Analysis of oral microbial community and Th17‐associated cytokines in saliva of patients with oral lichen planus

Oral lichen planus (OLP) is a chronic inflammatory disorder of oral mucosa of unknown cause. Microbial infection and dysimmunity appear to play important roles in its pathogenesis. In this study, differences in genetic profiling of salivary microbial communities in two subtypes of OLP and healthy controls were evaluated by means of PCR‐denaturing gradient gel electrophoresis (DGGE). Additionally, ELISA was used to investigate the possible role of Th17 in lesion formation by detecting two related cytokines IL‐17 and IL‐23 in the saliva of OLP patients. When the DGGE profiles were analyzed, the bacterial populations were found to be significantly less rich in subjects with reticular and erosive OLP than in healthy controls. There was significantly less microbial diversity, as denoted by the Shannon index, in saliva samples from subjects with erosive OLP than in those from healthy controls. Cluster analysis and principal component analysis showed that the DGGE profiles formed distinctly group‐specific clusters. Salivary concentrations of IL‐17 in subjects with erosive OLP group were significantly higher than in those with reticular OLP and healthy controls. What's more, significantly positive correlations were observed between salivary IL‐17 concentrations and disease clinical scores. Microbial richness and diversity was negatively correlated with salivary IL‐17 concentrations. These results suggest there is significantly less salivary bacterial diversity and complexity in subjects with OLP han in healthy controls and that the shifted community composition is closely related to an immune cytokine, IL‐17.     

NF-kappaB-dependent cytokines in saliva and serum from patients with oral lichen planus: a study in an ethnic Chinese population

To access the potential roles of NF-kappaB-dependent pro-inflammatory cytokines, including tumor necrosis factor-alpha (TNF-alpha), interleukin-6(IL-6), and interleukin-8(IL-8), in oral lichen planus (OLP), and to explore whether salivary detection of these cytokines is a better monitoring tool than serum for OLP, 30 Chinese patients with OLP and 30 age-sex-matched controls were enrolled in this study. Salivary and serum levels of cytokines were screened by enzyme immunoassay, and both salivary and serum levels of these cytokines were significantly increased in OLP patients in comparison with healthy subjects. Positive statistical correlations existed between these cytokines in serum and saliva. With regards to the subtypes of this illness, only salivary IL-8 levels in erosive form were remarkably inclined than that in reticular form. The results suggest that saliva-based test may provide a more cost-effective adjunctive tool for the detection of pro-inflammatory cytokines in OLP, and salivary IL-8 analysis may be a promising biomarker for OLP severity.     

口腔扁平苔藓患者血清白介素-17、P物质、趋化素水平与疾病活动和免疫功能的关系研究

摘要 目的：研究口腔扁平苔藓（OLP）患者血清白介素-17（IL-17）、P物质（SP）、趋化素（Chemerin）水平与疾病活动和免疫功能的关系。方法：选取我院2019年3月~2021年5月期间收治的117例OLP患者作为观察组,其中非糜烂型69例,糜烂型48例。另选取同期来我院体检的健康志愿者60例为对照组。对比对照组及不同分型患者血清SP、IL-17、Chemerin水平,对比不同分型患者的疾病活动相关评分和免疫功能指标,应用Pearson相关分析血清SP、IL-17、Chemerin水平与疾病活动相关评分和免疫功能指标的相关性。结果：不同分型OLP患者血清SP、IL-17、Chemerin水平均高于对照组（P<0.05）,且糜烂型OLP患者的血清SP、IL-17、Chemerin水平高于非糜烂型OLP患者（P<0.05）。糜烂型OLP患者的CD3⁺、CD4⁺低于非糜烂型OLP患者（P<0.05）,糜烂型OLP患者的CD8⁺、免疫球蛋白M（IgM）水平、OLP网纹-糜烂-溃疡疾病活动积分（REU）总分高于非糜烂型OLP患者（P<0.05）。糜烂型与非糜烂型OLP患者间免疫球蛋白A（IgA）、免疫球蛋白G（IgG）水平对比差异无统计学意义（P>0.05）。Pearson相关性分析结果显示,血清SP、IL-17、Chemerin水平与IgM水平、REU总分均呈正相关（P<0.05）。结论：OLP患者血清IL-17、SP、Chemerin水平异常升高,三者共同参与了OLP患者的病情进展及免疫功能调节。     

