Immunohistochemical localization of growth factors fibroblast growth factor-1 and fibroblast growth factor-2 and receptors fibroblast growth factor receptor-2 and fibroblast growth factor receptor-3 in normal oral epithelium,epithelial dysplasias,and squamous cell carcinoma

OBJECTIVES: Fibroblast growth factors (FGFs) and their receptors (FGFRs) have been identified in a variety of carcinomas, but there are few studies concerning their presence in oral cancers. The objective of this study was to determine whether FGF-1, FGF-2, and high affinity receptors FGFR2 and FGFR3 are present in the pathogenesis of oral epithelial dysplasias and oral squamous cell carcinoma. STUDY DESIGN: Sections from formalin-fixed, paraffin-embedded samples of oral normal mucosa (n = 14), epithelial dysplasia (n = 20), carcinoma in situ (n = 10), and squamous cell carcinoma (n = 12) were tested for cytoplasmic staining by standard in situ immunohistochemistry with antibodies for FGF-1, FGF-2, FGFR2, and FGFR3. RESULTS: Staining for FGF-1 is decreased or lost in the development of epithelial dysplasia and carcinoma. Staining for FGF-2 showed increased intensity (although not statistically significant) in oral epithelial dysplasias and squamous cell carcinomas and showed a significant increased expression in the upper layers of dysplasias and stratum spinosum-like cells in squamous cell carcinomas. Staining for FGFR2 showed a statistically significant increase in intensity in all layers of epithelial dysplasias and squamous cell carcinomas. Staining for FGFR3 was found in the upper stratum spinosum cells of normal and dysplastic epithelium and well-differentiated squamous cells in squamous cell carcinomas, with a statistically significant increase in staining intensity in dysplastic and carcinomatous tissues. CONCLUSIONS: The loss of FGF-1 is consistent with loss of differentiation in dysplasias and some squamous cell carcinomas. Changes in the localization of FGF-2 and FGFR2 into upper epithelial layers with increasing dysplasia suggest increased mitotic potential of high level cells. The co-localization of FGF-2 and its high affinity receptors in neoplastic tissues suggests an autocrine mechanism of influence on carcinogenesis.     

Immunohistochemical study during healing of free palatal mucosa grafts on plastic-embedded samples

This immunohistochemical study describes changes in the histology and in the distribution of the basement membrane components laminin and collagen IV as well as of the cytokeratins (CK)1/2/10/11, CK5/6, CK13, CK14, CK17, CK19 during the take of free split mucosa (epithelial and connective tissue) transplants in humans up to 36 months post-operative. Histology showed a flattening of the epithelial layer within the first 2 weeks after grafting, followed initially by an increase (25-30 layers, week 6) and later on by a decrease of cell layers in the epithelium (15-20 layers, week 20). From that time onwards, clear stratification and reteridges as signs of differentiation were present. Up to day 14 of graft take, the linear staining patterns for laminin and collagen IV were interrupted, which was not observed at any later stage. During this early interval CK5/6, CK1/2/10/11, CK14 and CK17 were expressed in all epithelial layers. The reactions for CK5/6 and CK1/2/10/11 were less intensive. At 6 weeks, CK1/2/10/11 stained the intermediate and superficial layers, being consistent with findings after longer graft take. CK5/6 reacted in the basal and intermediate cell layers, and CK13, CK14 and CK17 reacted in all layers. In the following period up to week 20, CK5/6 were found in the parabasal cells of the intermediate cell layers and the basal cells. CK14 staining was confined to these cell layers too, but also showed some reaction in the superficial layers. CK13 and CK17 were still bound to all layers. At 7 months post-operative, CK5/6 and CK14 were seen in their typical localisation in the basal cell layer and the parabasal cells of the intermediate layers, CK17 was seen mainly in the intermediate layer and CK13 was seen in focal areas of all layers. Anti-CK19 reacted only with single basal and parabasal cells up to week 20. These results suggest that during healing of mucosal autografts there is a sequence of changes in the expression of cell biological differentiation markers that may involve an epithelio-connective tissue interaction before the typical patterns for the donor side were observed again on the gingiva or mucosa of the hard palate.     

