[Effects of Fusobacterium nucleus derived outer membrane vesicles on claudin-4 expression in oral epithelial cells].

PURPOSE: To investigate the effect of outer membrane vesicles (OMVs) secreted by Fusobacterium nucleatum (F.n) on Claudin-4 of human oral keratinocytes (HOK) and oral epithelial barrier function. METHODS: Fusobacterium nucleatum was cultured under anaerobic conditions. The OMVs were extracted by dialysis and characterized by nanosight and transmission electron microscopy (TEM). HOK were stimulated with OMVs at different mass concentrations(0-100 µg/mL) for 12 h, and stimulated with 100 µg/mL OMVs for 6 h and 12 h respectively. The expression of Claudin-4 at gene and protein level was analyzed by RT-qPCR and Western blotting. Inverted fluorescence microscope was used to observe co-localization of HOK and OMVs and localization and distribution of Claudin-4 protein. Human oral epithelial barrier was constructed by Transwell apical chamber. Transepithelial electrical resistance(TER) of barrier was measured with a transmembrane resistance measuring instrument(EVOM2), and the permeability of the barrier was evaluated by transmittance of fluorescein isothiocyanate-dextran(FD-4). Statistical analysis was performed with GraphPad Prism 8.0 software package. RESULTS: Compared with the control group, the expression of Claudin-4 at protein and gene level in the HOK of OMVs stimulated group was significantly reduced (P＜0.05), and immunofluorescence showed that the continuity of Claudin-4 fluorescence among cells was destroyed. OMVs stimulation decreased TER value of oral epithelial barrier(P＜0.05) and increased the transmittance of FD-4(P＜0.05). CONCLUSIONS: OMVs derived from Fusobacterium nucleatum may damage oral mucosal epithelial barrier function through inhibiting the expression of Claudin-4.

[Correlation between low expression of Hsp90 protein in keratinocytes and the number of small extracellular vesicles and its potential clinical significance].

PURPOSE: To investigate the correlation between the level of heat shock protein 90(Hsp90) and the amount of small extracellular vesicles(sEVs) in keratinocytes. METHODS: Human keratinocytes(HaCaT) were cultured in vivo and divided into wild-type group, short hairpin RNA interference group (shRNA group, low expression of Hsp90), and 17-Allylamino-17-demethoxygeldanamycin group (17-AAG group, Hsp90 protein inhibitor). sEVs were isolated from culture system by ultracentrifugation, and their morphological characteristics were observed under transmission electron microscopy (TEM). Western blotting was applied to identify the biological characteristics of sEVs. The number of sEVs particles was detected by nanoparticle tracking analysis (NTA). GraphPad Prism8.0 software was used to analyze the difference in the number of sEVs among the groups by t test (non-parametric Mann-Whitney U test). RESULTS: HaCaT-derived sEVs, obtained by ultracentrifugation, were consistent with the criteria of morphological and biological identification. No expression of Hsp90 protein was detected in HaCaT-derived sEVs. When interfered with Hsp90-shRNA, the number of sEVs were significantly increased. On day 5, the sEVs number of shRNA-interfering group was (177.4±4.18)×108(n=3), while that of vector group was (82.34±4.83)×108(n=3), and the difference was statistically significant (P＜0.0001). After 5 days of inhibition with 17-AAG, the sEVs number of 17-AAG group was （652.5±26.73）×108(n=3) and that of control group was (262.22±5.44)×108(n=3), the difference was statistically significant (P＜0.000 1). CONCLUSIONS: Low expression of Hsp90 protein can promote the secretion of sEVs in HaCaT cells. sEVs may be involved in the transfer of molecules between epithelial cells and immune cells.

Management of oral leukoplakia by ablative fractional laser-assisted photodynamic therapy: A 3-year retrospective study of 48 patients.

OBJECTIVES: This study aimed to review the results of oral leucoplakia (OL) using ablative fractional laser-assisted photodynamic therapy (AFL-PDT) and to further evaluate the risk factors for recurrence and malignant transformation. MATERIALS AND METHODS: Forty-eight patients diagnosed with OL using histopathology were enrolled in this study. All patients received one session of AFL-PDT. Therapeutic efficacy was evaluated 1 month posttreatment. Follow-up was scheduled every 3 months in the first year and every 6 months thereafter. RESULTS: An overall positive response rate of 87.5% (42/48) was achieved, including 62.5% (30/48) complete responses and 25.0% (12/48) partial responses. During the 3-year follow-up period, the recurrence and malignant transformation rates were 37.5% (18/48) and 8.3% (4/48), respectively. Lesions on gingiva/palate seemed to be associated with recurrence (p < 0.001; odds ratio [OR]: 1.64, 95% confidence interval [CI]: 1.13-2.37). The severity of epithelial dysplasia (p = 0.02; OR: 2.93, 95% CI: 1.96-4.42) and recurrence (p = 0.016; OR: 3.14, 95% CI: 2.04-4.84) were associated with a predisposition to malignant transformation. CONCLUSIONS: AFL-PDT is an effective management of OL, but requires close follow-up. OL lesions on the gingiva/palate are predisposed to recurrence. OLs that recur with moderate/severe epithelial dysplasia have a higher risk of transforming into oral squamous cell carcinoma.