Frequency of micronucleated exfoliated cells in oral lichen planus

OBJECTIVE: The purpose of this study was to determine the frequency of micronucleated exfoliated cells (MEC) in atrophic and erosive oral lichen planus (OLP). MATERIALS AND METHODS: Twenty-two patients with atrophic and/or erosive OLP participated in this study. Lesions were scored ranging from 0 (no lesion) to 5 (large erosion) according to the severity and assessed for MEC. Exfoliated cells were obtained by swabbing the lesions and normal-appearing mucosa adjacent to the lesions. Swabbing was also performed in age-sex-matched normal individuals. Five hundred exfoliated cells were screened for nuclear anomalies including micronuclei, karyorhexis, pycnosis, and chromatid clumping. RESULTS: The severity score of OLP ranged from 2 to 4 with the average of 2. The frequency of MEC in OLP patients was 3.79% and 0.37% in the lesions and normal-appearing mucosa, respectively. In normal individuals, the frequency of MEC was also 0.37%. Using a paired t-test, it was found that the MEC frequency in the OLP lesions was significantly elevated (p<0.01) as compared to that in normal-appearing mucosa adjacent to lesions and that in normal individuals. There were no statistically significant differences in the MEC frequency of the three severity scores as analyzed by Kruskal-Wallis one way analysis of variance on ranks (p>0.05). CONCLUSION: This study revealed an increase in micronuclei in OLP lesions. The results indicate genotoxic damage in atrophic and erosive OLP.     

Association of HLA-te22 antigen with anti-nuclear antibodies in Chinese patients with erosive oral lichen planus

The purpose of this study was to determine the frequencies of the presence of serum anti-nuclear antibodies (ANA) and smooth muscle antibodies (SMA) in 76 patients with oral lichen planus (OLP), in 77 patients with other oral mucosal diseases, and in 41 healthy control subjects. HLA phenotypes in some of the patients with OLP and recurrent aphthous ulcers (RAU) were determined to show whether there was an association of HLA antigens with the presence of autoantibodies. Indirect immunofluorescence techniques with mouse liver or stomach as the substrate were used to detect the serum ANA or SMA, respectively. The B lymphocytes isolated from peripheral blood were used for HLA typing by means of a standard microcytotoxicity assay. We found that the positive rate of serum ANA in patients with OLP (29%, p < 0.01), especially in patients with erosive OLP (34%, p < 0.001), was significantly higher than that in the normal control subjects (5%). The frequency of serum SMA in patients with OLP (20%, p < 0.01), in patients with RAU (17%, p < 0.01), or in patients with oral squamous cell carcinomas (41%, p < 0.001) was also significantly higher than that in normal control individuals (0%). In the erosive OLP group, the HLA-Te22 antigen occurred more frequently in patients with positive ANA (75%, p < 0.05) than in those with negative ANA (25%). We conclude that there is an association of HLA-Te22 antigen with ANA in Chinese patients with erosive OLP.     

Microbiological and immunological features of oral candidiasis

Candida albicans(C. albicans) is the major infectious agent of oral candidiasis, and both innate immunity and cell-mediated immune response participate in the control of the fungal infections. The aim of this study was to correlate the clinical forms of oral candidiasis with the number of colony forming units (CFU) of C. albicans in saliva and to characterize T cell response in patients with oral candidiasis. Participants included 75 subjects: 36 with lesions of candidiasis and 39 without lesions of oral candidiasis. A 2-ml sample of saliva was collected from all subjects for microbiological analysis. Cytokine levels were determined by ELISA in supernatants of peripheral blood mononuclear cells of 25 patients with oral candidiasis, after in vitro stimulation with C. albicans antigens. In 48% of patients, no association was observed with denture use. C. albicans was detected in the saliva of 91.7% of patients with oral candidiasis, and there was an association between the number of CFU and the presence of oral lesions. A type Th1 immune response was observed in supernatants of peripheral blood mononuclear cells stimulated with C. albicans antigens. In contrast, IL-5 and IL-10 levels were very low or undetectable. Together, this study shows an association between clinical forms of oral candidiasis and the number of colonies of C. albicans in saliva, and that a systemic immune response characterized by the production of TNF-alpha and IFN-gamma is observed in patients with oral candidiasis.     

