8-Nitroguanine formation in oral leukoplakia, a premalignant lesion.

Oral leukoplakia is a premalignant lesion associated with development of oral cancer. To clarify the mechanism of development of oral carcinogenesis from leukoplakia, we examined DNA damage in oral epithelium of biopsy specimens of patients with leukoplakia by immunohistochemical methods. Histological changes, such as epithelial dysplasia and infiltration of inflammatory cells were observed in oral tissues of leukoplakia patients. A double immunofluorescence labeling study demonstrated that the accumulation of mutagenic 8-nitroguanine, an indicator of nitrative DNA damage, and 8-oxo-7,8-dihydro-2'-deoxyguanosine, an indicator of oxidative DNA damage, was apparently observed in the oral epithelium of patients with leukoplakia, whereas little or no immunoreactivity was observed in normal oral mucosa. Expression of inducible nitric oxide synthase (iNOS) was also observed in oral epithelium of leukoplakia patients. Immunoreactivity of 3-nitrotyrosine, an indicator of nitrative stress, was observed in oral epithelial cells and colocalized with 8-nitroguanine. Moreover, proliferating cell nuclear antigen and p53 were expressed in 8-nitroguanine-positive epithelial cells in the basal layer. These results suggest that iNOS-mediated nitrative stress contributes to development of oral carcinogenesis from leukoplakia through DNA damage as well as oxidative stress.     

Prognostic significance of cyclin D1, p27 and p63 expression in oral leukoplakia

Studies on the expression of genes regulating cell proliferation and apoptosis is of importance in relation to understanding the severity of the process and the possibility of malignant transformation. In the present study immunohistochemical demonstration of cyclin D1, p27 and p63 has been added to our previous investigations on Ki-67, p53 and apoptosis index. Clinical and pathological immunohistochemical studies on oral leukoplakias of 18 patients were performed. Clinically homogenous, non-homogenous or nodular, and erythroleukoplakia were distinguished. Pathologically the grading was made according to the degree of dysplasia. Immunoperoxidase reactions for cyclin D1, p27 and p63 were carried out, and the positivity was expressed in per cent, considering 1000 epithelial cells. Expression of cyclin D1 increased in parallel with the severity of leukoplakia. p27 expression was 14-16% in homogenous and nodular leukoplakias, whereas in erythroleukoplakia it decreased to 1-2%. p63 expression was 10% in average in homogenous, and 5% in nodular leukoplakias. While in erythroleukoplakias it increased to 20 per cent. The characteristic cyclin D1, p27 and p63 phenotype in various forms of leukoplakia may be considered as prognostic factors.     

NUMB在口腔白斑和鳞癌中的表达变化及意义

目的:研究NUMB在口腔白斑和鳞癌中的表达及意义。方法:采用免疫组织化学法检测88例口腔正常黏膜、口腔白斑及口腔鳞癌石蜡包埋组织中NUMB蛋白的表达。结果:NUMB在口腔正常黏膜(95.7%)、口腔白斑(75%)及口腔鳞癌(4.7%)中均有阳性表达但是表达频率依次降低。NUMB在各个组中的阳性表达率具有统计学差异(P0.05)。结论NUMB阳性表达率随口腔组织恶性程度增高而减少的趋势提示该基因可能在口腔正常黏膜到口腔白斑再到口腔鳞癌的转化中起作用,NUMB有可能成为早期发现癌变的分子生物学标志之一。     

Improvement in the risk assessment of oral leukoplakia through morphology-related copy number analysis

Oral leukoplakia is the most common type of oral potentially malignant disorders and considered a precursor lesion to oral squamous cell carcinoma. However, a predictor of oral leukoplakia prognosis has not yet been identified. We investigated whether copy number alteration patterns may effectively predict the prognostic outcomes of oral leukoplakia using routinely processed paraffin sections. Comparison of copy number alteration patterns between oral leukoplakia with hyperplasia(HOL,n=22) and dysplasia(DOL, n=21) showed that oral leukoplakia with dysplasia had a higher copy number alteration rate(86%)than oral leukoplakia with hyperplasia(46%). Oral leukoplakia with dysplasia exhibited a wider range of genomic variations across all chromosomes compared with oral leukoplakia with hyperplasia. We also examined a retrospective cohort of 477 patients with oral leukoplakia with hyperplasia with detailed follow-up information. The malignant transformation(MT, n=19)and leukoplakia recurrence(LR, n=253) groups had higher frequencies of aneuploidy events and copy number loss rate than the free of disease(FD, n=205) group. Together, our results revealed the association between the degree of copy number alterations and the histological grade of oral leukoplakia and demonstrated that copy number alteration may be effective for prognosis prediction in oral leukoplakia patients with hyperplasia.     

