The sequential demonstration of non-specific esterase reactivity Ia antigen and Thy-1 antigen in murine epidermal sheets

Immunofluorescence demonstrating Ia and Thy-1 antigens and non-specific esterase (NSE) enzyme histochemistry were performed sequentially on sheets of ear epidermis from CBA, C3H.OH, A/J, and Balb/c mice. The same areas of epidermis were photographed after each reaction and individual cells identified and compared. Thy-1+ dendritic cells expressed neither Ia antigen nor NSE reactivity. No cell was found to express Ia antigen or NSE alone: thus all Langerhans cells (LC) in normal murine epidermis appeared to co-express Ia antigen and NSE reactivity. LC expressing greatly increased amounts of Ia antigen were occasionally seen apposed to Thy-1+ cells suggesting that these cells may be immunologically active--perhaps involved in antigen presentation.     

Expression of TGF-beta1, Smad7 and cell apoptosis in epithelium of oral lichen planus

OBJECTIVE: To examine the expression of TGF-beta1, Smad7 and cell apoptosis in oral lichen planus (OLP) and to evaluate the possible pathogenesis of oral lichen planus. METHODS: Immunohistochemical technique was used to study the expression of TGF-beta1 and Smad7 in the epithelia cells of 17 OLP cases and 7 normal oral mucosa (NOM). TUNEL was used for detecting the cell apoptosis in 17 OLP cases and 7 NOM. RESULTS: TGF-beta1 was moderately positive in the epithelia cells of OLP. All the epithelia cells in OLP showed strong cytoplasmic staining. The expression of TGF-beta1 and Smad7 were significantly increased in OLP compared with that in NOM (P < 0.05). Cell apoptotic index (AI) was remarkably increased in epithelia cells in OLP cases, and the cell apoptosis was localized in basal and suprabasal epithelial layers. There was a positive correlation between TGF-beta1 expression and cell apoptosis in the epithelia of OLP (r = 0.69, P <0.05). CONCLUSIONS: High expression of TGF-beta1 and Smad7 in the epithelia of OLP suggests that TGF-beta1-Smad7 signal pathway was disturbed in oral lichen planus. The imbalance of TGF-beta1-Smad7 pathway may contribute to the mechanisms of cell apoptosis of epithelial cells in OLP.     

口腔黏膜癌前病变和口腔鳞状细胞癌中Stat3的表达及意义

目的研究口腔黏膜癌前病变和口腔鳞状细胞癌(OSCC)中细胞信号传导和转录激活因子(Stat3)的表达及意义。方法采用免疫组化方法分别检测9例正常口腔黏膜,口腔扁平苔藓(OLP)和白斑(OLK)共8例,OLP、OLK伴异常增生22例,OSCC19例中Stat3的表达。结果Stat3阳性表达分布于细胞质和细胞核内。正常口腔黏膜、单纯增生、异常增生和OSCC中Stat3阳性表达率分别为11.11%(1/9)、12.50%(1/8)、59.09%(13/22)和84.21%(16/19)。OSCC与正常口腔黏膜、单纯增生、异常增生相比,差异有显著性(P<0.05);异常增生与正常口腔黏膜、单纯增生相比,差异有显著性(P<0.05);而单纯增生和正常口腔黏膜相比差异无显著性(P>0.05)。结论Stat3与口腔黏膜癌变的发生发展有着密切关系,对Stat3表达的研究将有助于口腔黏膜癌前病变癌变的检测和OSCC的早期诊断。     

Staining patterns of rodent squamous epithelia by monoclonal anti-keratin antibodies

Anti-keratin staining patterns were examined by immunofluorescence in the epidermis, oral mucosa and forestomach of rats and mice by monoclonal antibodies AE1 and AE2. In contrast to human tissues enzymatic pretreatment of sections was necessary even when fresh-frozen tissue was used, suggesting masking of the antigens in vivo. AE1 stained the cytoplasm of spinous cells in most epithelia, whereas basal cell staining varied. AE2 showed suprabasal cytoplasmic staining in epidermis, forestomach and palate, whereas the other epithelia were stained only in keratohyalin granules and membranes of cornified cells. In some epithelia a small number of irregularly distributed basal cells stained positive with AE2, indicating heterogeneity in the basal cell compartment. Thus, the anti-keratin staining pattern varies both regionally and, as earlier shown in human epithelium, with the stage of maturation of cells within a given epithelium. The present study provides a basis for further studies of epithelial differentiation during normal and pathologic development.     

