Junya Kanehisa

A band of F-actin containing podosomes is involved in bone resorption by osteoclasts

Isolated rabbit osteoclasts cultured on devitalized thin bone slices excavate resorption lacunae that can be visualized with brightfield or phase-contrast microscopy. Superimposition of the brightfield images of such resorption lacunae and the fluorescence images of the corresponding osteoclasts after fixation and staining with rhodamine-conjugated phalloidin revealed that a bright fluorescent band of F-actin-containing podosomes precisely outlined the resorption lacunae in stationary osteoclasts. When the resorption lacunae were being extended laterally, the clearly delineated band of podosomes corresponded to the advancing edge of the resorbing osteoclast and the most recently excavated part of the lacunae. Reshaping and reorganization of the bright bands preceded development of the lateral boundary of the lacunae. Podosomes forming these bands were highly dynamic, changed in size and location, and appeared and disappeared continuously. Their lifespan varied between 2 and 12 min. Similar bands were also seen in vivo in bone-resorbing osteoclasts on the endocranial surface of growing calvariae. Podosomes disappeared in osteoclasts treated with calcitonin, resulting in the disruption of the fluorescent bands. Our results suggest that podosomes are an essential part of the resorption apparatus of osteoclasts.

In vitro bone resorption by isolated multinucleated giant cells from giant cell tumour of bone: Light and electron microscopic study

The behaviour of multinucleated giant cells (GCs), obtained from a giant cell tumour of the tibia and cultured on glass coverslips or on devitalized bone slices, was studied using light and electron microscopy. Monitoring the GCs on bone slices by phase-contrast microscopy revealed that they had removed calcified bone matrix resulting in excavation of lacunae, with subsequent lateral extension and perforation of the bone slices. Electron microscopy demonstrated for the first time that the GCs responsible for exavating lacunae had two specific membrane modifications, ruffled border and clear zone, and showed basically similar cytoplasmic fine structures to those of osteoclasts. Fluorescence images of the GCs on glass and on bone after rhodamine-conjugated phalloidin staining revealed that most of the GCs had an intensely fluorescent peripheral band composed of a number of F-actin dots called podosomes. Some GCs showed unusual arrangements of podosomes suggesting abortive attempts at GC formation. We have demonstrated that the band structure of the GCs cultured on bone is intimately involved in bone resorption. Two stromal cell types could be recognized. The predominant type, which seemed to be the only neoplastic element because of its proliferative capability, showed quite different fine structural and cytoskeletal features from the GCs. The other type, which was much less frequent and seemed not to proliferate, had morphological similarities to the GCs, and seemed to be their precursor. Importantly GCs cultured on bone and the osteoclasts share common structures for adhesion to and resorption of bone, strongly supporting the view that the GCs of the giant cell tumour of bone are potentially active bone resorbers and can be regarded as osteoclasts.

Salivary fibronectin in man: An immunoblotting, radioimmunoassay and immunohistochemical study

An antiserum and monoclonal antibody against plasma fibronectin recognized a 230-kDa, intact fibronectin molecule when 1000-fold diluted human plasma was subjected to immunoblot analysis. Immunoblot analysis of the parotid saliva demonstrated that the rabbit antiserum to human plasma fibronectin bound to five molecules (200, 110, 85, 75 and 65 kDa), other than the 230-kDa, intact fibronectin molecule, while the mouse monoclonal antibody recognized only the 230-kDa molecule. The 230-kDa type was not found in whole saliva with either the antiserum or monoclonal antibody. The antiserum reacted with 85-, 75- and 33-kDa molecules, and the monoclonal antibody recognized 75-, 33- and 20-kDa molecules in the whole saliva. Radioimmunoassay revealed that the mean +/- SD of fibronectin concentration in parotid saliva was 2.5 +/- 1.4 ng/ml (n = 20) and 149.8 +/- 46.2 ng/ml (n = 30) for whole saliva. Immunoperoxidase staining with rabbit antiserum and the mouse monoclonal antibody showed positive cytoplasmic staining of cells in the intralobular and interlobular ducts in parotid, submandibular, and sublingual glands. No acinar cells were stained.

Lectin-binding glycoconjugates and immunoreactive proteoglycans in resorption pits freshly excavated by isolated rabbit osteoclasts

Staining with FITC-conjugated concanavalin A, wheatgerm agglutinin and peanut agglutinin demonstrated that abundant sugar residues are present in resorption pits produced in vitro and over the cell surface of rabbit osteoclasts resorbing bovine femoral bone slices. After chondroitinase ABC digestion the stubs of chondroitin 4-sulphate and dermatan sulphate could be detected in the resorption pits by monoclonal antibodies.

Marginal periodontitis and the immune system. III. Differences in peripheral blood lymphocyte subsets before and after treatment in adult periodontitis patients

The lymphocyte subsets in the peripheral blood of 8 individuals, aged 25 to 57 yr, with moderate to advanced adult periodontitis were analyzed before and after treatment by means of flow cytometry, and then compared with the findings in 20 normal adults (aged 30 to 45 yr). The results were as follows: the percentage of OKT-8+ (CD 8) in the patients (20.25 +/- 7.15%) was lower than that in normal individuals (31.50 +/- 6.03%, P less than 0.01) while the OKT-4/OKT-8 ratio (2.34 +/- 1.18) was higher than the corresponding ratio in the controls (1.23 +/- 0.37); however, there was no significant difference in the percentages of OKT-3+ (CD 3), OKT-4+ (CD 4) and Leu-12+ (CD 19) between patients and normal subjects. The percentages of OKT-3+, OKT-4+, OKT-8+, Leu-12+ and OKT-4/OKT-8 after the periodontal therapy did not show a significant difference compared with the data obtained before treatment. These results indicate that the decrease in suppressor/cytotoxic T-cell population may be a kind of pathological disposition of the initiation and/or course of adult periodontitis.

Immunopathological study of periodontal disease. VII Bacterial enzyme and periodontal disease (1) Bacterial chondroitinase ABC and hyaluronidase in dental plaque.

Protews vulgarisおよびStreptococcus hyplurolyticus由来のchoudroitinase ABCとhyaluronidaseを抗原とする螢光標識家兎抗血清を作製し, 50名の健全志願者の各歯列の歯垢中のbacterial chondroitinase ABCとhyaluronidaseの存否を螢光抗体法によって検討した。chondroitinase ABCは50名全員に, また, hyaluronidaseは44名に検出され, その螢光patternはいずれもfibrillarあるいはgranularまたはdiffuce patternとして観察された。このような螢光patternは両酵素が糸状菌や球菌によって産生されることを示すとともに, それが歯垢中に放出されることを明示するものである。そして, 歯垢中のこれら細菌酵素は内皮上皮を破壊しつつ深部に達し, 歯周組織を直接的に傷害することを示唆するものである。