Elevated serum fibrinogen levels in Chinese patients with minor recurrent aphthous stomatitis: An observational study.

Fibrinogen is a protein that reflects systemic inflammation and regulates the immune response to disease. However, there is a scarcity of data on fibrinogen in recurrent aphthous stomatitis (RAS). We aimed to test the hypothesis that fibrinogen is involved in the aetiology of RAS. Between November 2016 and November 2018, we included 109 minor RAS patients and 29 age- and sex-matched controls in a single-center, observational study. Their clinical history and ulcer manifestations led to the diagnosis of minor RAS. The ulcer severity score (USS) was used to assess disease severity, and fibrinogen was also collected. We conducted three analyses: Analysis 1 (comparison of fibrinogen levels between patients and controls), Analysis 2 (comparison of fibrinogen levels between high and low USS patients) and Analysis 3 (comparison of fibrinogen levels between before and after anti-inflammatory treatment in patients). The fibrinogen levels in the 109 minor RAS patients were statistically higher than in the 29 controls (mean [SD], 2.6 [0.5] vs. 2.3 [0.3]; Student's t-test, p < 0.001). However, there were no significant differences in fibrinogen levels among the 43 patients with high USS and the 39 patients with low USS (mean [SD], 2.7 [0.5] vs. 2.6 [0.4]; Student's t-test, p = 0.278). Furthermore, fibrinogen levels were significantly higher before anti-inflammatory treatment in comparison to those after anti-inflammatory treatment in the 35 paired patients (mean [SD], 2.6 [0.4] vs. 2.5 [0.4]; Student's t-test, p = 0.026). Interestingly, fibrinogen levels were significantly higher in the 35 paired patients after anti-inflammatory treatment compared to the 29 control subjects (mean [SD], 2.5 [0.4] vs. 2.3 [0.3]; Student's t-test, p = 0.026]. Fibrinogen may play a role in the aetiology of RAS and may be a drug target for RAS treatment. Clinicians should be alert that high serum fibrinogen levels might be associated with the risk of RAS.

Cardiovascular diseases, risk factors, and ulcer relapse in older adults with aphthous stomatitis.

OBJECTIVES: To test the hypothesis that cardiovascular diseases and risk factors are associated with ulcer relapse in after-retirement patients with recurrent aphthous stomatitis. SUBJECTS AND METHODS: This retrospective cohort study analyzed the data of 40 minor recurrent aphthous stomatitis patients aged 55-75 years, admitted to Oral Medicine Clinic at one university hospital in China between 2016 and 2018. The diagnosis of minor recurrent aphthous stomatitis was made based on the history and manifestation of oral ulcers. The ulcer relapse was evaluated after a 5-week anti-inflammatory treatment, and the history of systemic diseases was collected. cardiovascular disease/metabolic risk referred to the presence of any cardiovascular diseases and metabolic cardiovascular disease risks. Associations among cardiovascular diseases, risk factors, and ulcer relapse were evaluated. RESULTS: The mean age of 40 patients with minor recurrent aphthous stomatitis was 62.4 years (SD 5.1), and 60% were women. The ulcer relapse rate was 37.5% (95% CI, 0.242-0.530). The proportion of cardiovascular disease/metabolic risk was higher in the relapse group than in the no-relapse group after 5-week anti-inflammatory treatment (Fisher's exact test, p = 0.041). CONCLUSIONS: According to this single-center experience, older patients with cardiovascular disease/metabolic risk may be more prone to oral ulcer recurrence. Nevertheless, larger prospective studies are needed to confirm our findings.

Dynamic salivary cytokine profile of recurrent aphthous stomatitis patients in thalidomide maintenance treatment.