Staining patterns of rodent squamous epithelia by monoclonal anti-keratin antibodies

Anti-keratin staining patterns were examined by immunofluorescence in the epidermis, oral mucosa and forestomach of rats and mice by monoclonal antibodies AE1 and AE2. In contrast to human tissues enzymatic pretreatment of sections was necessary even when freshfrozen tissue was used, suggesting masking of the antigens in vivo. AE1 stained the cytoplasm of spinous cells in most epithelia, whereas basal cell staining varied. AE2 showed suprabasal cytoplasmic staining in epidermis, forestomach and palate, whereas the other epithelia were stained only in keratohyalin granules and membranes of cornified cells. In some epithelia a small number of irregularly distributed basal cells stained positive with AE2, indicating heterogeneity in the basal cell compartment. Thus, the anti-keratin staining pallern varies both regionally and, as earlier shown in human epithelium, with the stage of maturation of cells within a given epithelium. The present study provides a basis for further studies of epithelial differentiation during normal and pathologic development.     

Cytokeratin patterns of human oral mucosae in histiotypic culture.

In a three-dimensional culture model, oral epithelial differentiation was investigated ultrastructurally and biochemically for cytokeratin expression. Epithelia from the hard palate, gingiva and alveolar mucosa grown on freely floating collagen lattices populated with fibroblasts from homotypic origins, and fed with medium containing 10% delipidized fetal calf serum for 21 days before analysis, stratified and differentiated to basal cuboidal cells, polyhydral spinous cells and elongated superficial cells. The epithelium of palatal origin had non-nucleated superficial cells resembling orthokeratinized cells. The upper spinous cells had keratohyalin-like granules. The corresponding cells of gingival and alveolar mucosal origins retained their nuclei and had smaller numbers of keratohyalin-like granules. Basal cell keratins (CK 5 and 14) and those of hyperproliferation (CK 6 and 16) were consistently found in all epithelia. Furthermore, simple epithelial keratins (CK 18 and 19) were variably expressed by cells from different oral origins. In epithelial cells from the alveolar mucosa, CK 13 and 19 formed major bands, which correlates with their expression in vivo. In contrast, these polypeptides were either absent or formed minor bands in extracts of gingival and hard palatal cells. Although in small quantities, keratins of terminal differentiation (CK 1, 2, 10 and 11) were detected in gels prepared from palatal epithelia. This expression correlates with the higher morphological differentiation of these cells in this model. The model is of interest for studies of epithelial differentiation, as the differentiation markers of keratinized epithelia (CK 1 and 10) were expressed by cells from palatal origin, and those of non-keratinized epithelia (CK 4, 13 and 19) were prominent in cells from alveolar mucosal origin.     

Immunohistochemical localization of Leu-M1 carbohydrate antigen in human oral squamous cell carcinoma

Twenty-six biopsy specimens of oral squamous cell carcinomas were examined by the avidin biotin peroxidase complex (ABC) method for the presence of an epithelial cell membrane bound lacto-N-fucopentaose III, known also as Leu-M1 or Lex antigen. In normal oral epithelium, Leu-M1 antigen was expressed on keratinizing epithelia in the stratum spinosum. In well-differentiated carcinomas the antigen was found on the cell membrane of nucleate cells in infiltrating epithelial islands. Such pattern in moderately well and in poorly differentiated carcinomas was minimally expressed and was associated with flattened squamous cells or otherwise recorded negative. Leu-M1 antigen immunoreactivity in normal oral epithelia and in carcinomas was comparable to that of blood group H-2 chain that were examined. It was concluded that the intensity of the reaction parallels the magnitude of differentiation of epithelia. Leu-M1 antigen can serve as a marker of differentiation in oral squamous epithelium.     

