Yoshinobu Shimizu

Dual Regulation of Osteoclast Differentiation by Periodontal Ligament Cells through RANKL Stimulation and OPG Inhibition

Periodontal ligament (PDL) cells play an important role in maintaining the homeostasis of periodontal tissues. However, it is not known how PDL cells contribute to osteoclastogenesis. In this study, we examined the consequences of cell-to-cell interactions between peripheral blood mononuclear cells (PBMCs) and PDL cells during osteoclastogenesis. PBMCs were co-cultured directly or indirectly with PDL cells for two to four weeks. PBMCs that were directly co-cultured with PDL cells formed significantly more resorption pits on dentin slices than did PBMCs that were cultured alone. However, soluble factor(s) produced from PDL cells inhibited the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells. Furthermore, PDL cells expressed both receptor activator nuclear factor kappa B ligand (RANKL) and osteoprotegerin (OPG) mRNA. In conclusion, PDL cells support osteoclastogenesis through cell-to-cell contact. PDL cells might regulate osteoclastogenesis by opposing mechanisms-stimulation of resorptive activity by RANKL and inhibition by OPG-thus affecting processes such as periodontitis and orthodontic tooth movement.

Compressive Force Induces Osteoblast Apoptosis via Caspase-8

Periodontal remodeling during orthodontic tooth movement is a result of mechanical stresses. The application of excessive orthodontic force induces cell death. However, the nature of compressive force-induced cell death is unclear. We examined whether the in vitro application of continuous compressive force would induce apoptosis in human osteoblast-like cells (MG-63 cells), and investigated the mechanism by which apoptosis was initiated. The cells became aligned irregularly, and cell viability decreased, indicating that the compressive force caused cell death. According to the TUNEL analysis, the number of apoptotic cells increased significantly in a time-and force-dependent manner. Caspase-3 activity increased with the magnitude of the compressive force, and this effect was reduced significantly by a caspase-8 inhibitor, whereas a caspase-9 inhibitor had no such effect. We conclude that the in vitro application of compressive force can induce apoptosis in MG-63 cells through the activation of caspase-3 via the caspase-8 signaling cascade.

Effect of chloroquine on the growth of animal viruses

The effect of chloroquine diphosphate on the growth of polio type 1, influenza, Newcastle disease, Sendai, vesicular stomatitis and vaccinia viruses was studied. Host dependency of the antiviral effect of chloroquine on Newcastle disease and Sendai viruses was shown using HeLa S 3 cells and primary chick embryo fibroblast cells. The antiviral effect of chloroquine was extensively studied in chick embryo cells infected with vesicular stomatitis virus. Chloroquine at a concentration of 12.5 μg per ml markedly reduced the virus yield. The drug did not affect the adsorption of vesicular stomatitis virus to chick embryo cells. No evidence for inhibition of the virus uncoating process by the drug was obtained in our present system. The addition of chloroquine at various times after the latent period induced immediate inhibition of the synthesis of progeny virus. Chloroquine inhibited selectively the synthesis of viral RNA without affecting that of cellular RNA or the synthesis of viral antigens.

Study of interferon production during pregnancy in mice and antiviral activity in the placenta

Although the mortality rate after herpes simplex virus type 2 inoculation was not significantly different between pregnant mice and nonpregnant mice, systemic interferon production was very high during late pregnancy compared with that in nonpregnant mice. Antiviral activity was detected in placentas from all noninfected pregnant mice (80 to 320 U/ml in 20% suspension). The antiviral activity had a broad spectrum and was also effective in the cells of other species; an antiviral effect was shown even if the cells were treated after challenge with a virus. In addition, this activity was not inactivated by antimouse interferon-neutralizing antisera. The molecular weight of this placental antiviral substance was estimated to be 200,000 to 450,000 daltons by gel filtration, and it was inactivated by heat, acid, and trypsin. Noninterferon antiviral activity (40 to 80 U/ml) was also detected in more than half the sera (61.5%) of noninfected mice in late pregnancy.

