Ieyoshi Kobayashi

Immunohistochemical study on the immunocompetent cells of the pulp in human non-carious and carious teeth

The condition of the pulp tissue was classified into seven groups according to the depth of carious lesions from stage (S) 0 (non-carious teeth) to S6 (exposed pulp). A substantial change in the infiltration of immunocompetent cells occurred between S3 and S4; all types were markedly increased in S4 as compared to S3, with a remarkable increase in the number of helper T lymphocytes, B-lineage cells, neutrophils and macrophages. Therefore, the pulpal immune reaction to carious stimuli could be classified into early (S1-S3) and advanced phases (S4-S6). In the early phase a cellular immunoresponse would be induced by T-lineage cells, and in the advanced phase the humoral immunoresponse is furthered by B-lineage cells concomitant with the destruction of pulp tissue by proteolytic enzymes released from infiltrating neutrophils and macrophages. Human dental pulp is thus equipped with a functional immune response that is sufficient as a biodefensive mechanism. Dental caries should be treated before S4.

Expression of p53 tumor suppressor gene in adenoid cystic and mucoepidermoid carcinomas of the salivary glands

Seventeen adenoid cystic carcinomas (ACCs) and 27 mucoepidermoid carcinomas (MECs) occurring in the salivary glands were analyzed for p53 tumor suppressor gene alteration (exons 5-8) and protein expression. The cell proliferation activity was also examined by Ki-67 immunohistochemistry. The p53 alterations were detected in three samples (17.6%) of ACC and in four samples (14.8%) of MEC, and were only found in carcinomas arising in the minor salivary glands. The occurrence of the p53 gene alteration is less frequent in ACC and MEC than that in other kinds of tumors, and therefore does not seem to play a critical role in the course of the tumorigenesis in ACC and MEC. All ACC samples arising from the minor salivary glands exhibiting p53 gene alterations showed recurrence/metastasis, thus suggesting a poor outcome of these patients. All ACCs and three out of four MECs samples with p53 gene alterations showed the lowest degree of p53 immunostaining ratio, thus suggesting that no correlation exists between the p53 gene alterations and the p53 immunostaining in these salivary gland carcinomas. No significant relationship was demonstrated between the immunostaining ratio of either p53 or Ki-67 and the morphological growth pattern or patient clinical course in the ACC samples. The p53 immunopositivity in MEC correlated to the histological grade. The Ki-67 immunostaining ratio was also significantly related to the histological grade and the clinical course in MEC.

Prevalence of Epstein–Barr virus in oral squamous cell carcinoma

Forty‐six samples of oral squamous cell carcinoma (OSCC) were evaluated for the prevalence of Epstein–Barr virus (EBV) infection by the polymerase chain reaction (PCR), Southern blot hybridization, and in situ hybridization (ISH). EBV DNA was detected in 7 (15·2 per cent) out of 46 samples by a combination of PCR and Southern blot hybridization methods. All seven positive samples showed well‐differentiated carcinoma, thus suggesting a possible relationship between EBV infection and the degree of differentiation of carcinoma tissue. Latent infection membrane protein 1 (LMP1) was detected immunohistochemically in six of the EBV‐positive OSCCs. However, no signal of the EBV‐encoded small RNA (EBER)‐1 was demonstrated by the ISH method. No significant relationship was observed between EBV infection and lymph node metastasis. A follow‐up study (range from 4·4 to 79 months; mean 34·9 months) showed no recurrence or death to occur in the EBV‐positive patients, which thus suggested a good prognosis for EBV‐positive OSCC patients. Copyright

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 distribution of BrdU- and TUNEL-positive cells during odontogenesis in mouse lower first molars

This study investigated the minute distribution of both proliferating and non-proliferating cells, and cell death in the developing mouse lower first molars using 5-bromo-2′-deoxyuridine (BrdU) incorporation and the terminal deoxynucleotidyl transferase-mediated deoxyuridine-5′-triphosphate (dUTP)-biotin nick end labeling (TUNEL) double-staining technique. The distribution pattern of the TUNEL-positive cells was more notable than that of the BrdU-positive cells. TUNEL-positive cells were localized in the following six sites: (1) in the most superficial layer of the dental epithelium during the initiation stage, (2) in the dental lamina throughout the period during which tooth germs grow after bud formation, (3) in the dental epithelium in the most anterior part of the antero-posterior axis of the tooth germ after bud formation, (4) in the primary enamel knot from the late bud stage to the late cap stage, (5) in the secondary enamel knots from the late cap stage to the late bell stage, and (6) in the stellate reticulum around the tips of the prospective cusps after the early bell stage. These peculiar distributions of TUNEL-positive cells seemed to have some effect on either the determination of the exact position of the tooth germ in the mandible or on the complicated morphogenesis of the cusps. The distribution of BrdU-negative cells was closely associated with TUNEL-positive cells, which thus suggested cell arrest and the cell death to be essential for the tooth morphogenesis.