Characterization of human dental pulp-derived cell lines

Aim  To establish and characterize different types of fibroblastic cell lines derived from dental pulp tissue.
Methodology  Human dental pulp tissue-derived cells were transfected with SV40 large T antigen by Lipofectamine transfection method. Geneticin (G418)-resistant cells were selected and different cell lines were established by a limiting dilution method. To characterize the lineages of cells, each clone was immunofluorescently stained by anti-fibroblast, anti-vimentin, anti-collagen type I and type III antibodies. Total RNA was extracted from each clone and subjected to a differential display experiment.
Results  By transfecting SV40 large T antigen, nine different cell clones were obtained. All these cell clones were positively stained by anti-fibroblast, anti-vimentin, anti-collagen type I and type III antibodies. With differential display experiment, eight different genes, the expression levels of these genes were varied amongst each cell clone, were detected. After sequencing and database search, one gene was revealed to be identical to T-cell marker, Thy-1. Thy-1 expression in dental pulp tissue was confirmed by immunohistochemical staining.
Conclusion  Fibroblastic cell lines derived from human dental pulp tissue possessed different gene expression profiles suggesting the existence of subpopulations.     

Expression profile of chemokines and chemokine receptors in epithelial cell layers of oral lichen planus

BACKGROUND: To understand the immunopathological features of oral lichen planus (OLP), we analyzed the expression of chemokines in the epithelial cell layers. Methods: Epithelia from OLP or healthy gingiva were collected by laser microdissection. The chemokine and chemokine receptor expressions in the epithelia were analyzed by DNA microarray. RESULTS: High levels of MIP-3alpha/LARC/CCL20 and its receptor CCR6 were expressed in the lesional epithelia. Furthermore, DC-CK1/CCL18, ELC/CCL19, SDF-1/CXCL12 and CXCR4 expressions were also increased. Immunohistologial analysis showed that high numbers of Langerhans cells (LCs) were present in the epithelia of OLP. Lesional epithelia also expressed high levels of the ligands specific for CXCR3 (e.g. MIG/CXCL9, IP-10/CXCL10 and I-TAC/CXCL11) and CCR5 (e.g. RANTES/CCL5). CONCLUSIONS: Infiltration of LCs is orchestrated by CCR6. Further, LCs residing in the lesional epithelia may be a mature phenotype. Moreover, infiltration of T cells in OLP could be mediated by signaling pathways through CXCR3 and CCR5.     

Lectin binding of rat gingival epithelia

Oral gingival epithelium (OGE), oral sulcular epithelium (OSE) and junctional epithelium (JE) were examined histochemically by using different lectins as markers for epithelial differentiation. The staining pattern of gingival epithelia was compared with that of the buccal and palatal epithelia. Binding of WGA had a uniform distribution in all the epithelia examined. A positive reaction was found in all the basal and spinous layers, but not in the cornified layer of the epithelia. BPA binding was seen in the lower spinous layer of OGE, OSE, buccal and palatal epithelia, and in most of the JE. The basal layer and the cells directly attached to the tooth surface at the apical part of JE were nonreactive with BPA. GS-I reacted with the basal and suprabasal layers of each epithelium and with the cells attached to the tooth at the apical part of JE. UEA-I reacted with the upper spinous layer of OGE, OSE and epithelia of hard palate, but not with any of the cells of the JE. Our results agree with previous data suggesting that OGE and OSE exhibit squamous differentiation similar to that of the masticatory epithelium of hard palate. Furthermore, our results suggested that the JE cells undergo differentiation equivalent to that of the suprabasal and lower spinous cells of OGE. The cells along the tooth surface at the apical part of JE, however, form a distinct population of cells with basal nature.     

