type.

[Transfer of bacterial drug resistance factor in artificial dental plaque].

Transfer of drug resistance factor was examined using so-called artificial dental plaque. Clinically isolated specimens of Enterococcus faecalis showing various kind of drug resistance were used as the donors. Streptococcus mutans BHT strain was used as the recipient and the plaque former. S. mutans formed artificial dental plaque on the surface of glass sticks after 7 to 10 day cultivation in brain heart infusion medium containing 5% sucrose. Each strain of E. faecalis was added to the plaque culture and co-cultured further 24 hours. Colonies were formed on drug-containing plates by 5 different mixed cultures with the frequencies of 10(-5)-10(-6) (kanamycin, KM) and 10(-4)-10(-8) (erythromycin, EM). Since these values were higher than that of the mutation frequency, we conclude that the resistant sublines were formed by a genetic transfer. Plasmid DNA derived from some transferred sublines was electrophoreted and the patterns were compared with those from the donor and the recipient. One KM-resistant subline showed a new band at the point of 40 kb which was not found in the plasmid DNA from the donor. It is suggested that the plasmid containing KM-resistance gene was modified molucularly. Sublines without new plasmid band were obtained also. There may be technical problems with it, or the transferred gene may be incorporated in a chromosome. Considering above results, the effect of bacterial transferable genes on oral hygiene were discussed.

Cell participation in immune response by immune ribonucleic acid. I. The role of T lymphocytes in immune response by immune RNA against T-dependent antigens.

T- and B-cell participation in the immune response induced by immune ribonucleic acid (iRNA) preparations against T-dependent antigens was studied using athymic nude, neonatally thymectomized (NT) and cyclophosphamide-treated (CY) mice. The iRNA(T + B) preparations were made from the spleen of BALB/c mice immunized with these antigens. Injection of the iRNA into nude or NT mice caused an increase in the number of specific rosette-forming cells (RFC) and of memory cells capable of responding to secondary stimulus with a small dose of the corresponding antigen. Injection with T-dependent antigens or with iRNA(T + B) did not cause any immune response in CY mice, suggesting depletion of the B-cell function. The iRNA(T) and iRNA(B) were prepared, respectively, from the thymuses of BALB/c mice and from the spleens of nude mice which had been immunized with T-dependent antigens. Injection of nude mice with both iRNA(T) and iRNA(B) caused an increase in the number of specific RFC and the secondary antibody formation response after boosting with a small dose of the corresponding antigen. Injection of iRNA(T) preparation into nude mice could induce the anamnestic response after boosting. However, neither of the iRNA(T) or iRNA(B) preparation could induce in nude mice the proliferation of the number of specific RFC. These results indicate the presence of at least two kinds of iRNA preparations against T-dependent antigens and that the cooperation of iRNA(T) and iRNA(B) was required for the induction of immune response against T-dependent antigens.

Cell preparation in immune response by immune ribonucleic acid. II. Independence of T lymphocytes in immune response against T-independent antigens.

The immune response of congenitally athymic (nude) mice induced by immune ribonucleic acid (iRNA) to lipopolysaccharides of Escherichia coli 0-55 (LPS) was studied. The thymus-independent nature of the immune response of mice to LPS was confirmed and nude mice responded to LPS in a manner similar to normal mice. An iRNA preparation extracted from the spleen of nude mice immunized with LPS could induce the proliferation of rosette-forming cells (RFC) in nude mice. iRNA preparations were insensitive to treatment with deoxyribonuclease and pronase, but were inactivated by ribonuclease treatment. The active fraction of the iRNA preparation had sedimentation values in a sucrose density gradient between 7 and 16 S and comprised only a small fraction of the total RNA present in the spleen cells, thereby indicating that the active moiety was one or more species of RNA. The anamnestic response was induced by treatment with iRNA made from the spleen of nude mice immunized with LPS. An increase in the number of rosette-forming cells (RFC), plaque forming cells (PFC) and formation of humoral antibody to LPS was seen after injection with a small amount of LPS 4 weeks after iRNA treatment.