The present study was to investigate the mechanisms of the enhancing effect of oral microorganisms on in vitro antibody formation by mouse spleen cells.
Sonicates of Actinomyces viscosus, Veillonella alcalescens, Fusobacterium nucleatum and Bacteroides melaninogenicus were used. AKR mice spleen cells were suspended in RPMI 1640 medium containing 10% heat-inactivated fetal calf serum. After adding the sheep red blood cells as an antigen and the sonicates, the cell suspensions were incubated for 4 days at 37°C in an atmosphere of 5% CO₂-95% air. The results were estimated by counting the numbers of direct plaque forming cells (PFC).
a) Experiment for macrophage factor: The mouse peritoneal macrophages were incubated with the sonicates for 24 hours and the immune response of the resultant supernatant (MP factor) on T·B-cells was examined.
b) Experiment for macrophage contact: The macrophages and spleen cells were incubated with the sonicates for 6 hours. After removing the macrophages, the immune response of remaining spleen cells was examined.
c) Experiment for T cell factor: T cells were incubated with the sonicates for 24 hours and the immune response of the supernatant (T cell factor) on B cells was examined.
d) Experiment for resistant spleen cells to irradiation: In vivo stimulation of the mice with the sonicates was carried out. Following irradiation of the whole body, the immune response of the resistant cells to irradiation with non-stimulated spleen cells was examined.
e) Experiment for mitogenicity: After 3 days of incubation of the mouse T-and B-cells with the sonicates in the culture plates, the uptake of ³H-thymidine into the immune cells for 24 hours was counted by liquid scintillation spectrometer.
The following findings were obtained:
1. The enhancement of antibody formation by the activations of macrophage, B cell proliferative response and irradiation-resistant cells to A. viscosus was found.
2. An increase in both T-and B-cell mitogenicity may enhance the antibody formation to the sonicates of V. alcalescens.
3. F. nucleatum may act as an adjuvant to enhance both the activation of macrophage and the responses of T cell, helper T cell and resistant cells to irradiation.
4. The immune effectiveness of the irradiation-resistant cells mediated, the direct activation of B cell and the enhancement of T cell mitogenicity was induced by B. melaninogenicus.

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This study was performed in two stages. The main purpose of the first experiment was to establish and to observe the bacterial plaque on the root surface in monkeys experimentally. Two adult male monkeys with normal dentition were employed and their 44 root surfaces of posterior teeth were used. A small plastic cover was fixed to the surgically denuded root surface. The cemental side of the cover had a groove to make room for plaque formation on the root surface. Surgical operation was made on each tooth 8, 7, 4, 3, 2 weeks and just before death of animals. The animals were fed with soft diet during the whole experimental period. Using light and electron microscopy, the bacterial plaque was found on the denuded cementum and the flora consisted of predominantly rod-shaped cells and some spirochetes 2 weeks or more after fixing the cover. By scanning electron microscopy, we also found the breakdown of collagen fibers which had remained on the surgically denuded cemental surface and calcified deposit on the cementum under the bacterial plaque.
The purpose of the secend experiment was to observe whether the bacterial plaque can survive and artificial pocket can be left after removal of the cover or not. Two adult female monkeys were used. Each cover was fixed to each tooth for 4-6 weeks in the same way, and they were removed 6, 5, 4, 3, 2, 1 week and 3 days before the animals were killed. We found the bacterial population on the root surface rapidly decreased immediately after the removal of the cover. But we also found a few rod-shaped cells and spirochetes on the root surface in the pocket and the deep pocket remained especially on the palatal surface of the upper posterior teeth even 6 weeks after removal of the cover.

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This investigation were performed to evaluate the relationship among thiol in incubated whole saliva and volatile sulphur compounds from incubated whole saliva and mouth air in three subjects groups. The subjects were divided into three groups based on clinical findings and intensity of odour by olfactory panel of trained judges, Group N: normal subjects, Group P-N: Periodontal patients without halitotis, Group P-H: periodontal patients with halitotis.
Volatile sulphur compounds from incubated whole saliva and mouth air were determined by gas chromatograph equipped with flame photometric detector and thiol in the supernatant of incubated whole saliva were determined by spectrophotometer.
Result were as flowed.
1) In periodotal patients, thiol in supernatant was detected prior to CH₃SH from incubated whole saliva, although they increased with time thereafter, there was 12-18 hours' difference in the time of their reaching their maximun quantity.
2) The thiol in the supernatant of incubated whole saliva and the methyl marcaptan produced from whole saliva from periodotal patients were of greater quantity than those from the normal. Furthermore Group P-H produced remarkably greater amount of thiol and methyl mercaptan than Group P-N. Also, in Group P-H a close correlation was confirmed between the highest values of the quantities of thiol and methyl mercaptan produced from incubated whole saliva.
3) Obvious correlationship was observed between the methyl mercaptan content in mouth air and the highest values of that produced from the incubated whole saliva.
4) In the periodotal patients, whereas no correlation was observed between the quantity of metyl mercaptan produced from the incubated whole saliva and P. I., there were a slight the correlation between pocket depth or bleeding and that, and a close correlation of that between discharge of pus and that. However, it was difficult to determine the correlation with G. I. coefficients being r=0.51.