OBJECTIVE: To dynamically compare the longitudinal (time axis) and transverse (between groups) differences of the salivary cytokines during thalidomide maintenance treatment of recurrent aphthous stomatitis. METHODS: A randomized, controlled, clinical trial was performed. After the initial prednisone treatment, thalidomide (50 mg/d vs. 25 mg/d) was used as a maintenance drug for 4 or 8 weeks. The salivary IL-4, 5, 6, 10, TNF-α, and IFN-γ were dynamically detected with a cytometric bead array. RESULTS: Overall, the level of six elevated salivary cytokines after prednisone treatment was significantly downregulated, remained low during thalidomide maintenance, and rebounded at recurrence. The effect of 50 mg/d thalidomide on the salivary cytokines was not superior to 25 mg/d medication. The relapse-free period following drug withdrawal was the longest in the subgroup using 25 mg/d thalidomide for 8 weeks. The order of magnitude of IL-6 was the most obvious, and at week 8, only the level of IL-6 in the group (25 mg/d thalidomide for 8 weeks) continued to decline compared with the other groups. CONCLUSION: Thalidomide maintenance treatment can effectively sustain low levels of salivary IL-4, 5, 6, 10, TNF-α, and IFN-γ of recurrent aphthous stomatitis patients. IL-6 displayed a good correlation with the disease and is expected to become an index for diagnosis and follow-up. CLINICAL RELEVANCE: Low-dose long-term thalidomide maintenance treatment was supported for recurrent aphthous stomatitis. TRIAL REGISTRATION: Trial registration number of ChiCTR-IPR-16009759 at http://www.chictr.org/index.aspx .

Preliminary analysis of the buccal mucosal fungal microbiome in oral lichen planus patients.

OBJECTIVE: To determine the structure and co-occurrence patterns of mucosal fungal community in oral lichen planus (OLP) patients. SUBJECTS AND METHODS: Mucosal swab samples from 20 OLP patients and 10 healthy controls (HCs) were collected and the mucosal mycobiomes were sequenced. The abundance, frequency, and diversity of fungi were analyzed, as well as the inter-genera interactions. The associations between fungal genera and OLP severity were further identified. RESULTS: At the genus level, the relative abundance of unclassified_Trichocomaceae was significantly decreased in the reticular and erosive OLP groups compared to HCs. Meanwhile, significantly lower levels of Pseudozyma were observed in the reticular OLP group compared to HCs. The negative:positive cohesiveness ratio was significantly lower in the OLP group than HCs, indicating a relatively unstable fungal ecological system in the OLP group. In the OLP group, the abundance of unclassified_Nectriaceae was significantly correlated with the reticulation/erythema/ulceration (REU) score. CONCLUSIONS: Compared to HCs, the decreased stability of fungal communities and the decreased abundances of two genera (unclassified_Trichocomaceae and Pseudozyma) on buccal mucosa were identified in OLP patients.

Preliminary analysis of mucosal and salivary bacterial communities in oral lichen planus.

OBJECTIVE: To characterize the bacterial community from different oral niches (buccal mucosa and saliva) in oral lichen planus (OLP) patients. SUBJECTS AND METHODS: This preliminary study analyzed site-specific (mucosa and saliva) microbial landscape of 20 OLP patients and 10 healthy controls. RESULTS: The microbial diversity was similar between OLP patients and healthy controls in both salivary and mucosal communities. However, the topological properties of co-occurrence networks of salivary and mucosal microbiome were different between healthy controls and OLP patients. SparCC analysis inferred three and five keystone taxa in the salivary and mucosal microbial networks of healthy controls, respectively. However, in the salivary and mucosal bacterial networks of OLP patients, only one hub OTU and three OTUs were identified as keystone taxa, respectively. In addition, analysis of community cohesion revealed that mucosal microbial community in OLP patients had lower stability than that in healthy controls. In final, correlation assay showed that the clinical severity of OLP was positively associated with the relative abundance of Rothia in saliva but negatively associated with that of Porphyromonas on mucosa. CONCLUSIONS: The salivary and mucosal bacterial communities of OLP patients differ in terms of composition, the genera associated with OLP severity, and co-occurrence patterns.

Effects of low-level laser therapy on burning pain and quality of life in patients with burning mouth syndrome: a systematic review and meta-analysis.