Possible salivary and serum biomarkers for oral lichen planus

There are few reports concerning the potential for clinical application of oxidative stress (OS) and collagen degradation markers in oral lichen planus (OLP) patients. We investigated the possibility of using some disease-related biomarkers in saliva and serum of OLP patients. Our study included 30 patients with OLP and 30 controls. We evaluated serum and salivary OS biomarkers including 8-OHdG, MDA, uric acid, TAC and GPx. We also investigated collagen degradation markers such as CTX I and MMP-8. We found significantly increased salivary levels of MMP-8 and CTX I in the OLP group compared to controls and significant differences between the OLP and control groups in serum and saliva for 8-OHdG, MDA (significantly increased), uric acid, TAC and GPx (significantly reduced). Currently there are no criteria for evaluating which OLP patients have a greater risk of malignant transformation. In addition to clinical surveillance, the serum and salivary biomarkers that we evaluated may be useful biomarkers for monitoring OLP patients in the future.     

抗菌肽对糜烂型OLP患者唾液中真菌的影响

抗菌肽经临床研究,发现具有抗生素不具备的优点,可对糜烂型口腔扁平苔藓(oral lichen planus,OLP)患者唾液中真菌有抑制和杀菌的作用,弥补了抗生素的缺点。通过使用不同浓度、不同种类的抗菌肽,作用于自糜烂型OLP患者口腔唾液标本中提取并经鉴定的菌种,对三种常见菌——白色念珠菌、光滑念珠菌、热带念珠菌进行抑菌试验。结果表明,抗菌肽使用前后的数值比对、菌落生长情况、最小抑菌值有明显变化。发现RISE-AP12对三种常见真菌的抑菌、杀菌效果优于其他的抗菌肽,在临床上有较大应用前景。     

Genetic linkage analysis of oral lichen planus in a Chinese family

Oral lichen planus (OLP) is a common oral inflammatory disease affecting about 1-2% of the general adult population. As with European families who are diagnosed with OLP, the Chinese family who we studied was diagnosed with a severe form of oral reticular and erosive lesions; moreover, two of the five affected individuals developed oral cancer at an early age. A whole-genome genotyping scan with linkage analysis was performed using the 10K SNP array to investigate the genetic susceptibility of the Chinese family to OLP, which revealed one maximal nonparametric LOD score of 2.32 (P = 0.0156) for SNP marker rs2372736, defined at the chromosome 3p14-3q13 region encompassing 19 SNPs. Blood samples were obtained from 10 members of the family, which included the grandmother, father and mother, and the children altogether. The grandfather is dead, but the family members remembered he also suffered from the same disease. Chromosome 3p14-3q13 was identified as the candidate gene region for OLP; this information provides a foundation for further identification of the gene responsible for OLP.     

Phenotypic variability and therapeutic implications of Candida species in patients with oral lichen planus

We investigated the prevalence and phenotypic variation of Candida species in oral lichen planus (OLP) and the therapeutic implications of our findings. Eighty patients with clinically and histopathologically confirmed cases of OLP (64 non-erosive, 16 erosive) and a control group of 80 healthy individuals with no predisposing factors for oral candidiasis were examined for evidence of Candida infection. Oral swabs and smears were obtained for cytology and culture. Identification, speciation and antifungal susceptibility tests of Candida isolates were performed using an automated microbial identification system. Fifty percent of erosive OLP cases, 28% of non-erosive cases and none of the controls showed evidence of Candida. Candida albicans was found predominantly in non-erosive OLP, while other Candida species were predominate in erosive OLP. Non-Candida albicans isolates (C. glabrata, C. krusei) were resistant to the commonly used antifungals, clotrimazole and fluconazole. Candida infection is common in cases of OLP. We recommend antifungal sensitivity testing prior to antifungal therapy for the erosive form of OLP.     