口腔粘膜白斑癌变的研究进展

口腔粘膜白斑是常见的口腔粘膜病,属于癌前病变。由于一些类型的口腔粘膜白斑可发展为癌变,因此对口腔粘膜白斑癌变的研究受到了越来越多的关注。本文从流行病学、癌变机制和治疗方面对近年来口腔粘膜白斑癌变的研究进展予以综述。     

口腔白斑组织癌变标志性基因筛查

口腔黏膜白斑病（oral leukoplakia,OLK）是最常见的口腔粘膜的癌前病变．OLK恶性转化率高达43%,因此加强其风险调查和监测对于该病的预防和治疗具有重要意义．本研究采用美国SuperArray公司的肿瘤基因芯片（OHS-802）检测口腔正常组织、OLK组织和口腔鳞状细胞癌（oral squamous cell carcinoma, OSCC)组织的肿瘤相关基因表达差异,旨在筛查与口腔黏膜白斑病发生和癌变相关的表达阳性基因,为探究OLK的发生及癌变机制奠定基础．将“正常组织→OLK组织→OSCC组织”连续递增2倍以上或递减2倍以上的基因确定为OLK组织癌变的标志性基因．SuperArray基因芯片、RT-PCR法和实时定量PCR结果显示,仅有3个基因（ACP-2、BCL-2、SOCS-3）表达水平连续下调2倍以上,有6个基因（CLK-3、CTNNB-1、FKBP-8、GDF-15、NF-1、XRCC-1）表达连续上调2倍以上．进一步经RT-PCR和实时定量PCR验证,其结果与SuperArray结果一致．本研究结果提示,ACP-2等9个基因可能是口腔正常黏膜组织经OLK转变为癌组织的驱动基因,可作为OLK组织、OSCC组织和口腔正常黏膜组织诊断的分子标志．     

MACC1和C-Met在口腔白斑和口腔鳞状细胞癌中的表达及意义

目的:探讨分析MACC1和C-Met在正常口腔黏膜、口腔白斑及口腔鳞状细胞癌中的表达及其临床意义。方法:采用免疫组化SP法检测20例口腔黏膜、20例上皮异常增生白斑、50例口腔鳞癌组织中的MACC1、C-Met蛋白的表达情况,采用X2和spearman等级相关分析对结果进行判定。结果:MACC1、C-Met蛋白在异常增生型白斑和口腔鳞癌中的阳性表达率分别为50%、76%,35%、66%,均明显高于正常口腔黏膜(17.6%,5.0%),差异均有统计学意义(P0.05)。MACC1和C-Met蛋白表达与口腔鳞癌的分期、淋巴结转移及分化程度密切相关(P0.05)。Spearman等级相关分析显示口腔白斑及口腔鳞癌中MACC1和C-Met的表达呈现正相关(P0.05)。结论:MACC1和C-Met在上皮不典型增生性白斑和口腔鳞癌中高表达,二者在口腔黏膜白斑的癌变和口腔鳞癌的发生发展中可能起重要作用。     

Candida biotypes in patients with oral leukoplakia and lichen planus

Prevalence of yeasts in 35 leukoplakia and 34 oral lichen planus patients was compared with that observed in persons without oral diseases. Serotype and morphotype were determined on Candida albicans isolates. Yeasts were isolated from the oral cavity specimens of 43.7% of the patients. C. albicans (serotype A) was the predominant species (76% in leukoplakia, 88.2% in lichen planus and 60.8% in healthy persons). Sixteen morphotypes were encountered on malt extract agar, being 732, 733, 734, 753 and 754 the most frequently found. Morphotypes SP1N and SP1Y were the most common on Sabouraud-trypheniltetrazolium agar (68.4% of the isolates from leukoplakia and 73.3% from lichen planus, but only 46.6% of the isolates from healthy oral mucosa showed SP1N morphotype). Presence of oral lesions was associated with a marked reduction in the yeast species and C. albicans biotypes, suggesting that C. albicans and particularly some of its biotypes, show a high potential of adaptation to the changes associated with the development of oral leukoplakia and lichen planus.     