凋亡相关蛋白Bcl-XL和Bak在口腔黏膜癌前病变和鳞癌组织中的表达

目的 :探讨凋亡相关蛋白Bcl-XL和Bak在口腔鳞状细胞癌组织发生、发展过程中的表达及意义。方法 :采用免疫组织化学SP法检测 8例正常口腔黏膜上皮、7例异常增生上皮和 4 2例鳞癌组织中Bcl-XL和Bak的表达。结果 :Bcl-XL和Bak在正常黏膜上皮中的表达分别有 12 .5 0 % (1/ 8)阳性率 ;Bcl -XL在鳞癌组织中的表达高于其在正常黏膜与异常增生上皮中的表达 (P <0 .0 5 ) ,差异有显著性 ;Bak在异常增生上皮和鳞癌组织中的表达明显高于正常黏膜 (P <0 .0 5 ) ,但在低分化鳞癌组的表达较高分化组明显降低 (P <0 .0 5 )。结论 :抑凋亡蛋白Bcl-XL上调导致异常增生上皮或 (和 )癌细胞积累。促凋亡因子Bak在代偿性过表达后 ,其作用随癌组织分化程度降低而减弱 ,癌细胞凋亡受限 ,恶性度增强。细胞凋亡的异常调控对口腔鳞癌的发生、发展有重要作用     

Ki-67 expression in non-tumour epithelium adjacent to oral cancer as risk marker for multiple oral tumours

Objective:  The aim of this study was to determine whether the differential assessment of epithelial proliferation is useful to diagnose premalignant fields and assess the risk of multiple tumours.
Material and methods:  We analysed 83 oral carcinomas with associated non-tumour epithelium classified as distant or close according to its distance (> or <1 cm) from the invasion point, and as squamous hyperplasia, mild, moderate, severe dysplasia or carcinoma in situ . Twenty-five healthy oral mucosa samples were used as controls. An immunohistochemical technique was applied using Mib-1. Ki-67 in premalignant epithelium was assessed in basal layer, parabasal layer, medium and upper third.
Results:  Parabasal expression was significantly higher or showed a tendency to be higher in close and distant epithelia with any histological grade than in the controls. Parabasal Ki-67 significantly differed between distant epithelia associated with multiple vs single tumours ( P  < 0.001) and between distant epithelia associated with multiple tumours vs controls ( P  < 0.001). This difference was not observed between distant epithelia associated with single tumours and controls ( P  = 0.175). The cut-off point that differentiated epithelia associated with multiple tumours was >50% of Ki-67 + parabasal cells in distant epithelia, which yielded 0.88 sensitivity and 0.79 specificity.
Conclusions:  The concept of a precancerous field may be linked to an increase in the proliferative activity of parabasal cells.     

Histochemical demonstration of lectinbinding sites and keratin in inflamed human gingiva

Histochemical demonstration of lectin-binding sites and keratin peptides in gingival epithelia was reported and differences in staining and distribution were compared to inner and outer gingival epithelia.
Gingival epithelia on the outer side exhibited zonal or regional distributions of lectin-binding, and the cytochemical staining was generally found in the cell coat and intercellular materials. Keratin protein was found frequently in the spinous cell, infrequently in the basal cells, and not at all in the superficial layer. The sugar residues in the cell coat of gingival epithelia probably were mannose, galactose, N-acetyl-galactosamine, and N-acetyl-glucosamine.
The inner side of the epithelia, crevicular, and pocket epithelia were characterized by irregular and incomplete staining for lectins. These epithelia also displayed less keratin staining compared to the outer gingival epithelia. These findings suggest the possibility of depolymerization of glycosaminoglycans in the cell coat by enzymes of either pocket bacterial origin or host tissue origin.     