BACKGROUND: Burning mouth syndrome (BMS) is a complex chronic pain disorder that significantly impairs patients' quality of life. Low-level laser therapy (LLLT) uses infrared or near-infrared light to produce analgesic, anti-inflammatory, and biological stimulation effects. The aim of this systematic review is to evaluate the effect of LLLT on burning pain, quality of life, and negative emotions in patients with BMS. METHODS: The PubMed, Embase, Cumulative Index of Nursing and Allied Health Literature (CINAHL), Cochrane Library, Web of Science, and Scopus databases were searched up January 2023 to identify relevant articles. All randomized controlled trials that were published in English and examined the use of LLLT treatment for BMS were included. The methodological quality of the included trials was assessed using the Cochrane risk of bias tool for randomized controlled trials (RCTs). A meta-analysis was performed to evaluate burning pain, quality of life, and negative emotions. Sensitivity, subgroup, and funnel plot analyses were also carried out. RESULTS: Fourteen RCTs involving a total of 550 patients with BMS met the inclusion criteria. The results showed that LLLT (measured by the Visual Analog Scale; SMD: -0.87, 95% CI: -1.29 to -0.45, P < 0.001) was more effective for reducing burning pain than placebo LLLT or clonazepam. LLLT improved quality of life (evaluated by the Oral Health Impact Profile-14; SMD: 0.01, 95% CI: -0.58 to 0.60, P = 0.97) and negative emotions (evaluated by the Hospital Anxiety and Depression Scale; SMD: -0.12, 95% CI: -0.54 to 0.30, P = 0.59), but these effects were not statistically significant. CONCLUSIONS: The meta-analysis revealed that LLLT may be an effective therapy for improving burning pain in patients with BMS, and producing a positive influence on quality of life and negative emotions. A long-term course of intervention, a larger sample size, and a multidisciplinary intervention design are urgently needed in future research. TRIAL REGISTRATION: PROSPERO registration number: CRD42022308770.

A Randomized controlled clinical trial on dose optimization of thalidomide in maintenance treatment for recurrent aphthous stomatitis.

BACKGROUND: Recurrent aphthous stomatitis (RAS) is the most common oral mucosal disease, and ulcer-free periods are a major concern for patients. Thalidomide has been shown to be an effective systemic drug in the treatment of RAS, but the value of undertaking a trial to evaluate various maintenance doses was warranted. METHODS: We performed this randomized controlled clinical trial with a two-stage design. Firstly, all the 125 cases of RAS received prednisone at a starting dose of 15 mg/d for one week as an initial therapeutic drug. Secondly, the 100 cases of RAS in the experimental group received thalidomide (50 mg/d vs. 25 mg/d) as a maintenance drug to observe its efficacy and safety. RESULTS: During maintenance medication at the fourth and eighth weekend, the two doses (50 and 25 mg/d) of thalidomide were equivalent in reducing the incidence of ulcers, ulcer number, and ulcer pain, respectively (all p > 0.05). Notably, the ulcer-free period in the group using 25 mg/d thalidomide for eight weeks was longer (mean, >3 months) than those in the other groups (all p < 0.05). Importantly, the total adverse events in the group using 25 mg/d thalidomide were significantly less than those in the group using 50 mg/d (p < 0.001). Moreover, the effect of 50 mg/d thalidomide on the levels of various salivary cytokines was not superior to 25 mg/d medication (p > 0.05). CONCLUSION: This dose optimization study concluded that 25 mg/d thalidomide had a long-term effect on extending the recurrence interval of RAS with better safety.

Changes in Th1/Th2-related cytokine expression in the saliva of patients with recurrent aphthous stomatitis before and after prednisone treatment.

