Architectural organization of human oral epithelium as visualized by keratin staining pattern in tobacco-associated leukoplakias

The keratin staining pattern in clinical normal buccal mucosa in smokers and non-smokers and in tobacco-associated leukoplakias histologically characterized by the chevron type of keratinization were studied. No differences in keratin staining pattern were found in normal buccal mucosa in smokers and non-smokers. In the tobacco-associated leukoplakias we found distinct differences in staining pattern between areas overlying connective tissue papillae and epithelial ridge areas indicating a lateral organization of oral epithelium related to the ridge system. It seems possible to explain this pattern in terms of current hypotheses of the proliferative conditions in squamous epithelia as previously studied in detail in the epidermis and tongue filiform papillae. We conclude, that our results confirm the presence of subpopulations of suprabasal and basal cells and support the presence of proliferative units in oral epithelium. Furthermore, smoking does not seem to influence the keratin staining pattern in clinically normal buccal mucosa.     

Cytokeratin pattern in normal and HPV infected oral mucosa in women with genital HPV infections

The distribution of cytokeratins Nos. 19 (CK 19), 14, 16 and 17 (CK2-27), and 8 and 18 (CK 60-61) in 96 oral mucosal biopsies taken from women with genital HPV infections were studied by immunohistochemistry, using polyclonal antibody CK 19, as well as monoclonal antibodies CK 2-27 and CK 60-61. White staining of the buccal mucosa after acetic acid application, which recently was shown to be affected mostly by smoking and age, could not be explained by differences in cytokeratin pattern. In HPV DNA-positive biopsies, the staining with CK 19 antibody in the basal cell layer was more intense than in HPV DNA-negative biopsies. The staining with CK 2-27 antibody was seen in 76% and 91% of the basal and superficial layers, respectively, even though these low molecular weight cytokeratins should be found mainly from the basal and parabasal cells. CK 60-61 staining was almost similar to that seen recently in normal genital mucosa. When trying to distinguish oral HPV infections from normal mucosa, CK 2-27 and CK 60-61 stainings were of no diagnostic value. The more efficient expression of CK 19 in HPV DNA-positive samples suggests that viral infection might accelerate the production of low molecular weight cytoskeletal protein. This could be interpreted as evidence that HPV might disturb the keratinocyte differentiation in the basal cells. As a result of the present study, CK 19 staining in oral mucosa needs to be further studied in regard to viral infections, because it may help to better understand the interaction between a virus and a host cell.     

单层上皮细胞角蛋白在口腔粘膜癌变过程中的表达

目的：探讨单层上皮细胞角蛋白ＣＫ１８和ＣＫ１９作为口腔癌前病变标志的可能性。方法：用ＬＳＡＢ免疫组化染色方法检测ＣＫ１８和ＣＫ１９在福尔马林固定,石蜡包埋的口腔正常粘膜,上皮单纯增生,轻度上皮导演增生,中度上皮异常增生,重度上皮异常增生和口腔鳞癌组织中的分布和表达强度,光镜观察染色切片,结果用秩和检验分析。     

Identification of the AgNORs, PCNA and ck16 proteins in oral lichen planus lesions

The expression of proliferating cell nuclear antigen (PCNA), cytokeratin 16 (ck16) and Ag nucleolar organizer regions (AgNORs) were assessed in 20 cases of lichen planus, 20 cases of keratosis and 20 cases of normal oral mucosa in order to evaluate the rate of keratinocyte proliferation in these tissues. Three hundred cells were counted in each sample: 100 basal cells, 100 suprabasal cells and 100 squamous cells. The mean number of AgNORs and the percentage of PCNA positive cells were calculated. Except from similar staining of suprabasal cells of lichen planus and keratosis, PCNA and AgNORs values were higher in all layers of lichen planus than in both keratosis and normal oral mucosa. The three groups showed similar ck16 immunostaining: all of the cells were positive, except those of the basal layer. The results suggest that the keratinocyte proliferation index is higher in lichen planus than in keratosis and normal mucosa. Besides, ck16 should not be used to differentiate the entities studied.     