氟化物作用下体外培养人牙胚内釉上皮细胞中Smad1、5表达的变化

目的观察不同浓度的氟化物对体外培养的人牙胚内釉上皮细胞中Smad1、5表达的影响,从细胞内信号转导水平观察氟对骨形成蛋白(BMP)信号转导的影响。方法Trowell法体外培养人牙胚,分别用10mg/L和25mg/L的氟化物刺激培养的牙胚,免疫组化观察结果。结果经图像分析显示,10mg/L组和25mg/L组从第4天开始,内釉上皮细胞中Smad1、5的表达均低于对照组。结论氟化物能抑制体外培养的人牙胚内釉上皮细胞中Smad1、5表达。提示氟可能通过抑制Smad1、5分子干扰上皮和间充质之间BMP正常的信号转导,进而使釉质的分化发育受到影响,可能是氟斑牙发生的细胞内机制之一。     

Culture and characterization of rat junctional epithelium

Junctional epithelium, which forms the interface between the gingival tissues and the teeth, is a unique nondifferentiating, nonkeratinizing simple epithelium with essentially no gradient of cytoplasmic alterations. The purpose of this study was to develop methods for obtaining and culturing junctional epithelial cells and characterizing these cells with monoclonal antibodies and by keratin analysis. Microsurgical methods were developed to obtain pure specimens of junctional epithelium from the interproximal molar regions of pathogen-free rats. Specimens of palatal and free gingival epithelia were also taken. The tissue explants were cultured by three methods; an explant technique using 3T3 fibroblasts as feeder cells, a procedure which used cloning rings to isolate primary epithelial outgrowths, and growth in small chambers coated with solubilized basement membrane. Specimens from the three oral epithelia grew well by all methods and appeared homogenous by phase contrast microscopy. Immunocytochemical analysis of fresh specimens by antikeratin antibody staining showed differences in the staining patterns of the three oral epithelia and suggested the absence of 54 and 40 kD keratins from these tissues. Junctional, palatal and gingival epithelial cells grown by the cloning ring technique stained positively with the monoclonal antibody 34βE12 which stains all stratified epithelia and identifies 66 and 57 kD keratins, but failed to stain with antibodies to vimentin and desmin which are intermediate filament proteins in mesenchymal and muscle cells, respectively, Junctional epithelial cells grown on solubilized basement membrane showed positive staining with antidesmoplakin antibodies I and II, which identify desmosomal plaque proteins, and negative staining with the keratin-specific monoclonal antibody AE2. Gingival cells grown by the same technique had the opposite staining pattern with these antibodies. Cells grown by the 3T3 feeder cell method were analyzed for their cytokeratin content by SDS polyacrylamide gel electrophoresis and immunoblotting using the keratin-specific monoclonal antibodies AEl and AE3. The keratin patterns in freshly explanted tissues all differed, indicating that these oral epithelia are indeed different in vivo. Further, the keratin patterns in extracts of subconfluent and confluent cultured oral epithelial cells differed from those in the freshly isolated tissues, but were similar for the different epithelia. Specifically, subconfluent cultures of the three epithelia had identical keratin patterns, but differed from those of confluent cultures which were themselves identical. This report presents methods for obtaining and culturing junctional epithelium, and data which indicate that junctional cells differ from other oral epithelia in cytokeratin and desmosomal protein composition.     

Patterns of expression of intermediate filaments in ameloblastoma and human fetal tooth germ

Monoclonal antibodies (Mab) were used to study the expression of cytokeratins and vimentin in various histological types of ameloblastoma and in human fetal tooth germ. The ameloblastoma and the tooth germ epithelia showed characteristics of both simple glandular and stratified squamous epithelial cells. Cytokeratin No. 18 was detected focally in most ameloblastomas studied but not in fetal odontogenic epithelia. Cytokeratins Nos. 8 and 19 were expressed in all epithelial elements of ameloblastomas and tooth germs. Only two tumors showed focally characteristics of keratinizing epithelia also seen in dental lamina but not in the enamel organ. All tumors except the granular cell ameloblastoma showed a variable coexpression of vimentin and cytokeratins in their neoplastic epithelia. A similar coexpression was detected in the stellate reticulum cells of the developing tooth. Ameloblastoma and human tooth germ epithelia share complex pattern of cytokeratin polypeptides together with coexpression of vimentin. The results strongly support the theory that ameloblastomas are of odontogenic origin and not direct derivatives of basal cells of oral epithelium or epidermis.     