Micronuclei and other nuclear anomalies in normal human buccal mucosa cells of oral cancer patients undergoing radiotherapy: a field effect

We evaluated micronuclei and other nuclear anomalies in exfoliated epithelial cells of the oral cavity on the side opposite the lesion targeted by radiotherapy and correlated them with radiation doses. Buccal smears were obtained from oral cancer patients undergoing radiotherapy with a cumulative dose of at least 1000 rad for 3 weeks and from controls matched for age, gender and habits. The exfoliated cells from the mucosa were collected using a cytobrush; smears were prepared, fixed in 80% methanol and stained using the Feulgen plus fast green method. The mean number of micronuclei and other nuclear anomalies/1000 cells was significantly greater in patients undergoing radiotherapy treatment, but the differences were not significant compared to radiation doses. It appears that radiotherapy has a potent clastogenic effect on buccal mucosal cells of oral cancer patients.     

Effect of EGCG on lipid absorption and plasma lipid levels in rats

Catechins, compounds derived from green tea, have been shown to reduce plasma cholesterol levels and the rate of cholesterol absorption. We investigated the dose response and the mechanism of action of epigallocatechin gallate (EGCG) on these parameters in rats. Wistar rats were fed a diet high in cholesterol and fat containing either none, 0.25% (0.2 g/day/kg BW), 0.5% (0.4 g/day/kg/BW) or 1.0% (0.7 g/day/kg BW) of EGCG. After 4 weeks of treatment, total cholesterol and low density lipoprotein plasma levels were significantly reduced in the group fed 1% EGCG when compared to the no treatment group. Plasma triglycerides and high-density lipoprotein levels did not change significantly. Following a single oral application of a liquid test-meal, intestinal cholesterol absorption in Wistar rats was 79.3% in the control group. In the group treated with 0.1 g/kg BW EGCG intestinal cholesterol absorption decreased to 73.7% and in the group treated with 0.5 g/kg BW of EGCG intestinal cholesterol absorption fell significantly to 62.7% (P = 0.005). Total fat absorption was very efficient in the control group (99.5% of the applied dose) and decreased significantly but moderately in the group treated with the highest doses of EGCG (0.75, 1 g/kg BW). In an in-vitro biliary micelle model, the addition of 55 microM to 1300 microM EGCG not only decreased cholesterol solubility dose-dependently in these micelles but also altered the size of the mixed lecithin/taurocholate/cholesterol micelles as demonstrated by light scattering. This study provides evidence suggesting that the cholesterol-lowering effect of green tea is mainly elicited by EGCG, one of the most abundant catechins contained in green tea. It is suggested that one of the underlying mechanisms by which EGCG affects lipid metabolism is by interfering with the micellar solubilization of cholesterol in the digestive tract, which then in turn decreased cholesterol absorption.     

The effect of environmental factors of the coking plant on the enzymatic activity of rabbit oral mucosa.

Histochemical methods were used to investigate the activities of some intracellular enzymes in the oral mucosa of the rabbits which had been kept in selected work-stands of a coking-plant for 3 months. The findings were compared with the results for the enzymatic activity of the oral mucosa of control rabbits. The epithelium of the oral mucosa of the experimental rabbits was found to be proliferated acanthotically; moreover, there occurred some other morphological changes of the mucosa which often resembled precancerous states of leukoplakia type. In comparison with the control group, the activities of the studied enzymes, i.e. reduced NAD dehydrogenase (NADD), glucose-6-phosphate dehydrogenase (G6PD), lactate dehydrogenase (LDS), succinate dehydrogenase (SD), cytochrome oxidase (CO), and phosphorylase a+b in the epithelium and the connective-tissue cells of the studied mucosa of the experimental animals were as a rule markedly lowered, this decline being especially pronounced for the three last-mentioned enzymes. It was only the proliferating stratum basale of the experimental rabbit epithelium that frequently exhibited enhanced activities of NADD and LDS. Besides, the activities of NADD, G6PD and LDS were of a markedly diverse intensity in the cells of the chaotically proliferating stratum spinosum of the experimental rabbit mucosa. The results point to the noxious modifying effect of chemical and physical agents of the investigated environment on the oral mucosa of the animals studied.     

