Tumor necrosis factor alpha-mediated tumor regression by the in vivo transfer of genes into the artery that leads to tumors.

We report that tumor necrosis factor (TNF) alpha induced a strong antitumor immune reaction when it was produced in arteries leading to tumors by gene transfer in vivo. We used a mouse model carrying a sarcoma-180 tumor in the right footpad and injected the fusogenic liposomes encapsulating the human TNF-alpha gene into the right femoral artery. Under this condition, human TNF-alpha was detected only in the artery leading to the tumor and in the tumor. There was a significant regression in tumor growth when the TNF-alpha gene was delivered into the right femoral artery, with 4 of 11 mice completely cured. No regression was observed when the TNF-alpha gene was delivered into the left femoral artery or into the tumor or when the luciferase gene was administered. Tumor regression was inhibited by the injection of anti-TNF-alpha, anti-CD4, or anti-CD8 monoclonal antibody, and CD8+ T cells accumulated in the tumors of TNF-alpha-treated mice. These results suggest that TNF-alpha expressed locally in the arteries leading to tumors efficiently suppresses tumor growth through reinforcement of an antitumor immune reaction. The significance of this phenomenon for cancer gene therapy was discussed.

Dentin matrix protein 1 is predominantly expressed in chicken and rat osteocytes but not in osteoblasts.

Although osteocytes are the most abundant cells in bone, little is known about their function, and no specific marker protein for osteocytes has been described. Dentin matrix protein 1 (DMP1) is an acidic phosphoprotein expressed in tooth organ and bone. Our previous work showed that in the chicken, which is not capable of forming tooth, DMPI messenger RNA (mRNA) is highly expressed in bone by Northern blot analysis. To clarify the significance of DMP1 expression in bone, the expression of DMP1 mRNA and its protein was examined in the chicken and rat. In the chicken, DMPI mRNA was detected only in bone tissues and was localized in osteocytes and preosteocytes but not in osteoblasts. Similarly, in the rat, DMPI mRNA was predominantly expressed in osteocytes and preosteocytes in bone matrix but not in osteoblasts located at the bone surface. Antiserum was raised against the peptide from rat DMP1, and the localization of DMP1 was examined by immunohistochemistry. In the development of bone, DMP1 was first detected in newly formed bone matrix after osteoblastic cells had been embedded within it. After the appearance of typical osteocytes, DMP1 was localized in the pericellular bone matrix of osteocytes, including their processes. These data show that DMP1 is a bone matrix protein specifically expressed in osteocytes and preosteocytes and suggest that DMP1 plays a role in bone homeostasis because of its high calcium ion-binding capacity.

mRNA expression and protein localization of dentin matrix protein 1 during dental root formation.

Dentin matrix protein 1 (DMP1) is an acidic phosphoprotein. DMP1 was initially detected in dentin and later in other mineralized tissues including cementum and bone, but the DMP1 expression pattern in tooth is still controversial. To determine the precise localization of DMP1 messenger RNA (mRNA) and the protein in the tooth, we performed in situ hybridization and immunohistochemical analyses using rat molars and incisors during various stages of root formation. During root dentin formation of molars, DMP1 mRNA was detected in root odontoblasts in parallel with mineralization of the dentin. However, the level of DMP1 mRNA expression in root odontoblasts decreased near the coronal part and was absent in coronal odontoblasts. DMP1 protein was localized along dentinal tubules and their branches in mineralized root dentin, and the distribution of DMP1 shifted from the end of dentinal tubules to the base of the tubules as dentin formation progressed. During the formation of the acellular cementum, DMP1 mRNA was detected in cementoblasts lining the acellular cementum where its protein was localized. During the formation of the cellular cementum, DMP1 mRNA was detected in cementocytes embedded in the cellular cementum but not in cementoblasts, and its protein was localized in the pericellular cementum of cementocytes including their processes. During dentin formation of incisors, DMP1 mRNA was detected in odontoblasts on the cementum-related dentin, where its protein was localized along dentinal tubules near the mineralization front. The localization of DMP1 mRNA and protein in dentin and cementum was related to their mineralization, suggesting that one of the functions of DMP1 may be involved in the mineralization of dentin and cementum during root formation.