Differential expression of Toll-like receptors in chronic hyperplastic candidosis

Introduction:  The first step in the host defense against oral candidosis is the recognition of Candida albicans through a set of germ-encoded pathogen recognition receptors, e.g. Toll-like receptors (TLRs). In man, 10 types of such receptors have been identified so far, of which only TLR2, TLR4, and TLR6 have been linked to mediating candidal ligands, e.g. zymosan.
Methods:  Biopsies from patients with chronic hyperplastic candidosis ( n  = 5), leukoplakia ( n  = 5), and healthy mucosa ( n  = 5) were immunohistochemically stained with antibodies to the TLRs (TLR1 to TLR9) to distinguish and compare the staining patterns of the epithelial layer in the three categories of tissues.
Results:  On analysis, the epithelium of all tissues was divided into three layers: basal, middle, and superficial. Two of the five chronic hyperplastic candidosis sections showed high numbers of hyphae compared to yeasts, which paralleled a decrease in the expression of TLR2 and an increase in the staining intensity of TLR4. Leukoplakia and healthy tissue sections demonstrated stronger immunostaining of TLRs, except TLR9 which showed weaker staining in some sections of the former, and in the basal layers of some sections of the latter.
Discussion:  This study supports the concept of negative regulation of TLRs that are either ligand-bound (e.g. in chronic hyperplastic candidosis), or not stimulated (in healthy tissue). It also augments the opinion that C. albicans , through its hyphae rather than blastospore, may utilize TLRs, i.e. TLR2, to evade the immune system of the host. Leukoplakia seems to be more immunologically alert, which reduces the chances of worsening the already-diseased tissue.     

Oral mucosal epithelial cells express the membrane anchored mucin MUC1

ObjectiveThe presence of a stable salivary pellicle (SP) is essential to provide a wet surface for the oral mucosal epithelia. The oral mucosa is covered by the SP which is suggested to be a mixed film of both salivary and epithelial components. Our aim was to analyse the presence of membrane-anchored mucin MUC1 in the oral mucosal epithelia.DesingThe presence of MUC1 was studied by immunohistochemical and immunoelectron microscopical methods in 19 buccal mucosal specimens. The localization and intensity of the epithelial expression were analyzed.ResultsStrong staining of MUC1 was found in the epithelial cells of intermediate and superficial layers. Some basal cells were shown faint expression. In the intermediate and superficial layers, the MUC1 expression was seen mainly on the upper cell surface. Furthermore, the expression of MUC1 was noted in the cytoplasm near the nucleus and in the rough granules. By electron microscopy, extracellular domain of membrane-anchored molecules extruded about 15–30 nm above the cell surface in the apical cells of the oral epithelium. Immunoelectron microscopic examination shows that MUC1 is mainly localized in the plasma membrane of epithelial cells and also in small vesicles (75–100 nm) just below the plasma membrane.ConclusionThe membrane-anchored MUC1 is expressed in the superficial layer of the oral mucosal epithelium, especially on the upper surface of epithelial cells. MUCI may be the anchoring protein of the salivary pellicle stabilization.     

Immunohistochemical localization of Toll‐like receptors 1–10 in periodontitis

Background/aim: In periodontitis, bacteria and pathogen‐associated molecular patterns are sensed by Toll‐like receptors (TLRs), which initiate intracellular signaling cascades that may lead to host inflammation. In this study, the expression and distribution of TLRs (TLR‐1 to TLR‐10) were immunohistochemically detected in gingival epithelium and connective tissue. Methods: Immunohistochemistry was used for the localization of TLRs in gingival tissue samples from 10 patients with chronic periodontitis and 10 healthy controls; these samples were obtained during routine periodontal flap operations and during extraction operations performed for retained wisdom teeth, respectively. For the evaluation, epithelial layers were stratified to basal, spinous, and superficial layers, and the percentages of TLR‐positive cells were determined. Results: Both healthy and periodontitis gingival tissues expressed all TLRs except TLR‐10. In patients with periodontitis, epithelial cells showed increased TLR expression towards the basal layer. Healthy controls showed more variable cellular TLR expression and distribution between the layers of epithelium. In the connective tissue, consistently higher TLR expression was found within the periodontitis group compared to the healthy group. Conclusions: For the first time, the cellular expression and distribution of TLR‐1 to TLR‐10 have been studied in periodontitis, indicating that TLR‐1 to TLR‐9 are differentially expressed both in connective tissue and epithelial layers. Except for TLR‐7 and TLR‐8, all the other TLRs showed statistically significant differences between patients with periodontitis and healthy controls, suggesting their involvement in the pathogenesis of periodontitis.     