Controlled trial of methylprednisolone pulses and low dose oral prednisone for the minimal change nephrotic syndrome.

In a multicentre, randomised, prospective trial 89 patients (67 children and 22 adults) with the minimal change nephrotic syndrome were treated with three intravenous pulses of methylprednisolone followed by low dose oral prednisone for six months (group given methylprednisolone) or with high dose oral prednisone for four weeks followed by low dose oral prednisone for five months (control group). Five patients in the group given methylprednisolone and one in the control group did not respond initially. The time to response was shorter in children treated with methylprednisolone. No significant differences between the two groups were observed in the number of patients who relapsed or number of relapses per patient per year. Patients given methylprednisolone tended to relapse earlier than patients in the control group. Side effects related to treatment were significantly fewer in the group given methylprednisolone than in the control group. These data suggest that a short course of methylprednisolone pulses followed by low dose oral prednisone is only marginally less effective than a regimen of high dose oral steroids but can improve the ratio of risk to benefit associated with treatment of the minimal change nephrotic syndrome.     

Human papillomavirus in oral leukoplakia is no prognostic indicator of malignant transformation

Background: Oral leukoplakia is considered as a premalignant lesion for the development of oral squamous cell carcinoma (OSCC); several risks factors have been reported to contribute to this step-wise carcinogenesis; including human papillomavirus (HPV). Nevertheless, few reports have analyzed both the HPV status and the genotype in a single individual who develops OSCC from pre-existing oral leukoplakia. In this study, we surveyed the HPV status, genotype and clinicopathological risk factors in cases of malignant transformation from pre-existing oral leukoplakia. Methods: HPV genomic DNA was detected by PCR (MY09/MY11 in conjugation with nested primer-GP05+/GP06+) from paraffin sections, and the genotype was determined by direct DNA sequencing. Fisher's exact test and logistic regression were used to analyze risk factors for malignant transformation of oral cavity leukoplakia. Results: One hundred and sixty-seven patients with oral leukoplakia were enrolled; including 12 who had malignant transformation from the pre-existing oral leukoplakia. HPV prevalence was 22.8% in cases with oral leukoplakia. The risk factor associated with malignant transformation was recurrence of leukoplakia after treatment (p = 0.03), nevertheless, HPV status was not statistically significant by logistic regression analysis. Among these 12 patients with malignant transformation from pre-existing oral leukoplakia, the status or genotype of HPV was chaotic; the oral habits of these patients might contribute to malignant transformation. Conclusions: Our data suggest that HPV in oral leukoplakia is no prognostic indicator of malignant transformation.     

A morphometric study of the protective effect of cryotherapy on oral mucositis in cancer patients treated with 5-fluorouracil

We investigated cytological changes in oral mucosa smears from patients treated with cryotherapy to determine whether cryotherapy prevented mucositis caused by 5-fluorouracil (5-FU) therapy. Patients with gastrointestinal malignancies were divided into four groups; control patients before 5-FU therapy, patients after 5-FU therapy without cryotherapy, patients with cryotherapy before 5-FU therapy and patients with cryotherapy after 5-FU therapy. Oral mucosa samples from all patients were assessed at the beginning and on day 14 of chemotherapy. We used exfoliative cytology to evaluate cellular changes in the oral mucosa that were caused by 5-FU. Smears from each patient were stained using the Papanicolaou method and analyzed using stereology. Smears were taken from each group before and after 5-FU infusion. We found that nuclear volume was decreased significantly in cells of the 5-FU therapy after cryotherapy patients compared to the 5-FU therapy before cryotherapy patients. We also found significantly decreased cytoplasmic volumes in the 5-FU therapy after cryotherapy patients compared to the 5-FU therapy before cryotherapy patients. The results of cytomorphometric estimations revealed that cryotherapy may be used to prevent damage to oral tissue and may decrease the frequency and duration of oral mucositis caused by 5-FU.     