细胞角蛋白CK19在口腔粘膜癌变过程中的变化

目的：探讨口腔粘膜癌变过程中CK19的变化。方法：收集正常口腔粘膜、上皮单纯增生、上皮异常增生和口腔鳞癌活检标本共53例，用免疫组化、电泳和Western杂交方法研究上皮中CK19的表达。结果：CK19表达于有上皮异常增生的复层鳞状上层和口腔鳞癌尤其是低分化鳞癌的癌细胞中。随病变程度加重，CK19表达的阳性率，表达强度和占细胞角蛋白构成比显著增加。结论：CK19表达于粘膜上皮的基底上层可作为口腔癌前病变的辅助标志，CK19表达增加是 口腔粘膜病变过程中的早期事件。     

颊癌及转移淋巴结角蛋白谱的分析研究

目的探讨口腔颊黏膜鳞状细胞癌及转移淋巴结角蛋白谱的特点。方法颊部鳞状细胞癌活检标本及转移淋巴结活检标本,提取角蛋白,进行SDS-PAGE电泳和利用免疫印迹(Western Blot)杂交方法研究转移角蛋白谱的表达。结果颊黏膜化鳞状细胞癌中出现了单层角蛋白上皮如CK18、19,而CK10表达缺失;淋巴结转移鳞状细胞癌中出现了单层角蛋白上皮如19等,并且出现了大量角蛋白片段。结论颊黏膜鳞状细胞癌变异性表达CK18、19,不表达CK 10。转移淋巴结和原发鳞状细胞癌的角蛋白谱是不同的,在口腔鳞状细胞癌淋巴结转移过程中,角蛋白谱发生了变化。     

颊黏膜中分化鳞状细胞癌角蛋白谱的分析

目的：研究口腔颊黏膜鳞状细胞癌角蛋白谱的特点.方法：颊黏膜中分化鳞状细胞癌活检标本,提取角蛋白,进行SDS-PAGE电泳和利用免疫印迹（Western-blot）杂交方法,研究颊黏膜中分化鳞状细胞癌中角蛋白谱的表达.结果：颊黏膜中分化鳞状细胞癌中出现了单层角蛋白上皮如CK18、19,而CK10表达缺失.结论：变异性表达的CK18、19以及不表达CK10,是颊黏膜中分化鳞状细胞癌的一个较为普遍的现象.     

Expression of keratin 14 and 19 mRNA and protein in normal oral epithelia, hairy leukoplakia, tongue biting and white sponge nevus

This study was undertaken to analyze keratin gene expression at both the mRNA and protein level in oral hairy leukoplakia (OHL). Comparisons were made with normal lingual epithelium from a similar site, tongue biting, normal buccal mucosa and another condition which disturbs oral epithelial differentiation, white sponge nevus. Combined immnunocytochemical and in situ hybridization studies for keratins 14 and 19 were carried out on 2 specimens of OHL from HIV-positive males and one sample each of the other cases. Keratin 14 protein expression was uniform throughout all the epithelia. In normal epithelia and in lesions other than OHL, keratin 14 mRNA was most strongly expressed in basal cells with weaker but still significant amounts in the spinous cell layer. In both cases of OHL there was weaker basal cell expression of keratin 14 mRNA and frequent absence in koilocytoid cells. Keratin 19 protein expression was heterogeneous in the basal layer ol all specimens with suprabasal staining of occasional groups of cells. Its mRNA was uniformly distributed in all cases. The findings indicate that keratin mRNA expression does not always parallel that of protein and that, in the case of keratin 14, expression may be influenced by the presence of EBV.     

Expression of keratins 8, 18, and 19 in epithelia of atrophic oral lichen planus

Keratins form intermediate filaments of the cytoskeleton in keratinocytes and have roles in cell structure, signaling, intracellular transport, and cell death. Oral lichen planus (OLP) is an oral inflammatory disease with derangements in basal keratinocytes and disruption of the basal membrane. Here, we focused on epithelial expression of keratins 8, 18, and 19 because these proteins are known to modulate cell death. Biopsies were taken from buccal oral mucosa of persons with normal oral mucosa (n = 10) or atrophic OLP (n = 10). Cultured normal oral keratinocytes (n = 4) showed expression of mRNA and protein for keratins 8, 18, and 19. Immunohistochemistry showed consistent staining for keratins 8 and 18 in basal keratinocytes of normal oral mucosa. In OLP, staining for keratin (K)8 was mostly negative and staining for K18 was weak. Keratin 19 was expressed irregularly in most biopsies of normal oral mucosa and not at all in OLP. Several mononuclear leukocytes in the cellular infiltrate showed membrane staining for K8 and K18. Positive staining for K16 confirmed partial collapse of the basal cell layer in OLP. The basal cell niche in OLP therefore appeared to be partly populated with keratinocytes demonstrating a higher degree of differentiation (K8⁻ K18⁻ K19⁻ K16⁺); consequently, such areas may be more susceptible to the action of cell death factors released from the cell infiltrate as a result of lacking the protective, normal keratin present in the basal epithelial cell layer of normal oral mucosa.     