Expression of dentin matrix protein 1 (DMP1) during fracture healing.

Dentin matrix protein 1 (DMP1) is one of the acidic phosphorylated extracellular matrix proteins called the SIBLING (small integrin-binding ligand, N-linked glycoproteins) family. Recent studies showed that DMP1 is expressed in the mineralized tissues and suggested that DMP1 is involved in the mineralization. We investigated the precise localization of DMP1 messenger RNA (mRNA) and protein during fracture healing. In situ hybridization demonstrated that DMP1 mRNA was strongly expressed in preosteocytes and osteocytes in the bony callus during intramembranous and endochondral ossification while DMP1 mRNA was not detected in osteoblasts and chondrocytes. During endochondral ossification, however, a low number of DMP1-expressing cells were identified in the cluster of hypertrophic chondrocytes. However, these DMP1-expressing cells were not hypertrophic and were likely to be osteoblast-lineage cells, which were embedded in the matrix of bone or cartilage, because type I collagen-expressing cells and invasion of capillary vessels were observed in the same area. Northern blot, in situ hybridization, and immunohistochemical analyses showed that DMP1 mRNA and protein expressions were increased until day 14 postfracture, when bony callus was formed, and then declined to a lower level during remodeling of the bony callus. Therefore, DMP1 is likely to play an important role in the mineralization of the bony callus.

Transfection of wild-type TP53 induces differentiation in human gingival carcinoma cells.

We investigated the effects of transfection of wild-type TP53 on the growth properties of a human gingival carcinoma cell line, KOSC-3, in which the TP53 gene is mutated at codon 248 and overexpressed. The wild-type TP53 expression plasmid, pCDM8-p53/neo and the control plasmid, pCDM8/neo, were each stably transfected into KOSC-3 cells by using the calcium phosphate method. The number of G418-resistant colonies from wild-type TP53-transfected cells was approximately half that from plasmid controls. Exogenous wild-type TP53 transcripts were identified in four of the 20 G418-resistant clones analysed by reverse transcription PCR. Although the growth rates of the wild-type TP53+ clones did not drastically change during log phase, their saturation density was significantly reduced. The wild-type TP53+ cells were morphologically flat and enlarged when cultured in vitro, and were less able to form colonies in soft agar. In nude mice, the wild-type TP53+ clones formed subcutaneous tumours with conspicuous keratinisation and notable cell death that was not manifested in the parental and plasmid control cells. These findings indicate that the wild-type TP53 gene, even when it coexists with a mutated form, may function as a growth suppressor and differentiation inducer under restricted conditions in gingival squamous cell carcinoma.

Ultrastructure of regenerated junctional epithelium after surgery of the rat molar gingiva.

To elucidate the ultrastructure of reconstructed dentogingival junction and cytological details of regenerated junctional epithelium (RJE) and to discuss the functional aspects of RJE through comparing its fine structure with normal structure, molar gingivae of 15 Wistar rats were examined by electron microscopy at 6, 8 and 12 weeks after electrosurgical gingivectomy. As early as 6 weeks after surgery, the epithelial architecture of dentogingival junction was reestablished at the light microscopic level, and RJE showed the ultrastructure indistinguishable from that before surgery. The cytoplasmic vacuoles, characteristic of rat JE, displayed the morphology, distribution and intimate relationship with lysosomes, all of which were quite identical to those in controls, and were regarded to represent "phagosomes." Since various findings specific to JE are apparently reproduced in RJE, it seems that those peculiar structures would be an expression due to the environment rather than to the predetermined nature. RJE also might play a defensive function through the endocytic-vacuolar system as suggested for normal JE. The presence of two types of cuticular structures, which were not conspicuous in normal JE, was also revealed between RJE and the tooth.

Penetration and uptake of colloidal gold-labeled concanavalin A in the junctional epithelium of the rat.