Immunohistochemical localization of Toll-like receptors 1-10 in periodontitis

Background/aim:  In periodontitis, bacteria and pathogen-associated molecular patterns are sensed by Toll-like receptors (TLRs), which initiate intracellular signaling cascades that may lead to host inflammation. In this study, the expression and distribution of TLRs (TLR-1 to TLR-10) were immunohistochemically detected in gingival epithelium and connective tissue.
Methods:  Immunohistochemistry was used for the localization of TLRs in gingival tissue samples from 10 patients with chronic periodontitis and 10 healthy controls; these samples were obtained during routine periodontal flap operations and during extraction operations performed for retained wisdom teeth, respectively. For the evaluation, epithelial layers were stratified to basal, spinous, and superficial layers, and the percentages of TLR-positive cells were determined.
Results:  Both healthy and periodontitis gingival tissues expressed all TLRs except TLR-10. In patients with periodontitis, epithelial cells showed increased TLR expression towards the basal layer. Healthy controls showed more variable cellular TLR expression and distribution between the layers of epithelium. In the connective tissue, consistently higher TLR expression was found within the periodontitis group compared to the healthy group.
Conclusions:  For the first time, the cellular expression and distribution of TLR-1 to TLR-10 have been studied in periodontitis, indicating that TLR-1 to TLR-9 are differentially expressed both in connective tissue and epithelial layers. Except for TLR-7 and TLR-8, all the other TLRs showed statistically significant differences between patients with periodontitis and healthy controls, suggesting their involvement in the pathogenesis of periodontitis.     

NGF and its receptors TrkA and p75NTR in the epithelium of oral lichen

Background:  Nerve growth factor (NGF) can through its receptors TrkA and p75^NTR convey signals for cell survival, differentiation and death. The aim of this study was to examine whether NGF can play a role in the pathology of oral lichen (OL).
Methods:  Sections from biopsies taken from patients with erythematous (ERY) OL and from volunteers with normal oral mucosa (NOM) were immunostained with antibodies against NGF, proNGF, TrkA, phosphorylated Trk, p75^NTR and phosphorylated Akt (pAkt) and expression of RNA coding for proNGF/NGF was investigated by in situ hybridization.
Results:  Both in ERY OL and NOM, cytoplasmic staining for NGF was seen in granular and upper spinous cell layers of the epithelium, whereas proNGF staining was seen in all epithelial cell layers. In situ hybridization showed that the proNGF protein was produced in the same cell layers. In OL, strong cytoplasmic stainings for TrkA and activated Trk (pTrk) were observed in all epithelial cell layers while these stainings were only weak in NOM. Basal keratinocytes in OL showed no or only weak cytoplasmic staining for p75^NTR, but in NOM there was a clear cell membrane staining. In OL, strong cytoplasmic and intermittent nuclear staining for pAkt was observed in spinous, granular and superficial layers, while basal and parabasal keratinocytes were negative. This staining was weak or absent in the entire epithelium of NOM.
Conclusions:  TrkA upregulation and activation in OL is one of the pathways that can activate pAkt and thereby rescue epithelial cells from untimely cell death.     

Differential expression of Toll‐like receptors in chronic hyperplastic candidosis

Introduction: The first step in the host defense against oral candidosis is the recognition of Candida albicans through a set of germ‐encoded pathogen recognition receptors, e.g. Toll‐like receptors (TLRs). In man, 10 types of such receptors have been identified so far, of which only TLR2, TLR4, and TLR6 have been linked to mediating candidal ligands, e.g. zymosan. Methods: Biopsies from patients with chronic hyperplastic candidosis (n = 5), leukoplakia (n = 5), and healthy mucosa (n = 5) were immunohistochemically stained with antibodies to the TLRs (TLR1 to TLR9) to distinguish and compare the staining patterns of the epithelial layer in the three categories of tissues. Results: On analysis, the epithelium of all tissues was divided into three layers: basal, middle, and superficial. Two of the five chronic hyperplastic candidosis sections showed high numbers of hyphae compared to yeasts, which paralleled a decrease in the expression of TLR2 and an increase in the staining intensity of TLR4. Leukoplakia and healthy tissue sections demonstrated stronger immunostaining of TLRs, except TLR9 which showed weaker staining in some sections of the former, and in the basal layers of some sections of the latter. Discussion: This study supports the concept of negative regulation of TLRs that are either ligand‐bound (e.g. in chronic hyperplastic candidosis), or not stimulated (in healthy tissue). It also augments the opinion that C. albicans, through its hyphae rather than blastospore, may utilize TLRs, i.e. TLR2, to evade the immune system of the host. Leukoplakia seems to be more immunologically alert, which reduces the chances of worsening the already‐diseased tissue.     