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Recently, the diagnosis and therapy of periodontal disease, especially chronic marginal periodontitis, have advanced remarkably.
In observing the postoperative process of a periodontal patient, it is considered necessary that observations of the oral conditions should be combined with the transposition of the alveolar bone.
Therefore, the author was concerned with the clinical findings, especially regarding the plaque Index (Pl. I.), gingival Index (G. I.) and periodontal pocket depth (P. D.), as radiographed in the alveolar bone both preoperatively and postoperatively.
The sample of this study was a consist of 52 patients (27 males and 25 females) who were treated in the periodontics of Nihon University Dental Hospital, ranging from 16 to 62 years in age. At the time their initial consultation, these subjects were classified into 1) vertical type and 2) horizontal type according to the resorption pattern of their alveolar bone. Concerning the latter type, they were further classified as P₁ P₂ and P₃ groupes based on Russell's Index.
The subjects were further divided into a group of toothbrushing instruction together with the removal of dental calculus and a group of flap operation. The former group was subjected to the standardized intraoral radiography at the initial construction, after 3 months, 6 months and 10 months, whereas the latter group were examined at the initial construction, 3 months after initial therapy, 3 months post-treatment, 6 months and 10 months postoperatively in conjuction with their clinical findings and radiographs. The results were as follows:
1. The patients who were instructed in toothbrushing and removal of their dental calculus were found to be reduced in Pl. I. and G. I. as well as P. D. proportionately to the passage of time.
But there were observed some differences according to the degree of alveolar bone resorption and clinical findings as above classified.
2. In the patients who were subjected to the flap operation, Pl. I. and G. I. as well as P. D., the values were observed to have been much reduced after operations. Therefore, no remarkable change was noted after the operations.
3. The height and change in the alveolar bone area were measured on the standardized dental radiographs. As a consequence, it was found that the height showed a tendency of increase postoperatively in common with the different types of periodontal diseases.
However, the area of P₂ and P₃ in the horizontal group decreased till about 3 months after the toothbrushing instruction and removal of dental calculus but this tendency did not change till 10 months of experiments.
As regards the horizontal P₁ and vertical groups, on the other hand, there was observed a tendency of an increase in the surface area from 3 months onward from the removal of dental calculus.
This was also true of the cases of flap operation after 3 months of the toothbrushing instruction and the removal of dental calculus. This tendency was particularly noteworthy with the vertical group.
4. The patients who were subjected to flap operation were divided, after 10 months, into those Pl. I. was less than 0.4 and larger than 0.4. of cases having the Pl. I. less than 0.4 was found to have the tendency of an increase.
As a result of statistical difference determination, there was confirmed a statistical difference at α=0.01 between the two groups.

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A hydroxyapatite (HA) with the crystallinity similar to enamel powder of teeth, determined by analyses of X-ray diffraction, was chemically synthesized. Commercial HA (Lot. 30307, Seikagaku-kogyo Co., Tokyo) was lower in the crystallinity (determined by X-ray diffraction) than that of enamel powder and higher than that of dentin powder. It was studied on the adsorption of submandibular-sublingual (SM-SL) salivary proteins by these two types of HA, synthesized HA (S-HA) and commercial HA (C-HA). Fifteen ml of SM-SL saliva was added to 3.4g of S-HA and C-HA, respectively. Eighty-seven percent of salivary proteins added was adsorbed by C-HA, whereas only 58% of those added was adsorbed by S-HA. There were three salivary proteins which adsorbed on to C-HA but not on to S-HA. These subunit molecular weight were estimated by poly a crylamide gel electrophoresis in sodium dodecyl sulfate (SDS) and β-mercaptoethanol to be approximately 70, 000, 60, 000, and 56, 000. These proteins could be eluted from C-HA by a low concentration of phosphate buffer. Two proteins with estimated subunit molecular weight of approximately 120, 000 and 30, 000 were tightly adsorbed by both HA specimens. A salivary glycoprotein with high molecular weight also adsorbed on to both HA specimens. SDS-polyacrylamide gel electrophoresis in the absence and in the presence of β-mercaptoethanol indicated that the glycoprotein is composed of subunits linked by disulfide bridges.