OBJECTIVES: To investigate the changes of T helper cell (Th)1/Th2-related cytokine expression in the saliva of minor recurrent aphthous stomatitis (RAS) patients before and after treatment with systemic prednisone. METHODS: A total of 101 patients with RAS and 15 participants with normal oral mucosa as controls were enrolled in this study. The levels of cytokine expression in the whole unstimulated saliva were examined using a multiplex bead-based cytometric bead array before and after prednisone treatment at a starting dose of 15 mg/day. RESULTS: The levels of salivary interleukin (IL)-4, IL-5, IL-6, IL-10, interferon (IFN)-γ, and tumor necrosis factor-α (TNF-α) in RAS patients were significantly higher than those of the normal controls (all P < 0.001). Importantly, the levels of salivary IL-6, IL-10, IFN-γ, and TNF-α in RAS patients were significantly decreased following prednisone treatment (all P < 0.001). Moreover, the IFN-γ to IL-4 ratio (mean: 26.9) was significantly (P < 0.001) decreased after treatment, which almost returned to normal (mean: 24.4; P > 0.05). CONCLUSION: This preliminary study demonstrates for the first time that prednisone exerts a significant therapeutic role against RAS through decreasing salivary cytokine levels and promoting a Th1/Th2 balance. CLINICAL RELEVANCE: Salivary cytokine profiles may provide a noninvasive, convenient, and effective approach to monitoring the course of RAS and may even be helpful to identify key pathogenic factors and potential mechanisms.

Elevated mean platelet volume in oral lichen planus and increased blood urea nitrogen level in its red-form: an observational study.

BACKGROUND: This retrospective observational study aims to assess platelet count, mean platelet volume (MPV), blood biochemical tests for liver and kidney function in Chinese oral lichen planus (OLP) patients. METHODS: Eighty pathologically confirmed OLP patients and 51 healthy controls were enrolled. Data on full blood count and biochemical tests were obtained from the electronic medical record system of the hospital. RESULTS: MPV was elevated in OLP patients compared to controls (10.68 ± 0.97 fL versus 10.33 ± 0.89 fL, P = 0.042) while platelet count showed no difference between them. Red-form OLP group had increased blood urea nitrogen (BUN, 5.24 ± 1.15 mmol/L versus 4.69 ± 0.98 mmol/L, P = 0.036) than white-form OLP group. By contrast, there were no differences between those two groups in the other variables including MPV, alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine. In terms of C-reactive protein (CRP), 92.5% of the OLP patients had a value of less than 3.48 mg/L. Besides, 75% of the OLP patients were overweight with body mass index (BMI) more than 25 kg/m2. CONCLUSIONS: These findings indicate MPV might play roles in inflammation in OLP. The red-form OLP might be associated with damage or reduction of kidney function.

Altered expression of CCN1 in oral lichen planus associated with keratinocyte activation and IL-1ß, ICAM1, and CCL5 up-regulation.

BACKGROUND: Emerging evidence indicates that CCN1 is a novel inflammation-regulated mediator involved in the pathogenesis of some immune-mediated inflammatory diseases. The objective of this study was to investigate the preliminary roles of CCN1 and its related cytokines IL-1ß, CCL5, and ICAM1 in oral lichen planus (OLP). METHODS: CCN1 expression levels in biopsies from OLP patients against normal oral mucosa (NOM) using immunohistochemistry (42 OLP vs 9 NOM) and RT-qPCR (20 OLP vs 20 NOM) were compared, respectively. The correlation of CCN1 and IL-1ß, CCL5, and ICAM1 expression was examined by RT-qPCR in tissue samples and an in vitro cell culture system using keratinocyte HaCaT cells incubated with lipopolysaccharides. RESULTS: Immunohistochemistry showed that CCN1 protein mainly expressed in the cytoplasm of epithelial keratinocytes of OLP. Consistently, RT-qPCR revealed that mRNA expression of CCN1 was increased in OLP compared with NOM (P < .05) and positively correlated with the high expression of IL-1ß, ICAM1, and CCL5 (P < .001), respectively. Importantly, an in vitro study showed that keratinocyte proliferation significantly (P < .05) increased by CCN1 stimulation. Moreover, IL-1ß, ICAM1, and CCL5 expression in keratinocytes stimulated by CCN1 was increased (P < .05), respectively. CONCLUSIONS: This preliminary study for the first time reported that altered expression of CCN1 was associated with high expression of IL-1ß, ICAM1, and CCL5 in OLP. And we demonstrated CCN1 promoted keratinocyte activation, as well as IL-1ß, ICAM1, and CCL5 production in keratinocytes. Our data indicated that the potential role of CCN1 and its related cytokines was involved in the pathogenesis of OLP.