口腔扁平苔藓中细胞角蛋白19的表达及意义

目的:探讨细胞角蛋白19(keratin 19,CK19)在口腔扁平苔藓(OLP)中的表达以及其病理意义.方法:采用免疫组化法检测17例OLP和14例正常颊黏膜中CK19的表达,采用RT-PCR方法分别在OLP患者和正常人颊黏膜中比较CK19 mRNA的表达.结果:正常颊黏膜中CK19均在基底细胞胞浆内呈强阳性表达,只有5例(29.4%)OLP基底细胞呈弱阳性表达,其余为阴性表达;RT-PCR结果显示在OLP组织中CK19 mRNA表达显著高于正常黏膜(P＜0.05).结论:CK19的表达下调与颊部OLP黏膜上皮更新能力下降密切相关.     

Effect of snuff on cytokeratin expression in oral vestibular sulcus epithelium

Differences in the expression of cytokeratins (CK) in specimens obtained from snuff-affected oral epithelium of the maxillary vestibular sulcus and clinically normal sulcular epithelium were studied by indirect immunofluorescence staining with a panel of monoclonal antibodies (MAbs), CK 14, a marker of stratified squamous epithelium, was not seen expressed in 3/11 of the snuff user's specimens. Terminal differentiation markers, typical of ramified epithelia (CK 1, 9, 10 and 11). were detected suprabasally in the snuff user's keratosis but not in the normal control epithelium. The use of snuff seemed to change the CK staining pattern of the mucosa so that it resembles more that of a cornfield type of epithelium. Simple epithelial-type CK were included in the study in order to establish the CK profile of the snuff-induced keratosis, for comparison with normal and dysplastic lesions. MAb to CK 7 and 19 showed reactivity in the basal cells and suprabasally, whereas the monospecific MAb anti-CK 7 showed suprabasal staining both in the control and affected epithelia. By using MAbs, we found no immunoreactivity against CK 18 either in normal or affected epithelia, whereas we found suprabasal reaction (5/11) against CK 8 in the snuff user's epithelia. The two MAbs demonstrating the expression of CK 19, normally confined to the basal cells of the stratified squamous epithelium, showed variable patterns of expression both in basal cells and suprabasally in the snuff lesions. The results show that use of oral snuff causes some alterations in the CK expression pattern of the affected epithelium. Whether the alterations are indicative of a premalignant change is, however, uncertain. The results encourage further studies on the subject.     

Expression of cytokeratins in odontogenic jaw cysts: monoclonal antibodies reveal distinct variation between different cyst types

Immunostaining with monoclonal antibodies was used to study and compare the cytokeratin content of odontogenic cysts and normal gingival epithelium. Two monoclonal antibodies, PKK2 and KA1, stained the whole epithelium in all cyst samples. In gingiva, PKK2 gave a suprabasal staining and KA1 reacted with all epithelial cell layers. Antibodies PKK1, KM 4.62 and KS 8.12 gave a heterogeneous staining in follicular and radicular cysts. In keratocysts and in gingiva PKK1 and KM 4.62 reacted mainly with basal cells and KS 8.12 gave a suprabasal staining. Antibodies reacting with the simple epithelial cytokeratin polypeptide No. 18 (PKK3, KS 18.18) recognized in gingiva only solitary cells compatible with Merkel cells. In a case of follicular ameloblastoma a distinct staining of tumor epithelium was revealed with these antibodies. In 2 follicular cysts, but not in other cyst types, a layer of cytokeratin 18-positive cells was revealed. KA5 and KK 8.60 antibodies, reacting exclusively with keratinizing epithelia, including normal gingiva, gave no reaction in radicular cysts, keratocysts and ameloblastoma. Two of the follicular cysts, were negative for PKK3 and KS 18.18, but reacted strongly with KA5 and KK 8.60. The present results show that odontogenic jaw cysts have distinct differences in their cytokeratin content. With the exception of some follicular cysts, they lack signs of keratinizing epithelial differentiation. Only follicular cysts appear to share with some types of ameloblastoma the expression of cytokeratin polypeptide No. 18.     