A complex of colloidal gold and concanavalin A (CG-Con A) with various biological properties and high ultrastructural resolution was applied into the sulcus of rat molar gingiva and traced with an electron microscope for three hours to examine the cytological changes occurring in the cells of the junctional epithelium (JE) during penetration of extrinsic irritants, and to determine the roles of JE cells in such a circumstance. While the penetration of CG-Con A was impeded on the surface of keratinized oral gingival/sulcular epithelium, CG-Con A penetrated swiftly through JE into the connective tissue. In the process of penetration, CG-Con A was taken up by lysosomal and vacuolar structures of JE cells in which degenerative changes were often provoked. Degeneration of JE cells was seen selectively in the second and/or third cell layers from the innermost cell layer of JE. It was assumed that JE cells by their phagocytic activity might participate in the first line of defense against extrinsic irritants. On the other hand, the phagocytic activity of JE cells seems also to be involved in tissue destruction, if the amount and/or toxicity of irritants exceed the dissimilating capacity of JE cells.

Effect of exogenously applied prostaglandin E2 on alveolar bone loss--histometric analysis.

The effect of prostaglandin E2 (PGE2) on alveolar bone resorption was examined in 8-week old Wistar rats by histometric analysis. One mg/ml PGE2 topically applied to gingival sulcus induced a marked increase in osteoclasts. The number of osteoclasts increased progressively and reached a maximum at 12 hours. Ultrastructurally, these osteoclasts were in active form with well developed ruffled borders and clear zones. The changes in numbers of osteoclasts after application of various concentrations of PGE2 were dose-dependent (0.001 to 1.0 mg/ml), but higher concentrations of PGE2 (2 mg/ml) were less effective. In addition, the number of osteoclasts in groups treated with both PGE2 and endotoxin was higher than those that received PGE2 only. These results indicate that bone resorption caused by PGE2 depends on activation and increase of osteoclasts, and suggests that endogenous PGE2 production by host cells stimulated by plaque-associated bacterial endotoxin may be an important pathogenetic factor in periodontal disease.

Immunohistochemical detection of prostaglandins E2, F2 alpha, and 6-keto-prostaglandin F1 alpha in experimentally induced periapical inflammatory lesions in rats.

The immunohistochemical localization of prostaglandin (PG) E2, PGF2 alpha, and 6-keto-PGF1 alpha (a stable metabolite of PGI2) was demonstrated in rat periapical inflammatory lesions induced by opening the pulp chamber. Two wk postoperatively, suppurative periapical lesions were formed, and active bone resorption was seen surrounding these lesions. Immunohistochemical examination showed that macrophages infiltrating in inflammatory tissue were positively stained for the examined PGs. In some lesions, wherein acute inflammatory changes subsided and proliferation of fibroblasts started, the fibroblasts were positively stained for 6-keto-PGF1 alpha. Osteocytes and osteoblasts were also positive for 6-keto-PGF1 alpha not only in experimental animals, but also in untreated animals. However the staining intensity of the PG in these cells was higher in periapical lesions than in normal condition. These findings suggested that the cellular sources of the PGs in the periapical lesions are mainly macrophages and fibroblasts, and that the PGs produced by these cells, and possibly osteoblast and osteocytes, may contribute to the osteolytic resorption of periapical lesions.

Immunohistochemical demonstration of prostaglandins E2, F2 alpha, and 6-keto-prostaglandin F1 alpha in rat dental pulp with experimentally induced inflammation.

Using formalin-fixed and EDTA-decalcified cryostat sections, the immunohistochemical localization of prostaglandin (PG) E2, PGF2 alpha, and 6-keto-PGF1 alpha (a stable metabolite of PGI2) was examined in normal rat and inflamed dental pulp. Inflammation was induced by opening the pulp chamber. There was no immunoreactivity for prostaglandins in normal dental pulp, whereas positivities for PGE2, PGF2 alpha, and 6-keto-PGF1 alpha were demonstrated in the cytoplasm of macrophages and endothelial cells in the inflamed dental pulp. In addition to these cells, numerous pulp cells and odontoblasts existing in the inflamed pulp and its apical noninflamed area also were intensely stained for PGF2 alpha. Such an area with positive cells gradually extended in an apical direction with the progression of inflammation. These findings suggested that PG production from these host cells is involved in development of inflammation of rat dental pulp.

Immunohistochemical localization of carbonic anhydrase isozyme II in rat incisor epithelial cells at various stages of amelogenesis.

