[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.

[Experimental study on the structure and immune cell phenotypes of the lymphoid tissues in oral lichenoid lesions].

PURPOSE: To evaluate the existence of tertiary lymphoid structures(TLS) in oral lichenoid lesions and its compositional characteristics of immune cells. METHODS: Tissue samples of normal oral mucosa, oral lichen planus (OLP) and oral lichenoid tissue reaction(OLTR) were collected, thirty cases in each group. Hematoxylin-eosin(H-E) staining was performed to identify the TLS-like structures, and immunohistochemistry (IHC) staining was applied to assess the structure and amount of infiltrating CD3+ T cells, CD19+, CD20+ B cells, CD21+ follicular dendritic cells (FDC), Bcl-6+ germinal centers, CD34+ PNAd+ venules and CD34+ Gp36+ micro lymphatic vessels in TLS of OLL. Histopathology and molecular markers were used to evaluate the morphological performance of TLS in OLL. Chi-square test (Fisher exact probability method) was applied to compare the proportion of TLS in each group; integral optical density (IOD) method was used to calculate the expression level of each molecular marker, nonparametric t test (Mann-Whitney U test) was employed to analyze their difference. Statistical analysis was performed with GraphPad Prism 7.0 software. RESULTS: In OLP group and OLTR group, 46.7% (14/30) and 23.4% (7/30) cases had TLS-like structures, respectively. The frequency of TLS-like structures was not correlated with the type of disease(P>0.05). Compared with the control group, the molecular markers in OLP group and OLTR group were highly expressed, and the expression of CD19, CD20, and CD21 in OLP group had morphological and structural characteristics of TLS. The expression of Bcl-6(mean and standard deviation of IOD were 15 498±15 108 vs. 1 841±2 276, P<0.0001), CD20 (13 067±9 049 vs. 7 695±5 159, P<0.05), CD21 (13 968±14 560 vs. 2 552±2 584, P<0.0001), PNAd (10 328±10 383 vs. 1 756±1 570, P<0.0001) and Gp36 (12 778±12 390 vs. 2 313±2 578, P<0.0001) showed significant differences between OLP and OLTR tissues, but it could not be used as the criteria for identifying the type of diseases without morphological characters. CONCLUSIONS: TLS exists in OLL lesions, mainly presented as non-classical forms. The classical forms can be occasionally found. CD20 and CD21 can be used as the biomarkers to identify the TLS in OLL. TLS can not be used as the diagnosing criteria for identifying OLP or OLTR.