Electron microscopic features of chronically inflamed human gingiva

Chronically inflamed human gingival was studied by electron microscopy, with particular emphasis on alterations in the tissues adjacent to the gingival sulcus. In spite of dilated intercellular spaces in the crevicular epithelium recognizable bacteria remained confined to the most superficial layers of the epithelium. Such spaces did, however, contain a variety of emigrating cell types, cellular debris, lysosomes and a granular precipitate. Lysosomes were also detected in the connective tissues and were released from disrupting neutrophils. Morphological variants of the plasma cell series formed the majority of the inflammatory cell population. The synthesis and extracellular release of proteins (immunoglobulins) was suggested in light of their morphological appearance.     

Expression of interleukin-8 and its receptor IL-8RA in chronic hyperplastic candidosis

INTRODUCTION: Neutrophils are the main opponents of Candida albicans in chronic hyperplastic candidosis. They migrate from the circulation to the epithelium where they form microabscesses. We therefore hypothesized that the neutrophil chemokine interleukin-8 (IL-8) might play a role in the neutrophil-Candida interaction. METHODS: Biopsies from patients with chronic hyperplastic candidosis (n = 10) were stained using the avidin-biotin-peroxidase complex protocol for IL-8 and IL-8 receptor A and were compared to healthy control mucosa (n = 3). A set of C. albicans agar sections was similarly analysed. RESULTS: In chronic hyperplastic candidosis lesions IL-8 was strongly expressed in both vascular endothelium and mucosal epithelium. Many resident and immigrant inflammatory cells, including intraepithelial neutrophils, were IL-8 receptor A positive. In addition, IL-8 (or an analogue) was found in the candidal mother cell in chronic hyperplastic candidosis and in agar, whereas the tips of the hyphae expressed IL-8 receptor A (or an analogue). CONCLUSION: IL-8 may play a role in the recruitment of neutrophils from the vascular compartment to the epithelial microabscesses. C. albicans may have developed an ability to sense IL-8. The IL-8 ligand-receptor interaction may help to direct the growth of the IL-8-receptor-containing tips of the hyphae away from the IL-8-producing candidal cell body (a centrifugal growth pattern to facilitate host tissue penetration). Later, this ability might help to keep the vulnerable hyphal tips away from areas with high concentrations of host IL-8 and candidacidal neutrophils. We suggest that this phenomenon, in contrast to chemotropism, is named chemophobia.     

Toombak-associated oral mucosal lesions in Sudanese show a low prevalence of epithelial dysplasia

Clinical ( n =281) and histopathological ( n =141) characteristics of toombak-associated oral mucosal lesions detected in an epidemiological study in northern Sudan in 1992/93 are described. The lesional site in the majority of toombak users was the anterior lower labial groove and the lower labial mucosa. 4 degrees (1–4) of clinical severity of lesions, similar to those used to characterise Swedish snuff-dipper's lesion, were applied. An association between the severity of mucosal lesions and a longer lifetime duration (>10 years) of toombak use was found, but the severity was not related to the daily frequency of the habit. Parakeratosis, pale surface staining of the epithelium and basal cell hyperplasia were commonly observed, but epithelial dysplasia was infrequent (10/141). The most significant observation was a PAS-positive amorphous deposit between the lamina propria and the submucosa, found in 25/141 biopsies. The clinical and histopathological features of toombak lesions are closely similar to Swedish moist snuff-dipper's lesions and this may reflect the high alkalinity of these products, resulting in an alkaline burn on the oral mucosa following chronic exposure. The low prevalence of epithelial dysplasia implies a low risk of malignant transformation. Nevertheless, the high concentrations of tobacco-specific nitrosamines present in toombak, and the high prevalence of oral cancer in Sudan, mandate biopsy and careful histopathological analysis of any such lesions detected in habitues.     