Carbonic anhydrase II (CAII) was purified from erythrocytes of male Sprague-Dawley rats, and its localization in rat maxillary incisor epithelial cells at various stages of amelogenesis was studied by means of immunoperoxidase staining using a rat CAII-specific monoclonal antibody. In the most apical portion of the incisor, some CAII immunoreactivity was localized in the outer or inner dental epithelium near the apical loop (i.e., the multiple layer of the outer dental epithelium and the posterior portion of ameloblasts facing the pulp). Immunoreactivity disappeared largely during the presecretory and secretory stages. CAII immunoreactivity appeared suddenly in ameloblasts during the transitional stage between enamel secretion and maturation. Immunoreactivity became intense in both ameloblasts and papillary cells during enamel maturation; the intracellular distribution of CAII was in the cytosol. The CAII signal in these cells was constant until the end of the maturation stage. These findings support the notion that the ameloblasts and papillary cells change into ion transport epithelial cells from the secretory to the maturation stage and that CAII in these cells plays an important role in the regulation of pH.

Non-calcifying Pindborg tumor with Langerhans cells.

A rare case of calcifying epithelial odontogenic tumor (CEOT) devoid of calcification is reported with histochemical, immunohistochemical and electron microscopic studies. The tumor occurred intraosseously in the left maxillary canine and premolar region of a 58-year-old man. The tumor chiefly consisted of scattered small islands of epithelial cells in an abundant fibro-myxoid connective tissue stroma. Among the nests, there were many spherical bodies of eosinophilic substance for which non-AA amyloid and non-keratin or basal lamina-like natures were demonstrated histochemically and immunohistochemically. In some nests, there were a few, occasionally several, cells positive for S-100 protein. Ultrastructurally, Langerhans cells with indented nuclei and Birbeck's granules were seen among tumor cells. The prognostic significance of the paucity of calcification and the presence of Langerhans cells in CEOT of which this is only the second description is discussed.

Clear cell tumors of minor salivary gland origin. An immunohistochemical and ultrastructural analysis.

Three cases of monophasic glycogen-rich clear cell tumors of palatal gland origin were examined immunohistochemically and ultrastructurally in attempts to characterize their cellular composition. Despite their histologic resemblances, the clear cells from each case showed different immunohistochemical features. In case 1 the extensive positivity for vimentin and S-100 protein, in addition to the focal expression of actin and glial fibrillary acidic protein, strongly suggested that the clear cells were myoepithelial in nature. In contrast, the clear cells from case 2 exhibited both keratin and epithelial membrane antigen positivity, as well as ultrastructural features that suggested that they were glandular epithelial in nature. In case 3 no special markers except for keratin could be detected, indicating the less differentiated nature of the clear cells. These results show the heterogeneity of the clear cell tumor group of minor salivary glands.

Verruciform xanthoma of the oral mucosa: a clinicopathological study with immunohistochemical findings relating to pathogenesis.

Verruciform xanthoma is an uncommon benign lesion with unknown aetiology and pathogenesis. In this study, we report ten cases of verruciform xanthoma and document their clinical and histopathological findings. An immunohistochemical investigation was performed using antibodies to macrophage, leukocyte common antigen, T lymphocytes, B lymphocytes, S-100 protein, lysozyme and alpha-1-antichymotrypsin. Our results were similar to the other reported cases. Eighty percent of our cases were found on the gingiva. Candidal hyphae were found in the superficial parakeratotic layers in five cases. The clinical diagnosis of the lesion ranged between papilloma and squamous cell carcinoma. It is important for clinicians to take into consideration the possibility of verruciform xanthoma in the differential diagnosis of papillary and granular lesions of oral mucosa. Immunohistochemically, all foam cells were strongly stained with antimacrophage antibodies. T lymphocytes were the predominant infiltrating lymphocytes in the lesion. Langerhans cells in the epithelia were fewer than those in corresponding normal tissue. Our immunohistochemical findings suggest that verruciform xanthoma is may be a local immunological disorder, with a cell mediated mechanism.

Florid cemento-osseous dysplasia with concomitant simple bone cysts: a case in a Japanese woman.