Protein disulfide isomerase family A member 3 expression is upregulated in tissue-derived extracellular vesicles in oral lichen planus and oral lichenoid lesions.

OBJECTIVES: The aim of this study was to identify the tissue-derived extracellular vesicles immunogen involved in the pathogenesis of oral lichen planus (OLP) and its variation oral lichenoid lesions (OLL). DESIGN: Six pooled tissue-derived extracellular vesicles from participants with OLP/OLL and three from healthy controls were isolated and enriched. The extracellular vesicles were characterized with transmission electronic microscopy, nanoparticle tracking analysis and western blotting. Proteins from extracellular vesicles were identified with proteomics analysis and differentially expressed proteins (DEPs) were further identified with a 2-fold (p < 0.05) increase or a 0.5-fold decrease. RESULTS: A total of 1805 peptides and 141 proteins were identified. Ten DEPs were further identified, with five upregulated proteins of fibrinogen alpha chain, lamin isoform A, 40S ribosomal protein, protein disulfide isomerase family A member 3 (PDIA3) and elongation factor 1-alpha 1, and five downregulated proteins of plakophilin-1, katanin p80 repeat-containing subunit B1, collagen alpha-3 chain, mitochondrial 2-oxoglutarate/malate carrier protein and guanine nucleotide-binding protein subunit gamma-12. PDIA3 was found to be immune-associated and to be involved in the antigen processing and presentation pathway. The upregulation of PDIA3 was confirmed in a verification cohort composed of three pairs of OLP/OLL-extracellular vesicles and healthy controls-extracellular vesicles with western blotting. CONCLUSIONS: Protein disulfide isomerase family A member 3 in extracellular vesicles may play a significant role in the local immune responses and the pathogenesis of oral lichen planus and oral lichenoid lesions.

A retrospective clinicopathological study on oral lichen planus and malignant transformation: analysis of 518 cases.

OBJECTIVE: To investigate the epidemiological and clinical characteristics of a relatively large cohort of patients with oral lichen planus (OLP) from eastern China. STUDY DESIGN: A total of 518 patients with histologically confirmed OLP in a long-term follow-up period (6 months-21.5 years) were retrospectively reviewed in our clinic. RESULTS: Of the 518 patients, 353 females and 165 males were identified. The average age at diagnosis was 46.3 years (range 9-81 years) with the buccal mucosa being the most common site (87.8%). At initial presentation, white lichen and red lichen was seen in 52.3% and 47.7% patients, respectively. Of these, 5 (0.96%) patients previously diagnosed clinically and histopathologically as OLP developed oral cancer. All of them were the females with no a history of smoking or alcohol use. CONCLUSIONS: Clinical features of eastern Chinese OLP patients were elucidated. Notably, approximately 1% of OLP developed into cancer, which provides further evidence of potentially malignant nature of OLP.

Malignant transformation of oral epithelial dysplasia: clinicopathological risk factors and outcome analysis in a retrospective cohort of 138 cases.

AIMS: To explore the usefulness of a new binary system of grading dysplasia proposed by the World Health Organization and to identify significant risk factors for malignant transformation in a long-term follow-up cohort of patients with oral epithelial dysplasia. METHODS AND RESULTS: A total of 138 patients with histologically confirmed oral dysplasia between 1978 and 2008 were reviewed retrospectively in our department. The mean follow-up period was 5.1 years. Of these dysplasias, 37 (26.8%) developed into cancer, with a mean duration of 4.6 years. Cox regression analysis revealed that high-grade dysplasia was an independent risk factor for transition, but age, gender, lesion site, diet habit, smoking and alcohol intake were not risk factors. High-grade dysplasia was associated with a 2.78-fold (95% confidence interval 1.44-5.38; P = 0.002) increased risk of transition, as compared with low-grade dysplasia. Consistently, high-grade dysplasia had a significantly higher incidence of malignancy than low-grade dysplasia by Kaplan-Meier analysis (log-rank test, P = 0.001). CONCLUSIONS: The utilization of high-grade dysplasia as a significant indicator for evaluating malignant transformation risk in patients with potentially malignant lesions is suggested; this may be helpful to guide treatment selection in clinical practice.