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Conventional mice (JCL: ICR strain) were inoculated intraorally with 1.0×10⁸ colony forming units (CFU) of S. mutans PK-1 and tested at appropriate interval for the establishment of this organism in the mouth and feces.
The results obtained were as follows:
1) The in vitro inhibition of indigenous microbial florae, as determined by antibiotic sensitivity disks on cultures of oral swabs and fecal samples, demonstrated that ampicillin was the most effective antibiotics in suppressing indigenous florae.
2) The S. mutans PK-1 could be established in 3 samples of 10 at 3 days after, in 9 samples of 10 at 14 and 28 days after and in all samples of 10 at 56 days after in the oral swabs of experimental group that had received ampicillin to supress indigenous florae. While, in the oral swabs of controll groups, the S. mutans PK-1 could not be established in 7 samples of 10 at 3 and 14 days after. But, the difference in two groups at 56 days after could not be found.
3) The amount of microorganisms at 28 days after in the oral swabs of experimental groups was statistically higer than the amount in control groups.

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We have already reported the positive correlation between calculus index and the protease activities in whole saliva. The purpose of this paper was to determine the origin of the proteases.
The saliva was collected in the siliconized tubes from the patients with gingivitis or marginal periodontitis before and after oral prophylaxis. The numbers of leukocyte and epithelial cell in the saliva were counted. The protease activity was determined by a fluorophotometer using hemoglobin as a substrate.
The protease activity was not detectable in parotid saliva, but a little in submaxillary saliva. However, the activity was observed in the whole saliva from edentulous persons. The oral prophylaxis reduced the protease activity (pH 8.5), the number of leukocyte and epithelial cell and calcium concentration. The positive significant correlation was observed between the protease activity and the number of epithelial cell, not of leukocyte.
These results suggest that the epithelial cell is a main source of the salivary protease.

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The purpose of the present investigation was to study the clinical and histopathological aspects in the progression of a periodontal lesion starting from a healthy periodontium.
One monkey weighing 3 kilos was used. During a pre-experimental period of 3 weeks the teeth of the monkey were cleaned once daily with toothbrush and rubber tip. At the end of the preparatory period the gingivae were in excellent clinical health and they were at quadrant divided into experimental and control groups. The silk ligatures were placed around the premolars and the molars on each side of the maxilla and a topical application of the sonicates from Actinomyces viscosus and Fusobacterium nucleatum was used in the mandibular left and the maxillary right sides for 4 hours once a week. The mandibular right side served as a control which was subjected to meticulous toothbrushing.
The plaque accumulation, the gingival inflammation, the pocket depth and the distance from the reference point to the gingival crest were assessed at regular intervals during an 3 month experimental period.
The animal was sacrificed after 3 months and a serial section of the premolars and the molars with surrounding tissues was prepared for the light microscopic examination. In the sections were measured (1) the size of the infiltrated connective tissue (ICT), (2) the distance from the cemento-enamel junction (CEJ) to the apical cells of the junctional epithelium (JE), and (3) the distance from CEJ to the marginal bone crest (BC).
The following findings were obtained:
1) At the start of the study no plaque and gingival inflammation could be detected. The gingivae around the teeth showed the presence of abundant amounts of plaque and the signs of pronounced inflammation after 1 month in the ligature and the ligature plus topical application groups. The plaque formation and the gingival inflammation developed with time. The control group did not show a significant increase in the Plaque and the Gingival Indices.
2) The pocket depth in both ligature and ligature plus topical application groups rapidly more increased than that in the other groups.
3) The distance from the reference point to the gingival crest in the three groups was significantly larger than that in the control group.
4) The size of ICT occupied 3.3% in the control group, 12.3% in the topical application group, 27.7% in the ligature group and 26.7% in the ligature plus topical application group.
5) The distance from CEJ to JE was similar in the topical application and the control groups. The “histological gingival recession” in the ligature and the ligature plus topical application groups was significantly larger than that in the control group.
6) In the biopsies from the ligature and the ligature plus topical application groups it was noted that BC was located slightly below the level in the control group.
It is apparent from the present study that the silk ligature and the topical application of the sonicates from oral microorganisms play an important role in the initiation of periodontitis.

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The etiology of the separation of the gingival epithelium from the underlying connective tissue in chronic desquamative gingivitis was studied in a 61-year-old female patient by conducting varying treatments and analyuing healing with the elapse of time by electron microscopy, with results as follows:
1) There was severe vacuolization in the stratum basale and the lower stratum spinosum affected by CDG and separation of connective tissue from stratum basale due to the destruction of the basal cells by the severe vacuolization.
2) An amorphous collagen-like material was observed widely in the connective tissue underlying stratum basale.
3) The amorphous substance appeared only in a certain period in the process of healing, and consisted of collagen-like material embedding fine filaments.
4) It was considered that this substance was formed by developed fibroblasts and involved in the formation of lamina densa and collagen fibrils.

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