白色念珠菌分泌型天冬氨酸蛋白酶在口腔扁平苔藓患者唾液中的表达

研究白色念珠菌分泌型天冬氨酸蛋白酶(SAP)家族在扁平苔藓患者唾液中的表达,进而探讨白色念珠菌病与扁平苔藓的关系。选取白色念珠菌携带者、白色念珠菌病患者、携带白色念珠菌的扁平苔藓患者、伴有白色念珠菌病的扁平苔藓患者各20例,念珠菌培养阴性的扁平苔藓患者10例。从唾液标本中直接提取RNA,进行RT-PCR。白色念珠菌携带者唾液中有SAP2、SAP4-6、SAP7的不同表达,携带白色念珠菌的口腔扁平苔藓患者唾液中有SAP2、SAP4-6、SAP7、SAP8、SAP9的不同表达,所有携带者中均未表现SAP1、SAP3和SAP10的阳性扩增。白色念珠菌患病者及伴有白色念珠菌病的口腔扁平苔藓患者唾液中有SAP1-10的不同表达。念珠菌培养阴性的扁平苔藓患者均无SAP1-10的阳性扩增。SAP9与扁平苔藓密切相关。     

Alteration of salivary glycopatterns in oral lichen planus

Background: Oral lichen planus (OLP) is one of the most common oral mucosal lesions affecting 0.5–2% of the adult population. It is difficult to distinguish between OLP and other oral mucosal diseases. Structural changes in the glycans of saliva proteins might be reliable indicators of OLP. However, little is known about the alteration of salivary glycopatterns during OLP.

Objective: We aimed to investigate the alterations of salivary protein glycosylation related to OLP.

Material and methods: Twenty-eight patients with OLP and 30 age- and sex-matched healthy volunteers (HVs) were enrolled in the test group to probe the difference of salivary glycopatterns using lectin microarrays. The lectin blotting were further utilized to validate the expression of certain glycans.

Results: The glycoproteins recognized by three lectins [Aleuria aurantia lectin (AAL); Phytolacca americana (PWM); Phaseolus vulgaris agglutinin (E?+?L), (PHA-E?+?L)] were mainly increasing in the saliva of OLP. Meanwhile, these glycoproteins also exhibited significant age-associated alterations.

Conclusions: This study provided a new basic insight into salivary glycopatterns in OLP and helped to develop new potential biomarkers for diagnosis of OLP.     

Serum and salivary levels of albumin as diagnostic tools for oral pre-malignancy and oral malignancy

The role of oxygen free radicals in the initiation, promotion and progression of carcinogenesis and the protective role of antioxidants has been a subject of much speculation. There are few studies that report evaluation of serum albumin and only one study in which salivary albumin was found and only one study that reports of salivary albumin in oral Leukoplakia and Oral Squamous Cell Carcinoma (OSCC). We evaluated serum and salivary albumin levels in normal individuals, patients with oral pre-malignancy and patients with oral malignancy, and we compared serum and salivary albumin levels in patients with oral pre-malignancy and oral malignancy. Our study comprised 45 subjects separated into three groups of 15: normal healthy, oral pre-malignancy and oral malignancy patients. Venous blood was drawn and unstimulated saliva was collected early in the morning. Albumin levels were estimated using the bromocresol green method. Serum albumin levels decreased in oral pre-malignancy and oral malignancy cases compared to healthy individuals. Salivary albumin levels increased in oral pre-malignancy and oral malignancy cases compared to healthy individuals. Our results suggest that albumin may play a role in early diagnosis and prognosis of oral pre-malignant and oral malignant tissues.     