[A retrospective analysis on the malignant transformation rate, time and risk factors of oral leukoplakia].

PURPOSE: A retrospective analysis was made on the oral leukoplakia (OLK) patients archived from 1978 to 2009 at the Department of Oral Medicine, Ninth People's Hospital, College of Stomatology, Shanghai Jiao Tong University, to analyze the malignant transformation of oral leukoplakia and its influential factors. METHODS: The data was studied with the SAS6.12 software package, using the methods of survival analysis and Cox regression to acquire malignant transformation rate (MTR) and malignant transformation time (MTT). RESULTS: According to the archived files, there were 576 cases of OLK with 350 males and 226 females. Among them, 66 cases (33 males and 33 females) developed OSCC, the total MTR was 11.46%. Two hundred and sixty-seven cases were suitable for survival analysis and Cox regression. The 1-year MTR was (0.40 ± 0.40)%, (0.86 ± 0.61)% (life-table method/product-limit method); the 2-year MTR went up to (2.84 ± 1.15)%, (2.93 ± 1.18)% and the 5-year MTR was as high as (11.28 ± 2.70)%, (11.31 ± 2.71)%. In Cox regression model, location of lesion and age were the main factors that affected OLK's transformation to OSCC. CONCLUSIONS: The MTR of OLK is related to the follow-up time; The lesion site and age are the most important risk factors. Supported by Research Fund of Science and Technology Commission of Shanghai Municipality (08DZ2271100), Shanghai Leading Academic Discipline Project (S30260) and Research Fund of Shanghai Municipal Education Commission (JDY-07061).

[Study on the expression and potential effect of PD-L1mRNA and PD-L2mRNA in oral lichen planus].

PURPOSE: To study the expression of PD-L1 and PD-L2mRNA in oral lichen planus(OLP) patients' peripheral blood and focal mucosa, and the different expression of target molecules in gene level in different clinical and pathological OLP groups. METHODS: The expression of PD-L1 and PD-L2 mRNA in OLP patients, which included 35 reticular and 25 atrophic-erosive OLP patients, 38 cases without dysplasia and 22 cases with dysplasia, was examined by real-time PCR. Peripheral blood and mucosa from 10 volunteers were used as control. All the results were analysed with Wilcoxon test by SAS.6.12 software package. RESULTS: The expression of PD-L2mRAN, but not PD-L1, was significantly higher in oral mucosa of OLP patients (P<0.01), while decreased in OLP patients' peripheral blood (P=0.0415). The gene expression of PD-L2 differed between different clinical types, and had highly significant correlation between the OLP patients' focal mucosa and peripheral blood(r=0.6976, P<0.0001). CONCLUSIONS: PD-L2 may have some potential effect on the pathogenesis of OLP at systemic and local level.

Malignant transformation of oral leukoplakia: a retrospective cohort study of 218 Chinese patients.

BACKGROUND: Oral leukoplakia (OL) is the best-known potentially malignant disorder. A new binary system to grade dysplasia was proposed by WHO, but the biological significance in predicting malignant transformation risk is unknown. The objective of this study is to estimate the rate of malignant transformation in a long-term follow-up cohort, explore the usefulness of the new binary system of grading dysplasia and identify significant risk factors of OL malignant transformation in China. METHODS: A total of 218 patients with clinical and histopathologic diagnosis of OL were retrospectively reviewed. They were selected among all archived files at the Department of Oral Mucosal Diseases, Ninth People's Hospital, Shanghai Jiao Tong University School of Medicine. The mean follow-up period was 5.3 years. RESULTS: Among 218 cases, 39 (17.9%) OL patients developed oral cancer, with a mean duration of 5.2 years. Cox regression analysis revealed that dysplasia was an independent risk factor for OL malignant transformation, but age, gender, lesion site, diet habit, smoking and ethanol intake were not risk factors. High-risk dysplastic OL was associated with a 4.57-fold (95% confidence interval, 2.36-8.84; P < 0.001) increased risk of malignant transformation, compared with low-risk dysplasia. Consistent with this result, high-risk dysplastic OL had significantly higher malignant incidence than low-risk dysplasia, particularly during the first 2-3 years of follow-up, by Kaplan-Meier analysis (Log-rank test, P < 0.001). CONCLUSIONS: The new binary system's function in predicting OL malignant transformation risk was investigated in this survey. The utilization of high-risk dysplasia as a significant indicator for evaluating malignant transformation risk in patients with OL was suggested, which may be helpful to guide treatment selection in clinical practice.