Yukiko Ishibashi

Immunohistochemical localization of cathepsins D and E in human gastric cancer: a possible correlation with local invasive and metastatic activities of carcinoma cells.

The immunohistochemical localization of cathepsins D and E in 44 cases of human gastric carcinoma, using antibodies specific for each enzyme, were investigated. Cathepsin D- and E- positive carcinoma cells were present in all samples. However, the staining intensity varied from cell to cell in the same carcinoma tissue as well as among samples. The most intense immunostaining of both cathepsins was often found in the cells, which were present at the advancing margin of the carcinoma tissues. The incidence of this peculiar localization of intensely stained carcinoma cells significantly correlated with the progression of the carcinoma tissue (D, P < .05; E, P < .01) and with occurrence of the lymph node metastasis (D and E, P < .05). There was no statistical significance between this localization and the histological type (differentiation) of the carcinoma tissues. Cathepsin-positive inflammatory cells infiltrated in and around the carcinoma tissue, and intensely stained inflammatory cells were often located in the stroma at the border of the carcinoma tissue. However, no statistical correlation was noted between the localization of cathepsin-positive inflammatory cells at the border and the stage of progression or the incidence of metastasis. These results indicated that cathepsins D and E in the carcinoma cells located at the advancing margin play an important role in the invasion and subsequent metastasis of human gastric carcinoma. Meanwhile, cathepsin-positive inflammatory cells seem to be less responsible for the biological behavior of carcinoma cells than those in the carcinoma cells themselves.

The proliferative activity in dysplasia and carcinoma in situ of the uterine cervix analyzed by proliferating cell nuclear antigen immunostaining and silver-binding argyrophilic nucleolar organizer region staining

The proliferative activity of dysplasia and carcinoma in situ (CIS) in the uterine cervix was examined using proliferating cell nuclear antigen (PCNA) immunostaining and silver-binding argyrophilic nucleolar organizer region (AgNOR) staining. A significant difference in the labeling index of PCNA immunostaining obtained from dysplastic cells in each histopathologic category was demonstrated between severe dysplasia and CIS. There was no significant difference among each category of dysplasia. The frequency of mitotic figures was highest in CIS, followed in decreasing order by severe, moderate, and mild dysplasia, and was closely correlated with the histopathologic classification. There was an intimate correlation between the PCNA and mitotic indexes in severe dysplasia and CIS. However, the mean numbers of AgNORs in each category of dysplasia and CIS were not significantly different, and there was no apparent relationship with the histologic classification. The PCNA index seemed to be a useful means of evaluating proliferative activity in dysplasia and CIS, and especially in distinguishing CIS from severe dysplasia. In addition, the present PCNA index suggests that a considerable alteration of the biologic behavior involving genetic changes occurs during the progression of carcinogenesis from severe dysplasia to CIS. However, AgNOR staining did not appear to reliably represent the proliferative activity in these lesions.

An immunohistochemical and ultrastructural study of vasomotor nerves in the microvasculature of human dental pulp

Immunohistochemical methods for S-100 protein and neurone-specific enolase showed two types of nerve endings around the pulp microvasculature: the free endings, which comprise the major neural component and are distributed in all types of microvessel such as arterioles, venules and capillaries; and the varicose endings. The varicose ending was a less frequent, minor component observed only in the arterioles. Both immunohistochemical and ultrastructural observations confirmed that the varicose endings were the terminal axons of efferent vasomotor nerves. Further extensive ultrastructural examinations on the vasomotor nerves added the following new findings to our previous reports. Vasomotor nerves sometimes ramified into more than two terminal axons around arterioles, and most of these ramified axons ended in the adventitia-media junction of the arteriolar wall; however, nerve endings occasionally penetrated into the media. These findings suggest an intimate structural association between vasomotor nerves and arterioles in regulating the arteriolar microcirculation in human dental pulp by stimulating smooth-muscle cells not only of the outermost but also of the inner layers. Furthermore, the deep penetration of terminal axons into the arteriolar wall seems to provide effective regulation of pulpal blood flow under physiological and pathological conditions.

