Preparation of monoclonal antibodies against dog gingiva.
As part of immunohistochemical studies of periodontal disease, we have been producing monoclonal antibodies related to the oral region for several years. The aim of the present investigation was to produce and characterize monoclonal antibodies to dog gingiva. The results were as follows:
1) Spleen cells from Balb/c mice immunized with dog gingival epithelial cells and myeloma cells were fused and followed by HAT selection. Fusion of Balb/c splenocytes immmunized with the epithelial cells resulted in 236 hybridomas. The incidence of hybrid formation was 236/348. Out of these 236 clones, 46 hybridomas produced immunoglobulins. Of them 23 hybrydomas produced antibodies being reactive with the frozen section of the dog gingiva.
2) Two monoclonal antibodies, MoAb D1 (IgM) and D2 (IgM) were finally established. MoAb D1 reacted with the cell membrane of epithelial cells of dog gingiva and MoAb D2 reacted with the cytoplasm of the cells. Both reacted with the capillaries in connective tissue. The epithelial tissue of other oral regions, such as lip, cheek, hard palate, tongue showed the pattern of reaction similar to that of gingiva. Futhermore, MoAb D1 reacted with the membrane parts of the surface epithelium of stomach, the sinusoidal capillary and Bowman's capsule, whereas MoAb D2 reacted with the cytoplasm of surface epithelium and parietal cells of stomach, hepatocytes, distal tubule and the thick ascending limb. However, these two antibodies did not react with human and monkey gingiva.
3) Proteolytic digestion using pepsin and trypsin did not reduce the reactivity of the epithelium with the MoAb D1, but trypsin treatment abolished the reactivity of the capillaries with MoAb D2. 4) Data from sodium dodecyl sulfate polyacrylamide gel electorophoresis and western blotting showed that MoAb D1 recognized an antigenic protein of 50 K dalton.
5) These two monoclonal antibodies will be available for studying pathological changes in periodontal tissues.

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Cortical motor areas of the jaw and orofacial regions were studied by intracortical micro-stimulation (ICMS:<30μA, 200μsec duration and 300 Hz in 40 msec train duration) in the cat lightly anesthetized with Ketamine-HCL. EMG activities produced by ICMS to the cerebral cortex were bi-polarly recorded from jaw and orofacial muscles, and the onset of latencies of EMG activities were analyzed. Furthermore, patterns of motor effects were examined by the ablation of jaw and orofacial motor areas. Jaw and orofacial motor effects were produced by ICMS delivered to anterior parts of the coronal and lateral sigmoid gyri (C area), and to the middle to the anterior part of the orbital gyrus (O area). Cytoarchitectonically, jaw and orofacial motor areas were restricted to areas 3a, 4 γ and6aβ in the C area, and to areas 43 and 6aβ in the O area. Different patterns of movements in jaw and orofacial regions were evoked from these two areas. In the C area one or, at most two, types of simple movements were produced by ICMS to one location, while in the O area more co-ordinated movements than those in the C area were produced and rhythmic jaw movement was evoked by repetitive stim-ulation (<30μA, 30 Hz and 200μsec duration) to the O area. The onset of latencies of the EMG activities of the facial muscles of the whisker pad and eyelid regions, evoked by ICMS to the O area were longer than those evoked by ICMS to the C area.

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Light and electron microscopic observations were carried out on the secretory granules of the labial gland of the lizard, Japalura polygonata polygonata. The results obtained are.
1) Japalura polygonata polygonata has superior and inferior labial glands, which can be described as a branched tubular type.
2) The glandular cells are arranged in a layer and appear to be columnar in shape.
3) The secretory granules of the superior labial gland stain intensely with naphthol yellow S and iron hematoxylin and are weakly positive with PAS and alcian blue.
4) The secretory granules of the superior labial gland can be divided into two types when examined with the electron microscope. One is of moderate electron density, while the other has a moderate dense core and a narrow less dense halo. The PA-methenamine silver method shows a positive reaction which is limited to the narrow halo of the latter granule.
5) The secretory granules of the inferior labial gland except those of the acini which are oriented superolaterally in the lower lip stain intensely with PAS and alcian blue, and they are also slightly positive with naphthol yellow S and iron hematoxylin.
6) The secretory granules of the inferior labial gland can be classified into six types by the degree of the electron density of their core and halo. One of these types has a tripartite structure. Almost all of the secretory granules are positive for the PA-methenamine silver reaction.
These findings suggest that the superior and inferior labial glands of Japalura polygonata polygonata have different characteristics from each other, because the secretory granules of the superior labial gland mainly contain protein, while those of the inferior labial gland contain mucopolysaccharides.