Intracellular localization of Porphyromonas gingivalis thiol proteinase in periodontal tissues of chronic periodontitis patients

OBJECTIVES: Porphyromonas gingivalis is a significant periodontal pathogen that has been shown in vitro to be able to invade gingival epithelial cells and grow intracellularly. The aim of the present study was to detect P. gingivalis in gingival tissues from chronic periodontitis (CP) patients. MATERIALS AND METHODS: Monoclonal antibodies specific to a cell membrane-bound thiol proteinase of P. gingivalis were used to detect the microbe in gingival tissues of CP patients (n = 13) by immunohistochemistry. The presence of P. gingivalis was also analysed by polymerase chain reaction (PCR). RESULTS: Immunohistochemical analysis of the periodontal tissues revealed positive staining for P. gingivalis thiol proteinase in 11 of the 13 patients. Positive staining was mainly located intracellularly in the perinuclear region of the cytoplasm in the periodontal epithelial cells and it could be detected throughout the whole depth of both pocket and oral epithelium. The sensitivity of immunohistochemistry was found to be comparable with that of PCR. CONCLUSIONS: Our results provide in vivo evidence of the ability of P. gingivalis to enter human gingival epithelial cells. Intracellular localization of P. gingivalis contributes to its evasion of the host immune surveillance and eventually increases its resistance to conventional treatments of periodontal diseases.     

Immunohistochemical localization of mast cells and mast cell-nerve interactions in oral lichen planus

OBJECTIVES: Mast cell mediators are likely to be involved in at least some aspects of the immunopathogenesis of oral lichen planus (OLP). The aim of this project was to map mast cell populations in OLP and identify possible sites of mast cell-nerve interactions.
MATERIALS AND METHODS: Monoclonal antibodies specific for tryptase and neurofilaments were used to identify mast cells and nerves respectively in an immunohistochemical study of OLP ( n = 25) and normal oral buccal mucosa (NOBM) ( n = 13) using a double-labelling protocol. Data analysis used paired t-test, multiway analysis of variance and Wilcoxon rank testS. RESULTS: Morphometric analyses showed the greatest mast cell density in the most superficial of the three depth layers examined in OLP, an increase of 130% compared with NOBM.Mast cells associated with neurofilaments ranged from 21.9% in OLP to 10.2% in NOBM.Mean epithelial thickness was significantly lower in OLP ( P < 0.001) but without a strong correlation with mast cell density.
CONCLUSIONS: Increased mast cell and mast cell-nerve interactions in OLP suggest both a controlling role over the lesional cell populations and a secondary role to the immune response once this becomes established.     

A possible CD1a Langerhans cell–mast cell interaction in chronic hyperplastic candidosis

AIMS: T lymphocyte-antigen-presenting cell (APC) interaction plays a central role in T lymphocyte activation and APC maturation. We therefore studied the CD1a-positive Langerhans cells with respect to receptor activator of nuclear factor kappa B ligand (RANKL)-positive cells in chronic hyperplastic candidosis (CHC). MATERIALS AND METHODS: Tissue sections of CHC were compared with leukoplakia and healthy oral mucosa using RANKL and CD1a monoclonal antibodies in an avidin-biotin peroxidase complex protocol. Two different antigen-retrieval protocols, pepsin preincubation and Tris-EDTA heat treatment, were used. RESULTS: CD1a-positive Langerhans cells were in healthy and leukoplakia epithelium found in the middle layer, but in CHC in all layers of the epithelium, at the basement membrane and as mononuclear round cells in the lamina propria. Use of pepsin digestion enabled studies of mast cells and their activation in the form of degranulation of RANKL. CONCLUSIONS: The numerical, morphological and topographical versatility of the CD1a-positive Langerhans cells in CHC can be clarified by dendritic cell (DC) recruitment into the epithelium. RANK-positive and RANKL-sensitive DCs have ample opportunity to interact with local T lymphocytes. Use of an optimized antigen-retrieval protocol enabled demonstration of an active engagement (degranulation) of mast cells, which represent a rapidly available source of soluble RANKL.     