Biological Behavior of Human Dental Pulp Cells in Response to Carious Stimuli Analyzed by PCNA Immunostaining and AgNOR Staining

The change in proliferative and metabolic activities of human dental pulp cells responding to carious stimuli was studied by means of immunohistochemical staining for proliferating cell nuclear antigen (PCNA) and silver-binding nucleolar organizer region (AgNOR) staining. We classified the pulp tissues into five groups according to the progression of dental caries, ranging from grade 0 (the pulp of noncarious teeth) to grade 4 (the pulp of perforated carious teeth). PCNA-positive pulp cells were detected only in advanced dental caries (grades 3 and 4), and the difference in immuno-positive rate was significant between the two grades (p <0.001). However, the mean number of AgNORs per nucleus increased even in the early phase of dental caries, significant differences being detected between grades 1 and 2 (p<0.005), 2 and 3 (p<0.005), and 3 and 4 (p<0.001). Our data suggested that the metabolic activity of dental pulp cells was enhanced in the early phase of dental caries. However, proliferation of pulp cells occurred later in small degrees during fully developed caries such as grades 3 and 4. The slow and weak response in cellular proliferation might contribute to the usual fragility of the pulp to various assaults including caries or pulpitis.

An immunohistochemical study of HLA-DR and α1-antichymotrypsin-positive cells in the pulp of human non-carious and carious teeth

The condition of the pulp tissue was classified into seven groups according to the progression of carious lesions from stages S0 (non-carious teeth) to S6 (exposed pulp). There was a small number of anti-HLA-DR antibody-positive cells in the pulp of the early carious teeth, and a markedly increased number at S5 and S6. The recruitment of a large number of anti-HLA-DR cells concomitant with a marked increase of other kinds of immunocompetent cells in the pulp of late-stage caries might indicate the occurrence of antigen presentation followed by both cell-mediated and humoral immune reactions. The number of anti-alpha 1-antichymotrypsin (ACT) antibody-positive macrophages showed a proportional increase with the development of caries, and these cells may be involved in protecting against the tissue damage caused by proteases released from inflammatory cells, as well as having a defensive role by phagocytosis of toxic micro-organisms and damaged tissue residues. Thus anti-HLA-DR and anti-ACT antibody-positive cells might participate in both an efficient immune system and a tissue-protective mechanism in the human dental pulp.

Ultrastructure of the neuromuscular junction of vasomotor nerves in the microvasculature of human dental pulp

Vasomotor nerves in human dental pulp were more closely related to arterioles than to venules. Most were composed of unmyelinated fibres, which were mainly adrenergic. They appeared close to arterioles that were surrounded by a few layers of contractile smooth-muscle cells. The smaller arterioles with a diameter of 10-15 microns received a more intimate innervation by vasomotor nerves than did the larger. These vessels occasionally showed much narrower neuromuscular junctions than previously reported. Most of these nerve fibres were identified as adrenergic by the presence of chromaffin-positive synaptic vesicles detected by ultrastructural enzyme histochemistry. Their function appeared to be to regulate the blood flow and/or the blood pressure by stimulating smooth-muscle cells, resulting in contraction and a change in the calibre of the vessels. Capillaries and venules, which have a higher permeability, received weaker innervation by the vasomotor nerves than did arterioles. The intimate relation between vasomotor nerves and arterioles is related to the function of dental pulp in normal and pathological conditions.

Expression of Set-α during morphogenesis of mouse lower first molar

The detailed in situ expression pattern of the Set-α gene has been studied. Previously we showed that Set-α is a differentially expressed gene in the embryonic mouse mandible at day 10.5 (E10.5) gestational age. Cells expressing Set-α were widely distributed in both the epithelial and underlying ectomesenchymal cells at E10.5. At E12, they were slightly aggregated in an area where tooth germ of the lower first molar is estimated to be formed. At E13.5, Set-α was strongly expressed in the tooth germ. At the cap stage, Set-α was expressed in the enamel organ and dental papilla. At the bell stage, Set-α was distinctly expressed in the inner enamel epithelial and dental papilla cells facing the inner enamel epithelial layer, which were intended to differentiate into ameloblasts and odontoblasts, respectively. Interestingly, Set-α was also expressed in several embryonic craniofacial tissues derived from the ectoderm. This study is the first report that Set-α is distinctly expressed in the developing tooth germ, and suggests that Set-α plays an important role in both the initiation and the growth of the tooth germ, as well as in the differentiation of ameloblasts and odontoblasts.

Significance of pH on differentiation of human erythroid cell lines

As physiological factors and compounds, BSA and/or higher pH of the culture medium could induce the erythroid differentiation of cells. Optimum pH values of the culture medium for higher spontaneous differentiation of KU-812 and K562 cells after 7 days cultivation were 7.5 and 7.6, respectively. The synergistic effects on the differentiation were observed by exposure to hemin under the higher pH condition. In the presence of BSA, 25% of KU-812 and 40% of K562 cells became benzidine positive. Synergistic effects of BSA and the higher pH of the medium were also observed.