Expression of cytokeratins in odontogenic jaw cysts: monoclonal antibodies reveal distinct variation between different cyst types

Immunostaining with monoclonal antibodies was used to study and compare the cytokeratin content of odontogenic cysts and normal gingival epithelium. Two monoclonal antibodies, PKK2 and KA1, stained the whole epithelium in all cyst samples. In gingivu, PKK2 gave a suprabasal staining and KA1 reacted with all epithelial cell layers. Antibodies PKK1, KM 4.62 and Ks 8.12 gave a heterogeneous staining in follicular and radicular cysts. In keratocysts and in gingiva PKK1 and K_M 4.62 reacted mainly with basal cells and K_s 8.12 gave a suprabasal staining. Antibodies reacting with the simple epithelial cytokeratin polypeptide No. 18 (PKK3, K_s 18.18) recognized in gingiva only solitary cells compatible with Merkel cells. In a case of follicular ameloblastoma a distinct staining of tumor epithelium was revealed with these antibodies. In 2 follicular cysts, but not in other cyst types, a layer of cytokeratin 18-positive cells was revealed. KA5 and K_k 8.60 antibodies, reacting exclusively with keratinizing epithelia, including normal gingiva, gave no reaction in radicular cysts, keratocysts and ameloblastoma. Two of the follicular cysts, were negative for PKK3 and K_s 18.18, but reacted strongly with KA5 and K_k 8.60. The present results show that odontogenic jaw cysts have distinct differences in their cytokeratin content. With the exception of some follicular cysts, they lack signs of keratinizing epithelial differentiation. Only follicular cysts appear to share with some types of ameloblastoma the expression of cytokeratin polypeptide No. 18.     

Cytokeratin profile of the junctional epithelium in partially erupted teeth

This study uses cytokeratins (CK) as markers to investigate the phenotype of the junctional epithelium (JE) in partially erupted human teeth. The gingival samples, which were clinically healthy, were carefully dissected from the teeth. Cryostat sections were cut for histological staining, immunofluorescence microscopy and gel electrophoresis. Cytokeratins were extracted after microdis-section. The basal and suprabasal epithelial cell markers, cytokeratins 4, 5, 13, 14 and 19 were detected with specific monoclonal antibodies. They showed that the junctional epithelium in erupting teeth has a complex topography. The cytokeratin immunohistochemical profile distinguished between the primary junctional epithelium (CK 5, 14 and 19 in basal and suprabasal cells and CK 13 faintly stained throughout the suprabasal layers) and the adjacent epithelium that had the same cytokeratin profile as the sulcular epithelium (CK 5, 14 and 19 in basal cells and CK 4 and 13 intensively stained in the suprabasal cells). Extraction, two-dimensional electrophoresis and western blotting showed that this transitional JE during eruption also contained CK 6, 16 and perhaps CK 4. Thus, the JE in erupting teeth shows patterns of CK distribution that are very similar to that of developing oral epithelia.     

Integrin expression in oral hairy leukoplakia and normal tongue epithelium

OBJECTIVE: To describe the expression of integrins in the epithelium of oral hairy leukoplakia (HL) and compare to that of normal lateral tongue epithelium. MATERIALS AND METHODS: Immunohistochemistry to identify integrins (alpha 2, alpha 3, alpha 5, alpha 6, alpha v, beta 1) was performed, using a standard biotin-streptavidin-peroxidase technique on five clinically and histologically confirmed frozen biopsy specimens of HL and five normal lateral tongue control tissues. RESULTS: Expression of integrins alpha 2, alpha 3, alpha 6, alpha v, beta 1 was seen both in HL epithelium and in normal control tissue. alpha 5 expression was not seen in HL or in control tissue epithelium. alpha 2 and alpha 3 were expressed mainly in the basal and suprabasal layers; alpha 6 expression was most intense on the basal surface of the basal cells, alpha v was expressed in the basal and suprabasal layers with more expression seen in the higher differentiated cell layers than the other integrins. beta 1 expression was seen in the basal and suprabasal layers only. No apparent difference between HL and normal oral mucosa was noted in the staining pattern of the various integrins. CONCLUSION: Integrins alpha 2, alpha 3, alpha 6, alpha v, beta 1 are expressed in HL and the expression pattern is not different from that of normal oral mucosa. alpha 5 is not expressed in HL or in normal oral epithelium.     