Enhanced T-cell proliferation and IL-6 secretion mediated by overexpression of TRIM21 in oral lesions of patients with oral lichen planus.

BACKGROUNDS: To explore the expression and functions of the tripartite motif-containing protein 21 (TRIM21) in oral lichen planus(OLP) lesions. METHODS: Paraffin sections of buccal mucosa samples from 15 cases of reticular oral lichen planus (OLP) patients and 10 healthy controls were used for immunohistochemistry to determine expression and distribution of TRIM21. Buccal mucosae from 11 OLP patients and seven healthy controls were analyzed by qPCR to quantify its gene expression. Peripheral blood mononuclear cells and CD3+ cells from four pairs of age- and sex-matched OLP patients and healthy controls were isolated for immunocytochemistry and culture. Following lentivirus-mediated overexpression of TRIM21 gene in CD3+ cells, CCK-8 was applied to evaluate cell proliferation. Cytokines including IL-2, IL-4, IL-5, IL-6, IL-10, TNF-α, and IFN-γ in the supernatants were measured by the cytometric bead array and verified by ELISA. RESULTS: A larger number of TRIM21-positive cells infiltrating the lamina propria were observed in OLP lesions by immunohistochemistry than those of healthy controls. Significantly higher transcription of TRIM21 was revealed by qPCR. TRIM21 overexpression in CD3+ cells significantly enhanced the proliferation and IL-6 secretion in CD3+ cells from 12 to 72 hours. CONCLUSION: Overexpressed TRIM21 in OLP may be a primary proinflammatory molecule rather than a secondary and inducible regulatory factor in immunopathogenesis of OLP.

Potential association between oral mucosal nevus and melanoma: A preliminary clinicopathologic study.

OBJECTIVES: To assess potential association between oral nevi (ON) and nevus-associated melanoma (NAM), in which melanoma cells coexist with nevus cells. METHODS: A total of 74 ON patients and 7 NAM patients were retrospectively reviewed. Comparative and regression analyses of clinical and histological data were performed between two groups. RESULTS: The mean age of the patients with ON was 36.5 years compared with that of 54.7 years of the patients with NAM (p = .008). Gender ratio was female predominance for ON (1.64:1 ratio) and male predominance for NAM (6:1 ratio). The most common location of ON and NAM was the palate (31.1%) and gingiva (71.4%), respectively. Univariate regression analysis revealed that elderly male patients (≥60 years) with junctional ON located on the gingiva correlate with higher risk of melanoma. Multivariate analysis revealed that junctional type of ON was an independent factor (adjusted OR, 38.32; 95% CI, 3.20-458.64; p = .004) associated significantly with increased risk for melanoma. CONCLUSIONS: The preliminary study for the first time elucidated the clinicopathologic features of a Chinese series of ON and evaluated the potential association between ON and NAM with a limited sample size. Further large multicenter studies are needed to confirm the findings.

Potential association between Fusobacterium nucleatum enrichment on oral mucosal surface and oral lichen planus.