Immunohistochemical evaluation of intermediate filament proteins in squamous papilloma and oral verrucous carcinoma

OBJECTIVE: Cytokeratins (CKs) are the intermediate filament proteins of the epithelium cells, which have become important markers of normal and abnormal cell differentiation. The goal of the present study was to investigate the expression pattern of CK 10, 13, 14 and 16 in oral verrucous carcinoma (OVC) and oral squamous papilloma (OSP). MATERIAL AND METHODS: Formalin-fixed paraffin-embedded sections from eight cases of each lesion were assessed. Immunohistochemistry was carried out using streptoavidin-biotin complex method. RESULTS: In OVC, CK 10 was expressed in suprabasal to superficial layers whereas in OSP mainly in superficial layer. CK 13 was detected in prickle and superficial cells in most cases of OVC and in suprabasal to superficial cells of OSP. All the cell layers of OVC reacted positively for CK 14 while basal and suprabasal layers of OSP were more pronounced for CK 14. Finally, CK 16 was observed in suprabasal to superficial layer in OVC and the majority cases in OSP showed only superficial reactive cells. CONCLUSIONS: CK 10, 13, 14 and 16 immunohistochemical profile emphasis the biological behavior of the studied lesions and confirm the use of these proteins as markers of differentiation.     

Differential invasion of Candida albicans isolates in an in vitro model of oral candidosis

The study assessed the ability of Candida albicans isolates to invade an in vitro oral tissue model. The extent and pattern of isolate invasion was then correlated with the infection origin of the isolate to identify characteristics that may be restricted to specific forms of oral infection, particularly chronic hyperplastic candidosis (CHC). Reconstituted human oral epithelium was infected with C. albicans isolated from normal oral mucosa (n = 4), CHC (n = 7), non-CHC oral candidoses (n = 4) and squamous cell carcinoma (SCC; n = 4). After infection for 24 h, histological analysis revealed yeast adhesion, hyphal extension, and invasion of the epithelium. Differential patterns of invasion were evident and, whilst consistent for a given isolate, did not relate to the infection origin of the isolate. Two principal patterns of invasion were evident and described as either a 'localised' or a 'uniform' distribution of invading hyphae. Several isolates also exhibited superficial infection with limited hyphal invasion. In conclusion, the use of the in vitro tissue model allowed the assessment of the invasive capabilities of isolates of C. albicans. However, the apparent differences in invasive characteristics did not appear to be related to the clinical origin of isolates.     

Expression of transforming growth factor-beta 1 (TGF-beta 1) in odontogenic cysts

AIM: To evaluate the positivity to transforming growth factor-beta 1 (TGF-beta 1) in different types of odontogenic cysts. METHODOLOGY: A total of 30 radicular cysts (RCs), 27 follicular cysts (FCs) and 28 odontogenic keratocysts (OKCs) were evaluated for immunohistochemical analysis of TGF-beta 1. TGF-beta 1 was evaluated in blood vessels, stromal cells (fibroblasts) and pluristratified squamous epithelium. TGF-beta 1 expression was determined by evaluating the number of positive elements. TGF-beta 1 expression was determined by evaluating 1000 cells in the pluristratified squamous epithelium (500 in the basal and parabasal layers, and 500 in the superficial layer) and 500 cells (the fibroblasts in the stroma) for each specimen, and counting the number of positive cells. The number of positive vessels was evaluated in 10 high power fields (HPF). The Chi-square test was used to evaluate differences between the two groups (RC + FC and OKC). A P-value <0.05 was considered to indicate statistical significance. RESULTS: A higher and statistically significant positivity was found in the basal-suprabasal epithelial layers (P=0.0011), superficial epithelium (P=0.053) and stromal cells (P=0.0002) of orthokeratotic and parakeratotic OKC as compared with RC and FC. CONCLUSIONS: These differences suggest that control of the cell cycle may be abnormal in orthokeratotic OKCs. These OKCs may have an intrinsic growth potential not present in other cyst types.     