Florid cemento-osseous dysplasia (FCOD) with concomitant simple bone cysts is not common. We report a case of FCOD involving three quadrants of the jaws and associated with two large cystic lesions of the mandible in a 40-year-old Japanese woman. Microscopic examination revealed unencapsulated fibroblastic proliferation with formation of bone and cementum, showing different developmental stages, and cystic lesions resembling a simple bone cyst.

Enhanced collagen phagocytosis by rat molar periodontal fibroblasts after topical application of lipopolysaccharide--ultrastructural observations and morphometric analysis.

To investigate the effect of lipopolysaccharide (LPS) on phagocytic activity of collagen fibrils by periodontal fibroblasts, we studied rat molar gingival connective tissue and periodontal ligament under light and electron microscopy after topical application of LPS (5 mg/ml in physiological salt solution (PS)) on the gingival sulcus. Phagocytic activity of collagen fibrils by fibroblasts was evaluated by counting the number of collagen-containing vacuoles inside fibroblasts that were present within a defined area (1200 microns2). Values obtained from fibroblasts in the subepithelial connective tissue, the region near the alveolar crest, and the middle region of periodontal tissue were compared. Periodontal ligament fibroblasts showed increased phagocytosis of the collagen fibrils from 3 hours to 1 day after topical LPS application, but no differences were observed in the gingival tissue. The intracytoplasmic vacuoles containing collagen fibrils were of various sizes and shapes, showing positive for acid phosphatase and/or alkaline phosphatase reaction. Collagen phagocytic activity of the fibroblasts in the middle region of the periodontal ligament also increased after PS treatment. However, this was significantly less than that observed in LPS-treated animals (p less than 0.01). This study indicates that LPS may enhance the degradation of collagen by stimulating the phagocytic activity of the periodontal ligament fibroblasts.

Jaw cysts with orthokeratinization: analysis of 12 cases.

The clinico-pathologic, immunohistochemical and radiological features of 12 jaw cysts with a prominent orthokeratinized epithelial lining were studied and compared with those of typical odontogenic keratocysts and dentigerous cysts. They differed significantly from odontogenic keratocysts in terms of biologic behavior and histopathologic findings. Although immunohistochemical staining of the epithelial linings for cytokeratins, EMA, CEA and involucrin has not shed any light on the histogenesis of these lesions, staining patterns for these markers were significantly different from those of odontogenic keratocysts and non-keratinized dentigerous cysts. Radiologically, nine cases appeared as dentigerous cysts; two cases, one with sebaceous differentiation, as non-dentigerous unilocular cysts, and the remaining one was exceptional as it showed multiple epidermal cysts with prominent dermal appendages histologically. It is suggested that most of the orthokeratinized jaw cysts may belong to clinico-pathological entities different from odontogenic keratocysts with the majority representing dentigerous cysts with orthokeratinization. The possibility of the existence of rare central dermoid or epidermoid cysts is also to be considered.

Immunohistochemical demonstration of prostaglandins in various tissues of the rat.

To demonstrate the tissue localization of prostaglandin (PG) E2, PGF2 alpha and 6-keto-PGF1 alpha (a stable metabolite of PGI2) various tissues, including decalcified periodontal tissue of 7-week-old male Wistar strain rats, were immunohistochemically examined using a streptavidin-biotin complex method. Besides tissue macrophages and endothelial cells in various tissues, hepatocytes, renal tubular cells, and parietal and chief cells in the gastric mucosa showed a positive reaction for the various PGs examined. PGs were demonstrated in the cytoplasm or in association with the cell membrane. We generally observed no difference between the localization patterns of PGE2-, PGF2 alpha-, and 6-keto-PGF1 alpha-positive cells in these tissues. However, in the periodontal ligament and alveolar bone, 6-keto-PGF1 alpha was localized in the cytoplasm of osteocytes, osteoblasts, cementocytes, and cementoblasts, while no reaction for PGE2 or PGF2 alpha was revealed in these cells. We demonstrated the immunohistochemical localization of PGs in various rat tissues including decalcified periodontal tissue and discuss the important roles of PGs in the modulation of their normal functions in these tissues.