Expression of p63 (TA and deltaN isoforms) in human primary well differentiated buccal carcinomas

Abnormalities in the p53 gene have been regarded as the most consistent genetic abnormalities detected in head and neck squamous cell carcinogenesis. Two new members of the p53 gene family, p73 and p63, have recently been identified. We investigated the expression of the two N-terminal p63 isoforms (TA and deltaN isoforms) in human primary well-differentiated buccal squamous cell carcinoma. Both TAp63 and deltaNp63 isoforms were detected in the basal/suprabasal layers of all of the five specimens of normal buccal mucosa. The deltaNp63 isoform was found in all of the 23 specimens of human primary well-differentiated buccal carcinoma whereas TAp63 isoform was absent in 18 (78.3%) of the 23 specimens. The immunostaining patterns of both TAp63 and deltaNp63 isoforms were similar in that the p63 positivity was noted mainly in the peripheral cells of tumor nests whereas negative staining was observed in the areas with keratin pearl formation. A higher number of T3-T4 patients and patients with recurrence showed negative staining of TAp63 than T1-T2 patients and patients without recurrence but the difference was not statistically significant. These results suggested that specific p63 isoforms were associated with human oral squamous cell carcinogenesis. The deltaNp63 isoforms might be involved in epithelial differentiation and proliferation in human oral carcinogenesis whereas there was evidence for a possible role of TAp63 under-expression in human oral tumorigenesis.     

Cytomorphometric analysis of the buccal mucosa of tobacco users.

OBJECTIVE: This study has been carried out to assess the effect of tobacco smoking and of betel quid chewing with tobacco on buccal mucosa by cytomorphometry, in a south Indian population. STUDY DESIGN: Cellular diameter (CD) and nuclear diameter (ND) of exfoliated buccal squames obtained from clinically normal appearing buccal mucosa of tobacco smokers, betel quid with tobacco chewers, and those with a combined habit, stained by the Papanicolaou method, were measured. Non-users served as negative controls and oral squamous cell carcinomas in tobacco users served as positive controls. One way ANOVA test of the values obtained followed by multiple range comparison with Tukey-HSD procedure (at p=0.05) was carried out. RESULTS: A statistically significant reduction in CD and increase in ND in smokers and those with a combined habit were observed. CONCLUSION: The use of tobacco in the form of smoking influences the cytomorphology of buccal mucosa.     

Oral lichen planus has a high rate of TP53 mutations. A study of oral mucosa in icelanD

Oral squamous cell carcinoma (OSCC) is a world-wide health problem. In addition to external exposure (smoking and alcohol), certain oral lesions may increase the risk of oral cancer (e.g. leukoplakia, erythroplakia, and oral lichen planus). TP53 has been implicated in OSCC, but there are limited studies of mutations in premalignant oral lesions. In this study, 55 samples from OSCC, 47 from hyperkeratotic (HK) oral mucosa, clinically diagnosed as white patches, 48 samples from oral lichen planus (OLP), and 12 biopsies from normal oral mucosa were studied immunohistochemically for expression of TP53 protein. From all the carcinoma samples and selected non-malignant samples showing moderate or strong TP53 protein expression, malignant cells or TP53-positive nuclei were microdissected and screened for mutations in exons 5-8 by constant denaturation gel electrophoresis. Moderate to strong TP53 protein staining was seen in 56% of OSCC, 32% of OLP but only in 13% of HK. All OLP samples showed a characteristic pattern of positive nuclei confined to the basal layer, whereas TP53 staining was seen in suprabasal nuclei in HK. Mutation rate was 11 out of 52 for OSCC, three out of 20 tested for HK and, remarkably, nine out 27 tested for OLP. There was no correlation between TP53 protein staining and TP53 mutations. No associations were found with anatomical sites or disease progression. The unexpectedly high mutation rate of OLP might explain the premalignant potential of this lesion.