细胞角蛋白18及其基因在牙源性角化囊肿衬里上皮中表达的意义

目的检测细胞角蛋白18（CK18）及其基因在牙源性角化囊肿（OKC）衬里上皮中的表达。方法选取32例OKC的衬里上皮组织,分别进行CK18、CK8和CK19单克隆抗体的免疫组织化学染色。对其中12例使用RT- PCR法检测CK18 mRNA,观察其在衬里上皮中的表达;同时使用CK18基因探针进行原位杂交,检测CK18 mRNA在衬里上皮细胞层的定位表达。结果在免疫组织化学染色中, 17例CK18蛋白在OKC衬里上皮的表层细胞层表达为弱阳性;27例CK18蛋白在棘细胞层上层染色为阳性;14例CK18蛋白在棘细胞层染色为阳性;所有标本基底细胞层染色呈阴性。RT- PCR法检测见4例CK18 mRNA表达为强阳性,8例表达为弱阳性。原位杂交法检测见8例CK18 mRNA在棘细胞层和棘细胞层上层呈阳性,4例在上皮基底细胞层和角化层呈阳性。CK8蛋白在所有32例OKC衬里上皮基底细胞层均有表达。CK19蛋白在23例OKC衬里上皮表层均有表达。结论CK18在OKC衬里上皮的表达由基底细胞层向棘细胞层迁移,CK18蛋白免疫组织化学染色阳性表达与CK18 mRNA原位杂交法阳性表达不同,提示CK18可能与衬里上皮的增殖活性有关,OKC衬里上皮中可能存在CK18蛋白和CK18 mRNA表达的调控因子。     

Expression of the cell cycle regulation proteins p53 and p21WAF1 in different types of non-dysplastic leukoplakias

Objectives

The aim of this study was to analyze the immunolabeling of two cell cycle protein regulators, p53 and p21^WAF1, in non-dysplastic leukoplakias with different epithelial alterations: acanthosis, hyperkeratosis and acanthosis combined with hyperkeratosis, and compare them with dysplastic leukoplakias.

Material and Methods

This was a prospective cohort study involving 36 patients with oral homogeneous leukoplakias. Excisional biopsies were performed and the patients remain under clinical follow-up. The leukoplakias were divided into four groups: 6 acanthosis, 9 hyperkeratosis, 10 acanthosis combined with hyperkeratosis, and 11 epithelial dysplasias. Paraffin-embebeded sections were immunostained for p53 and p21^WAF1. Five hundred cells from the basal layer and 500 from the parabasal layer were counted to determine the percentage of positive cells. A qualitative analysis was also carried out to determine the presence or absence of immunohistochemical staining in the intermediate and superficial layers. Groups were compared with ANOVA (p<0.05). Pearson''s correlation coefficient was used to test for associations between the two markers, p53 and p21^WAF1.

Results

No leukoplakia recurred and no malignant transformation was observed whitin a follow-up period of 3-6 years. The mean percentage of p53 staining in the basal and parabasal layers was similar in all groups. p21^WAF1 staining differed between layers was as follows: in the basal, only 3 to 4% of cells were stained, while in the parabasal, between 16 and 28% of the epithelial cells were stained in the four different studied groups with no statistically significant difference (p>0.05).

Conclusions

Our findings failed to differentiate the non-dysplastic lesions by means of p53 and p21^WAF1 immunostaining, notwithstanding similar profiles between non-dysplastic and dysplastic leukoplakias were observed.     

Reliability of toluidine blue application in the detection of oral epithelial dysplasia and in situ and invasive squamous cell carcinomas

OBJECTIVE: The objective of this study was to evaluate the reliability of in vivo staining with toluidine blue in the detection of oral epithelial dysplasia, in situ carcinoma, and invasive squamous cell carcinomas in potentially malignant epithelial lesions (PMELs) and superficial oral ulcerations suggesting malignancy. STUDY DESIGN: Fifty patients with PMELs and superficial oral ulcerations suggestive of malignancy were selected from those treated at the Oral Medicine Service, Faculty of Dentistry, Araraquara, Brazil. All lesions were submitted to staining with an aqueous solution of 1% toluidine blue, followed by biopsy and histologic analysis. The sensitivity, specificity, and positive and negative predictive values were calculated. RESULTS: Histologic diagnosis revealed that 14% of the lesions analyzed were in situ carcinoma and invasive squamous cell carcinomas, 12% were epithelial dysplasias, 13% were keratosis, 40% were lichen planus, and 8% were other benign lesions. The sensitivity of the staining was 77%, the specificity 67%, and the positive and negative predictive values 43.5% and 88.9%, respectively. CONCLUSIONS: Staining with toluidine blue was demonstrated to be highly reliable in the detection of in situ carcinoma and invasive squamous cell carcinoma, because false-negative results for the lesions did not occur. Toluidine blue staining is an adjunct to clinical judgment and not a substitute for either judgment or biopsy.