Surface characteristics of cells from different layers of keratinized and non-keratinized oral epithelia

Portions of clinically healthy non-keratinized and keratinized oral epithelia were removed from adult vervet monkeys (Cercopithecus aethiops). The epithelium was separated from the underlying lamina propria by trypsin digestion, following which the epithelial cells were separated from the various epithelial layers. These were examined with the light and scanning electron microscope. Cells from non-keratinized epithelia have surface microplications while those from keratinized epithelia show microvilli and pits. Two distinct types of cell are therefore present. It is suggested that the different surface appearances are related to different types of mechanical adhesion between the cells.     

Interstitial and Langerhans' dendritic cells in chronic periodontitis and gingivitis

The aim of the present study was to compare quantitatively the distribution of dendritic cell subpopulations in chronic periodontitis and gingivitis. Fourteen biopsies from patients with chronic periodontitis and fifteen from patients with gingivitis were studied. An immunoperoxidase technique was used to quantify the number of Langerhans' cells (CD1a) and interstitial dendritic cells (factor XIIIa) in the oral and sulcular and junctional/pocket epithelia and in the lamina propria. A greater number of factor XIIIa+ dendritic cells in the lamina propria and CD1a+ dendritic cells in the oral epithelium were observed in gingivitis compared to the periodontitis group (p = 0.05). In the sulcular and junctional/pocket epithelia and in the lamina propria, the number of CD1a+ dendritic cells was similar in the gingivitis and periodontitis groups. In conclusion, the number of Langerhans' cells in the oral epithelium and interstitial dendritic cells in the lamina propria is increased in gingivitis compared to periodontitis, which may contribute to the different pattern of host response in these diseases.     

Lectin-binding in premalignant lesions during submandibular gland carcinogenesis

Complex carbohydrates in premalignant lesions of mouse submandibular gland tumors were examined by the lectin-peroxidase conjugate method. Peroxidase-conjugated lectins of PNA, RCA-1, DBA, SBA, UEA-1 and WGA were used to detect specific sugar residues of complex carbohydrates in premalignant lesions during experimental carcinogenesis. Marked reduction of PNA and SBA bindings occurred in duct-like structures and cystic lesions which were transformed from granular convoluted tubule cells. Premalignant lesions bound slightly to PNA, RCA-1, DBA, SBA and WGA and manifested increased UEA-1 binding. Squamous metaplastic epithelia of premalignant lesions manifested increased binding to PNA, RCA-1 and SBA as compared to those of duct-like structure and cystic epithelia.     

Lectin-binding in premalignant lesions during submandibular gland carcinogenesis

Complex carbohydrates in premalignant lesions of mouse submandibular gland tumors were examined by the lectin-peroxidase conjugate method. Peroxidase-conjugated lectins of PNA, RCA-1, DBA, SBA, UEA-1 and WGA were used to detect specific sugar residues of complex carbohydrates in premalignant lesions during experimental carcinogenesis. Marked reduction of PNA and SBA bindings occurred in duct-like structures and cystic lesions which were transformed from granular convoluted tubule cells. Premalignant lesions bound slightly to PNA, RCA-1, DBA, SBA and WGA and manifested increased UEA-1 binding. Squamous metaplastic epithelia of premalignant lesions manifested increased binding to PNA, RCA-1 and SBA as compared to those of duct-like structure and cystic epithelia.