Computer aided morphometric analysis of oral leukoplakia and oral squamous cell carcinoma

We compared the changes in the cells in the basal layer of normal mucosa, oral leukoplakia with dysplasia and different grades of oral squamous cell carcinoma (OSCC) using computer aided image analysis of tissue sections. We investigated three morphometric parameters: nuclear area (NA), cell area (CA) and their ratio (NA:CA). NA and NA:CA ratio showed a statistically significant increase from dysplasia to increasing grades of OSCC. Nuclear size was useful for differentiating normal tissue, potentially malignant leukoplakia and OSCC.     

p53 Expression in the carcinogenesis in the oral mucosa

Hyperplastic lesions of the oral mucosa such as leukoplakia and oral lichan planus can eventually develop into squamous cell carcinomas. In the clinical treatment of these lesions it would be very important tobe able to predict the biological behaviour of an individual lesion. In 64 hyperplastic lesions and 85 squamous cell carcinomas of the oral mucosa, the expression of the mutant tumor suppressor gene p⁵³and the grade of dysplasia of the lesions.     

A histochemical comparison of methyl green-pyronin,and hematoxylin and eosin for detecting apoptotic cells in oral squamous cell carcinoma,oral leukoplakia,oral submucous fibrosis and normal oral mucosa

Abstract

Analysis of apoptotic cells in oral pathological states could be useful for determining the rates of tissue turnover, which would help determine prognosis. The use of histochemical stains such as hematoxylin and eosin (H & E) and methyl green-pyronin (MGP) can provide a simple and cost-effective method for detecting apoptotic cells. We compared the efficacy of MGP and H & E for detecting apoptotic cells in oral squamous cell carcinoma (OSCC), oral leukoplakia (OL), oral submucous fibrosis (OSMF) and normal oral mucosa (NOM). Ten cases each of OSCC, OSMF, OL and NOM were retrieved from the archives and two serial sections were stained, one with H & E and the other with MGP. Apoptotic cells were identified at 100 x magnification and the apoptotic index was calculated. Apoptotic cells were distinguished more readily in MGP stained sections than in those stained with H & E. Also, the apoptotic cell count was greater in OSCC compared to OL, OSMF and NOM. We concluded that MGP staining can be used as a routine, cost-effective method for detecting apoptotic cells.     

Immunohistochemical analysis of substance P containing nerve fibres and their contacts with mast cells in the diabetic rat's tongue

Sensory neuropathy is common symptom of the diabetes mellitus and the prevalence of oral lesions is higher in diabetic patients. The distribution of substance P was studied immunohistochemically in streptozotocin induced diabetic rat's tongue. The morphological association of sensory nerves (substance P immunoreactive) with mast cells (nerve fibre-mast cell contact) was monitored. The substance P nerve fibre mast cell contacts were very scanty in control tongue. The number of substance P nerve terminals and mast cells was significantly increased (p < 0.05) in diabetes mellitus after 4 weeks of the treatment compared with the control tongue. The number of mast cell nerve contacts was even more significantly increased (p < 0.001) in diabetes. The distance between nerve fibres and mast cells was about 1 mm and very often less than 200 nm. In some instances, the mast cells were degranulated in the vicinity to nerve fibres. Increased number of mast cell nerve contacts in neurogenic inflammation might cause vasoconstriction and lesions of the oral mucosa, so some disorders such lichen planus, leukoplakia and cancer might frequently develop in diabetes mellitus.     

成人口腔黏膜念珠菌病的多因素分析

为探讨影响成人口腔黏膜念珠菌病发生的因素,选取口腔黏膜念珠菌病、复发性阿弗他溃疡、扁平苔藓、舍格伦综合征、白斑、天疱疮、类天疱疮患者及口腔黏膜健康者为研究对象,详细记录731个样本的年龄、性别、吸烟、口腔卫生情况、抗生素使用、义齿、口腔黏膜疾病、全身疾病,对结果进行多元Logistic回归分析,得到影响口腔黏膜念珠菌病发生的因素有4类,分别为发病前使用抗生素、口腔卫生情况、口腔黏膜疾病、全身疾病,即多种因素的作用导致口腔黏膜念珠菌病的发生。