OBJECTIVE: We determined the bacterial community structure of the buccal mucosa in patients with oral lichen planus (OLP) and evaluated the potential association of Fusobacterium nucleatum with OLP. SUBJECTS AND METHODS: We collected buccal mucosal swab samples of patients with OLP (n = 20) and healthy controls (n = 10) and performed 16S rRNA gene sequencing and real-time PCR to determine potentially different bacteria. Damaged and adjacent non-damaged mucosal swab samples of 25 OLP patients were used to detect the amount of F. nucleatum by real-time PCR. RESULTS: Compared with healthy controls, enrichment of Fusobacterium and Granulicatella was more abundant in patients with OLP (p = .0146 and 0.0034). The abundance of Fusobacterium and F. nucleatum was significantly enriched on buccal mucosa of patients with OLP compared with healthy controls (p = .0043 and 0.0235). Compared with adjacent non-damaged buccal mucosa of OLP patients, the amount of F. nucleatum in the damaged mucosa was significantly increased (p = .001). We examined third-level KEGG pathways for bacteria on mucosal surface and found that genes controlling sporulation and ether lipid metabolism were enriched in patients with OLP. CONCLUSIONS: A high amount of F. nucleatum may be associated with OLP. Further studies are required to investigate the precise association of F. nucleatum with OLP.

Down-regulation of miRNA-27b-3p suppresses keratinocytes apoptosis in oral lichen planus.

Oral lichen planus (OLP) is considered a precancerous lesion with no known cure. Recent studies reported that abnormal regulation of apoptosis was involved in the pathogenesis of OLP. Next generation sequencing was used to screen the candidate microRNAs and genes in biopsies from patients with OLP and healthy mucosa. Human oral keratinocytes were transfected into the related oligonucleotides of miR-27b-3p/cyclophilin D and their control groups. Apoptosis was detected by TdT-mediated dUTP nick end labelling and flow cytometry. The levels of mRNA and protein were detected by quantitative PCR, Western blots, and enzyme-linked immunosorbent assays, respectively. Luciferase assays were performed to detect the luciferase activities of miR-27b-3p and cyclophilin D. Here, we showed that basal epithelium apoptosis was reduced and the miR-27b-3p levels were decreased in clinical OLP samples. We also found that down-regulation of miR-27b-3p inhibited epithelial keratinocyte apoptosis by up-regulating cyclophilin D expression. Moreover, cyclophilin D increased the protein stability of Bcl2 through direct binding, and Bcl2 suppressed caspase9/3 activation and cytochrome C release. Taken together, these data showed that miR-27b-3p regulated keratinocyte apoptosis through cyclophilin D/Bcl2 signalling, suggesting the miR-27b-3p regulated the pathogenesis of OLP.

Downregulated miR-27b promotes keratinocyte proliferation by targeting PLK2 in oral lichen planus.

BACKGROUND: MicroRNA-27b (miR-27b) was recently found to be significantly downregulated in oral lichen planus (OLP). However, evidence of the function of miR-27b in OLP remains limited. METHODS: Initially, miR-27b expression in OLP was verified using the quantitative real-time polymerase chain reaction (qRT-PCR). Functionally, gain-/loss-of-function studies were then conducted using miR-27b mimics/inhibitor to investigate cell growth in human oral keratinocytes (HOKs). Mechanistically, subsequent miRNA target analyses including a starBase database analysis and a luciferase reporter assay were performed to predict and validate the direct target, respectively. In addition, overexpression/knockdown assays of target(s) of miR-27b were performed to investigate its functional significance and qRT-PCR and western blotting were used to evaluate the target(s) of miR-27b mRNA and protein levels, respectively. RESULTS: MicroRNA-27b was significantly downregulated in OLP tissues when compared with healthy control tissues. Bioinformatics predicted that Polo Like Kinase 2 (PLK2) might be a potential target of miR-27b, while the luciferase reporter assay results showed the direct inhibition of the plk2-3'untranslated region by miR-27b. Moreover, functional analysis indicated that downregulated miR-27b caused an increase in cell growth in HOKs, and correspondingly, overexpression of PLK2 promoted HOK proliferation. CONCLUSIONS: There were aberrant expressions of miR-27b and PLK2 in OLP tissues. Decreased miR-27b may have induced cell proliferation by increasing the levels of PLK2 in HOKs, which provides a new perspective into the potential mechanisms underlying OLP development.

Aberrant expression of interleukin-22 and its targeting microRNAs in oral lichen planus: a preliminary study.