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The adrenergic nerve terminals in the rat submandibular gland, with the conventional method of fixation, contained many small round clear or granular vesicles; whereas, with the chromaffin reaction method of Tranzer and Richards, frequent tubular or flattened vesicles were observed in addition to the small round granular or clear vesicles. The factors influencing the occurrence of tubular or flattened vesicles after the chromaffin reaction method were examined.
The fixation procedure of chromaffin rection method was divided into three steps; prefixation, storage in the reaction medium and postfixation. Total of 11 experimental series were set up replacing some of these steps with corresponding ones used in conventional fixation method. The tubular or flattened vesicles were exclusively observed in the experimental series using 0.2M chromate buffer as the storage medium. With 0.08M chromate buffer which has the same osmolarity with 0.1M cacodylate buffer used in conventional fixation method, tubular or flattened vesicles could not be observed. On the other hand, the variation of the pH (from 6.0 to 7.2) of the buffer did not alter the profiles of synaptic vesicles; whereas, many synaptic vesicles were stained with dense chromaffin-reaction-products at pH 6.0 but not at pH 7.2.
In conclusion, the tubular or flattened vesicles may be an artefact resulting from high osmolarity of the storage buffer used in the chromaffin reaction method.

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Adherence of a Gram positive, anaerobic bacterium Propionibacterium ances to epithelial cells was examined using an in vitro assay system. The adherence of three strains ATCC 6919, 11827, and 11828 of P.acnes to human buccal epithelial cells was dependent on bacterial concentration, incubation time, and pH. Heat treatment of the bacterial cells decreased the subsequent bacterial adherence to the epithelial surfaces but the bacterial adherence was not completely eliminated by heat treatment. Trypsin added to an adherence assay system significantly inhibited the adherence of P. acnes ATCC 11828.
Scanning electron microscopy demonstrated projecting structures up to 300mμ long on the surface of strain ATCC 11828 associated with the bacterial attachment to the surface of the epithelial cells. Negative staining technique and an ultrathin section of a strain ATCC 11828 adherent to an epithelial cell also showed fuzzy structures mediating the bacterial binding to the epithelial cell. These results suggest that P.acnes adheres to human buccal epithelial cells by the heat-resistant fuzzy strurtures on the surface of the bacteria.

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A possible bone resorptive activity of various cytokines has been investigated by a bioassay with the use of the release of previously incorporated ⁴⁵Ca from fetal rat long bones in organ culture. Supernatant media from human peripheral blood mononuclear cells stimulated with phytohemagglutinin was found to contain a bone resorptive activity. When an IL-1-like factor was partially purified from the culture media of activated murin peritoneal macrophages or rabbit peritoneal polymorphonuclear leukocytes and then tested for their bone resorptive activity, they showed a definite activity in a dose-dependent fashion. Based on these results, several purified cytokines or Escherichia coli-derived recombinant cytokines of human or mouse origin were further examined for their bone resorptive activity. Human recombinant interleukin1 (IL-1/β) and tumor necrosis factor (TNF) showed a bone resorptive activity, while T cell products, interleukin2 (IL-2), interferon γ(IFNγ) and colony stimulating factor of granulocytes (G-CSF) of human origin showed no activity. In addition, interleukin 3 (IL-3), a factor promoting multipotent hematopoietic stem cells in bone marrow has been found to be effective on bone resorption. These results suggest that multiple cytokines may be involved in regulation of bone resorption by probably modulating the differentiation of bone marrow cells.

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We attempted to examine whether tissue culture media could support the growth of Trichomonas tenax (T. tenax).The effectiveness of the use of specific antiserum and complement to eliminate the accompanying bacteria was also examined. The tissue culture media such as both RPMI 1640 and Eagle's minimum essential medium (MEM) with serum were capable of supporting the growth of the trichomonad and even the serum-free synthetic media permitted more or less the growth. The growth of the bacteria was inhibited by the treatment with a combination of the antiserum and complement for 48 hr of incubation. It had been previously reported that only non-synthetic media such as Egg-Yolk could support the growth of the trichomonad. Therefore, it is very convenient that the synthetic medium could permit the growth of the cells. In addition, these culture media were used in the study of the interaction of cultured cells and trichomonad because they support the growth of both the T. tenax and the cultured cells. Although our present experiment using antiserum and complement failed to support the growth of the trichomonad without accomapnying bacteria, the experiments may provide a useful culture system for the study of the biological characteristics of the trichomonad with accompanying bacteria.

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Scanning electron microscopic observations of the dorsal tongue surface of mildly zinc deficient chickens revealed that the number of tangle-shaped filiform papillae normally located on the anterior dorsal tongue surface were fewer in number than in control chickens. The posterior tongue mucosal surface of both control and zinc deficient chickens was almost smooth with no lingual papillae. In the control chickens higher magnification showed relatively indistinct microridges and micropits widely distributed on the epithelial surface on the back of the tongue. In contrast, the epithelium of the same area in the chickens on a low zinc diet showed widespread, distinct microridges. Transmission electron microscope observations revealed that the stratum corneum of the dorsal tongue mucosa just beneath the filiform papillae, were clearly reduced in mildly zinc deficient chickens when compared with the control chickens. A relatively large number of nuclei could be recognized up to the stratum corneum in the mildly zinc deficient chickens. In the posterior dorsal tongue mucosa, a thin electron dense layer, which could be clearly observed in the normal chickens was indistinct in the mildly zinc deficient chickens and adjacent cells were firmly fixed to each other almost up to the dorsal surface of the tongue by numerous desmosomes.

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