Specific Inactivation of Herpes Simplex Virus by Silver Nitrate at Low Concentrations and Biological Activities of the Inactivated Virus

The infectivities of herpes simplex virus types 1 and 2 were inactivated by silver nitrate at concentrations of 30 μM or less, which did not affect at all the infectivities of hemagglutinating virus of Japan, vesicular stomatitis virus, poliovirus, vaccinia virus, and adenovirus. The inactivated virus retained the capability of adsorbing to the cell, with an adsorption kinetics quite similar to that of intact virus, and of inducing the concanavalin A agglutinability in the infected cells, whereas it lost completely the capability of producing viral antigens and other cytopathic changes.

Suppression of osteoclast-like cell formation by periodontal ligament cells

When human peripheral blood lymphocytes (PBL) were cocultured separately with periodontal ligament (PDL) cells, osteoblasts (OB), and gingival (GIN) cells using a membrane filter (millicell), the formation of osteoclast (OC)-like cells was clearly suppressed in PBL cocultured with PDL cells, but was enhanced in PBL cocultured with OB and GIN cells. This suppression and enhancement of OC-like cell formation was found in all of the 50% coculture fluids. When PBL were cocultured with PDL or GIN cells for 4, 6, and 8 days, a smaller number of PBL (when cocultured with PDL cells) and a larger number of PBL (when cocultured with GIN cells) were observed. It was also confirmed that the nonadherent type of PBL was suppressed by coculture with PDL cells, while both the nonadherent and adherent types of PBL were increased by coculture with GIN cells. When PBL cocultured with PDL cells for 4 days were analyzed by flow cytometry, an increase of Mac-1-positive cells was not observed, although an increase of Mac-1-positive cells was clearly shown in PBL cocultured with OB. Although the DNA-synthesizing activity of PBL was clearly observed after treatment with macrophage-colony stimulating factor (M-CSF) or granulocyte macrophage-colony stimulating factor (GM-CSF), this activity was clearly suppressed by the addition of a 50% coculture fluid of PBL/PDL cells. Moreover, the number of OC-like cells shown in PBL treated with M-CSF or GM-CSF was clearly suppressed by the addition of a 50% coculture fluid of PBL/PDL cells. These results indicate that PDL cells may produce some suppressing factors of OC precursor cells, and this function of PDL cells may act to protect alveolar bone from bone resorption caused by the bite force.

Bone remodeling with resorbable bioactive glass and hydroxyapatite.

Particulate resorbable bioactive glass and resorbable hydroxyapatite did not inhibit the growth of periodontal ligament cells and osteoblasts in cultures. Both cell types promoted the activation of alkaline phosphatase around the surface of bioactive glass particles. When bioactive glass and hydroxyapatite were cultured with peripheral blood lymphocytes/monocytes, the number of nonadherent and adherent cells was the same as observed with a control. Resorbable bioactive glass activated the cells necessary for bone formation better than hydroxyapatite and suppressed the formation of osteoclasts for bone resorption. Resorbable bioactive glass and resorbable hydroxyapatite were found to be harmless to periodontium cells and lymphocytes/monocytes.

Protection of OK-432, a Streptococcus pyogenes preparation, against lethal infection of mice with herpes simplex virus

We have studied the protective effect of OK‐432, a biological response modifier (BRM) of Streptococcus pyogenes origin, on the lethal infection of mice with herpes simplex virus (HSV)‐1. A single intraperitoneal (i.p.) injection of more than 10 μg of OK‐432, when given at least two days before the infection, gave a marked effect yielding nearly 100% protection against ordinarily lethal infection. The protection was independent of the amount of infected virus inoculated. When given after the infection, the agent even at the maximal dose (100 μg), produced only a marginal effect. A single i.p. administration of OK‐432 augmented the natural killer (NK) activity of peritoneal exudate cells and spleen mononuclear cells in mice 2 to 3 days after injection of OK‐432, coinciding with the times when it induced a survival effect on HSV‐infection. Treating OK‐432‐treated mice with a combination of an anti‐macrophage agent, silica, and an anti‐NK cell agent, anti‐asialo GM1 serum, before infection diminished the antiviral effect of OK‐432. The OK‐432 protection against HSV infection was also markedly diminished in athymic nude mice. Thus, the protective effect of OK‐432 on lethal HSV infection seems to be based on the activation of NK cells, macrophages, and T lymphocytes.