BACKGROUND: Oral lichen planus (OLP) is a T cell-mediated autoimmune disease involving oral mucosa. Interleukin-22 (IL-22) as the signature cytokine of T helper 22 cells is increasingly recognized as a key regulator in various autoimmune diseases. Our previous study reported that IL-22 immunoexpression in OLP was significantly increased compared with the normal controls. METHODS: The objective of this preliminary study was to compare the IL-22 expression levels in oral biopsies from patients with OLP (n = 50) against normal oral mucosa (n = 19) using RT-qPCR and Western blot, identify the potential targeting miRNAs of IL-22, and examine the miRNA expression levels in OLP. RESULTS: Interleukin-22 expression level in OLP was significantly increased compared with the normal controls. The Dual-Luciferase reporter assay system in human embryonic kidney 293 (HEK293) cells demonstrated that miR-562 and miR-203 were the target miRNAs of IL-22, which was consistent with predictions from bioinformatics software analyses. Interestingly, miR-562 expression in OLP was significantly decreased, but miR-203 expression in OLP was significantly increased compared with the normal controls. CONCLUSION: This preliminary study for the first time reported that aberrant expression levels of miR-562 and miR-203 were associated with high expression of IL-22 and demonstrated the target relationship between miRNAs and IL-22 in HEK293 cells. Our data indicated that IL-22 and its targeting miRNAs contribute to the pathogenesis of OLP. Further studies are required to investigate the regulatory pathways of IL-22 and miR-562 and miR-203 in OLP.

[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.

The implications of gene mutations in salivary DNA for noninvasive diagnosis of head and neck cancer with a focus on oral cancer.

DNA-based liquid biopsy as a diagnostic strategy of head and neck squamous cell carcinoma (HNSCC) has emergingly gained momentum. In this letter, we identified 6 studies contained 274 patients with HNSCC focused on gene mutations in salivary DNA. We observe that the incidence of DNA mutations with at least one gene mutated ranges from 63% to 95.9%, and the most frequently examined gene mutations are TP53, CDKN2A, PIK3CA, FAT1, and NOTCH1. Meanwhile, studies have demonstrated that saliva had a greater sensitivity and much higher quantitative values than plasma in both tumor DNA count and variant allele frequency. Interestingly, more tumor-derived mutations were detected in salivary DNA among patients with tumors arising in oral cavity compared to in oropharynx, larynx, and hypopharynx. Collectively, it is feasibility to identify somatic mutations in driver genes using saliva samples to noninvasively diagnose HNSCC, especially in oral cavity cancer and even at early stages of the disease. Larger well-designed studies are needed to consolidate the evidence.

Methicillin-Resistant Staphylococcus aureus Membrane Vesicles Inhibit the Proliferation and Induce the Apoptosis of Epithelial Cells.

Staphylococcus aureus, or methicillin-resistant Staphylococcus aureus (MRSA), is the predominant pathogen in skin and soft tissue infections (SSTIs), and MRSA membrane vesicles (MVs) play a pivotal role in bacterial pathogenesis and the modulation of the host immune response. We aimed to investigate the interaction between MRSA MVs and epithelial cells. In this study, MVs were isolated from an MRSA culture supernatant using the ELD method, comprising an electrophoretic technique used in combination with a 300 kDa cut-off dialysis bag. The proteomic analysis of the MRSA MVs via mass spectrometry showed that shared and distinct proteins exist in the MVs from clinical MRSA isolates with different genetic backgrounds, such as health-care-associated MRSA (HA-MRSA) and community-associated MRSA (CA-MRSA). These MRSA MVs were found to suppress the proliferation and increase the apoptosis of HaCaT cells. We conducted qPCR array, quantitative real-time PCR (qRT-PCR), and Western blotting (WB) analyses, and the results indicated that BCL2 antagonist/killer 1 (Bak1) may be involved in the apoptosis of HaCaT epithelial cells. Our findings suggest that MRSA MVs inhibit the proliferation and induce the apoptosis of epithelial cells.