T-box transcription factor Brachyury expression is correlated with epithelial-mesenchymal transition and lymph node metastasis in oral squamous cell carcinoma

The prognosis of patients with oral squamous cell carcinoma (SCC) is influenced by the presence of lymph node metastasis. Epithelial-mesenchymal transition (EMT), a process that involves events that convert adherent epithelial cells into individual migratory cells that can invade the extracellular matrix, is critical for cancer progression. Recently, the T-box transcription factor Brachyury was reported to promote EMT in human carcinoma cell lines. We analyzed the relationship between EMT (assessed by staining for E-cadherin and Vimentin) and the expression of Brachyury in association with lymph node metastasis in oral SCC. Oral SCC biopsy specimens (152 cases) were examined immunohistochemically for the expression of E-cadherin, Vimentin and Brachyury. Expression of Brachyury was correlated with EMT (p=0.035) and was significantly associated with lymph node and distant metastasis (p<0.05). Logistic regression analysis showed that Brachyury and EMT were predictive factors for lymph node metastasis (odds ratio 4.390 and 5.936, respectively) and that EMT was a predictive factor for distant metastases (odds ratio 11.786). Our findings present clinical evidence for an important role of Brachyury in EMT in oral SCC, and suggest that Brachyury and EMT patterns are useful prognostic markers.

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.

Possible functional involvement of thymosin beta 4 in developing tooth germ of mouse lower first molar

We examined the detailed in situ expression pattern of thymosin beta 4 (Tβ4) in the developing mouse mandibular first molar. Tβ4 mRNA was expressed in the presumptive dental epithelium at embryonic day 10.5 (E10.5) and in the thickened dental epithelium at E12. An in situ signal was observed in the invaginated epithelial bud at E13, in the enamel organ at E14 and E14.5, and in the primary enamel knot (PEK) at E14.5. The signal was localized in the epithelial cells of the outer layer of the enamel organ at E15 and E15.5. No signal was found in the PEK at these stages. Tβ4 mRNA was expressed in the inner enamel epithelium, cervical loop and dental lamina at E16 and E17. The expression of Tβ4 mRNA was observed in the polarized inner epithelial cells at E18, newborn day 1 (N1) and N2. However, the signal intensity decreased markedly at N3. We herein report for the first time that Tβ4 is distinctly expressed in developing tooth germ, and it may also play functional roles in the initiation, growth and differentiation of tooth germ.

Localization of Activated Caspase-3-positive and Apoptotic Cells in the Developing Tooth Germ of the Mouse Lower First Molar

This study examined the immunohistochemical detection of activated caspase-3, and its association with apoptosis, during tooth morphogenesis of the mouse lower first molar. The distribution of cells positive for caspase-3 closely corresponded with the localization of the terminal deoxynucleotidyl transferase-mediated deoxyuridine-5′-triphosphate-biotin nick end labelling (TUNEL)-positive apoptotic cells through the developmental course of tooth germs from embryo day 12 (E12) to E19, thus showing that the apoptosis occurring in the developing odontogenic tissue was induced by the activation of the caspase family. The specific distribution pattern of apoptotic cells in the developing odontogenic epithelial tissue from the initiation (E12) of tooth germ to the completion of tooth crown morphology (E19) also suggests that apoptotic events are related not only to a deletion of functionally suspended cells, but also participate in initiation and the completion of tooth morphogenesis. Electron microscopic examination revealed that apoptotic cells were present in the primary enamel knot, and these apoptotic cells were phagocytized by neighbouring odontogenic epithelial cells, thus indicating the prompt disposal of any dead cells by epithelial cells.

STAT3 signal transduction through interleukin-22 in oral squamous cell carcinoma

Interleukin (IL)-22 is a member of the IL-10 family. Its main targets are epithelial cells, not immune cells. We examined IL-22 signal transduction in oral squamous cell carcinoma (OSCC) cells. Immunohistochemical staining revealed that IL-22R was expressed more highly in OSCC compared to normal regions. An IL-22R signal was also observed in metastatic OSCC cells in the lymph node. RT-PCR showed that the human OSCC cell lines MISK81-5, HSC-3, HSC-4, SAS and SQUU-B expressed IL-22 receptor chains. Immunoblotting showed that IL-22 induced a transient tyrosine phosphorylation of STAT3 (pY705-STAT3) in MISK81-5 cells. The change in the serine phosphorylation of STAT3 was subtle during the examination periods. Simultaneously, pY705-STAT3 activation in HSC-3 cells was undetectable after IL-22 stimulation. Immunocytochemistry demonstrated that IL-22 induced the translocation of phosphorylated STAT3 into the nucleus of MISK81-5 cells. IL-22 temporarily upregulated the expression of anti-apoptotic and mitogenic genes such as Bcl-x, survivin and c-Myc, as well as SOCS3. IL-22 transiently activated ERK1/2 and induced a delayed phosphorylation of p38 MAP kinase, but negligibly involved the activation of NF-κB in MISK81-5 cells. MISK81-5 and SQUU-B cells treated with IL-22 showed mild cellular proliferation. MISK81-5, HSC-4 and SAS cells treated with IL-22 downregulated the keratinocyte differentiation-related genes compared with unstimulated cells. Conversely, STAT3 suppression by STAT3 siRNA strongly disrupted the down-regulation of these genes by IL-22, but it did not significantly affect the activation of ERK1/2 by IL-22. The OSCC cells used in this study upregulated the expression of SERPINB3/4 (SCCA1/2), well-known SCC markers, following treatment with IL-22. These results indicate that IL-22 differentially activates the STAT3 signaling system depending on the type of OSCC. IL-22 may therefore play a role in tumor growth, cell differentiation and progression through STAT3-dependent and -independent pathways.