A comparison of ghrelin, glucose, alpha-amylase and protein levels in saliva from diabetics

During the past decade, many salivary parameters have been used to characterize disease states. Ghrelin (GAH) is recently-discovered peptide hormone secreted mainly from the stomach but also produced in a number of other tissues including salivary glands. The aim of this work was to examine the relationship between active (aGAH) and inactive (dGAH) ghrelin in the saliva and other salivary parameters in type II diabetic patients and healthy controls. Salivary parameters were assessed in a single measurement of unstimulated whole saliva from 20 obese and 20 non-obese type II diabetes patients, and in 22 healthy controls. Total protein and alpha-amylase were determined by colorimetric methods, and glucose by the glucose-oxidase method. Saliva aGAH and dGAH levels were measured using a commercial radioimmunoassay (RIA) kit. Salivary concentrations of aGAH and dGAH ghrelin were more markedly decreased in obese diabetic subjects than in the two other groups. Glucose and alpha-amylase levels were higher in diabetic subjects than in controls. Furthermore, there were correlations between GAH levels and BMI, and between GAH and blood pressure. However, there was no marked variability in saliva flow rates among the groups. These results indicate that measurement of salivary GAH and its relationship to other salivary parameters might help to provide insight into the role of ghrelin in diabetes.     

Estimation of salivary nitric oxide in oral precancer patients

Abstract

The role of nitric oxide (NO) in the initiation, promotion and progression of cancer has been the subject of speculation and conflicting reports in the literature. The high incidence of oral cancer and precancer has been linked to tobacco chewing and smoking habits; NO is considered an indicator of tobacco-related diseases. We compared salivary NO levels in oral precancer and normal patients. Unstimulated whole saliva was collected from 15 patients with oral precancer (group 1) and 15 healthy age and sex matched subjects (group 2). Salivary nitrite levels were estimated using a colorimetric method and a spectrophotometer. The salivary nitrite concentration of group 2 (median = 4.21 μg/ml) was significantly less than for group 1 (median = 12.91 μg/ml). We have added evidence concerning involvement of NO in the pathogenesis of oral cancer, but whether it is a potentially carcinogenic agent at the concentration at which it is present in oral precancer patients requires further evaluation.     

TNF-alpha levels and TNF-alpha gene polymorphism in type I Gaucher disease

The objective of this pilot study was to determine the levels of Tumor Necrosis Factor (TNF)-alpha and TNF-alpha gene polymorphism as a marker of inflammation among patients with type I Gaucher disease as well as to ascertain the relationship between this cytokine and parameters of disease severity and other measures of inflammation. Levels of TNF-alpha and genotyping for the -308 G-->A polymorphism in the promoter of the TNF-alpha gene were performed in 17 patients with type I Gaucher disease. TNF-alpha levels were compared with the promoter gene polymorphism, and with hematological and other clinical parameters of Gaucher disease. Eight patients (47.1%) were homozygotes (A/A) for the TNF-alpha polymorphism, six patients (35.3%) had the wild type (G/G), and three patients (17.6%) were heterozygotes (G/A). A significant correlation was found between serum TNF-alpha levels and TNF-alpha genotypes for homozygotes versus heterozygotes patients (p = 0.02), with patients homozygous for the polymorphism having the lower levels of serum TNF-alpha relative to heterozygotes with the highest levels. No correlation was found between TNF-alpha genotypes and chitotriosidase levels, a putative biochemical marker for Gaucher disease severity. Because a significant correlation was found between homozygosity for a common promoter polymorphism of TNF-alpha and milder expression i.e. non-neuronopathic form, of Gaucher disease (versus the neuronopathic forms), this may be suggestive of an association between genetic variability in TNF-alpha and phenotypic expression in Gaucher disease. Larger studies will be required.     

Blood concentrations of tumor necrosis factor-alpha in malignant lymphomas and their decrease as a predictor of disease control in response to low-dose subcutaneous immunotherapy with interleukin-2

Tumor necrosis factor-alpha (TNF-alpha), a cytokine provided by both immunomodulating and inflammatory activities, has been described to be abnormally increased in the blood of patients affected by malignant lymphomas, particularly NHL. However, the biological and clinical significance of TNF-alpha secretion in malignant lymphomas is still controversial. The present study was carried out to further define TNF-alpha secretion in untreated malignant lymphomas and during low-dose IL-2 immunotherapy. The study included 80 malignant lymphoma patients, 54 of whom were affected by HD and the other 26 by NHL. The mean TNF-alpha serum concentrations observed in untreated lymphoma patients were significantly higher than those seen in the healthy controls, without significant differences between HD and NHL. Moreover, both HD and NHL lymphoma patients at clinical stage III-IV showed significantly higher mean TNF-alpha levels than those at clinical stage I-II. Finally, patients with systemic symptoms had higher mean TNF-alpha concentrations than those without any systemic symptoms, even though statistical significance was observed only for NHL patients. In a second study we have evaluated changes in TNF-alpha levels in seven evaluable lymphoma patients (NHL: 6; HD: 1)--who did not respond to conventional therapies--during subcutaneous low-dose IL-2 (3 MIU/day, 6 days/week for 4 weeks). Long-term stable disease was achieved in four patients with NHL, whereas the other three progressed. In patients with stable disease the mean TNF-alpha concentrations significantly decreased during treatment, whereas they increased in progressing patients. This study, by showing an abnormally enhanced TNF-alpha secretion in both NHL and HD patients with advanced disease and systemic symptoms and a decrease in its levels in patients who achieved disease control on IL-2 immunotherapy, appears to confirm the unfavorable prognostic significance of enhanced TNF-alpha levels in malignant lymphomas.     

Obestatin is present in saliva: alterations in obestatin and ghrelin levels of saliva and serum in ischemic heart disease

Ghrelin and obestatin are a single gene products and are a multiple functional peptides that regulates energy homeostasis, and food intake. In the present work, we studied the secretion of ghrelin and its co-secreted peptide obestatin in 44 patients with ischemic heart disease with that of 27 healthy matched controls. Here we first conducted using an immunohistochemistry assay to screen whether human salivary glands have any obestatin immunoreactivity. Then, serum and saliva obestatin and acylated ghrelin levels were determined by using Radioimmunoassay. Our immunohistochemical analysis demonstrated that obestatin was localized in the striated and excretory duct of human salivary gland. We also report for the first time that obestatin, like ghrelin, is present in human salivary gland and saliva. No evidence of the role of obestatin or ghrelin saliva levels in the context of ischemic heart disease was found. Salivary ghrelin and obestatin levels are correlated in controls with the blood levels. Determination of salivary values could represent a non-invasive alternative to serum ones that can be useful in clinical practice.     

Analysis of the factors motivating HCV-infected patients to accept interferon therapy

ABSTRACT: BACKGROUND: The aims of this study were to analyze factors motivating the acceptance of interferon (IFN) therapy and to clarify the prevalence of oral mucosal diseases in hepatitis C virus (HCV)-infected Japanese patients treated with IFN. FINDINGS: A total of 94 HCV-infected patients who were admitted to our hospital for IFN therapy were asked questions regarding their motivation to accept IFN therapy and were investigated for the presence of oral lichen planus (OLP) before and during IFN treatment. Recommendation and encouragement from other people were the most common factors motivating the acceptance of IFN therapy (49/94, 52.13%). The other motivators were independent decision (30.85%), economic reasons (5.32%), and others. According to multivariate analysis, three factors - sex (male), retreatment after previous IFN therapy, and independent decision to accept IFN therapy - were associated with patients after curative treatment of hepatocellular carcinoma (HCC). The adjusted odds ratios for these three factors were 26.06, 14.17, and 8.72, respectively. The most common oral mucosal lesions included OLP in 11 cases (11.70%). One patient with OLP had postoperative squamous cell carcinoma of the tongue. The rate of sustained virological response (SVR) was 45.45% in cases with OLP and 54.55% in cases without OLP. There were no patients who discontinued IFN therapy because of side effects such as oral mucosal diseases. CONCLUSIONS: We should give full explanation and recommend a course of treatment for a patient to accept IFN therapy. The system to support liver disease as well as oral diseases is also necessary for patient treated for IFN therapy.