Buccal Mucosa Cells as a Potential Diagnostic Tool to Study Onset and Progression of Arrhythmogenic Cardiomyopathy

In arrhythmogenic cardiomyopathy (ACM) pathogenic variants are found in genes encoding desmosomal proteins and in non-desmosomal genes, such as phospholamban (PLN, p.Arg14del variant). Previous research showed that plakoglobin protein levels and localization in the cardiac tissue of ACM patients, and PLN p.Arg14del patients diagnosed with an ACM phenotype, are disturbed. Moreover, the effects of pathogenic variants in desmosomal genes are reflected in non-cardiac tissues like buccal mucosa cells (BMC) which could serve as a promising new and non-invasive tool to support diagnosis. We collected the BMC of 33 ACM patients, 17 PLN p.Arg14del patients and 34 controls, labelled the BMC with anti-plakoglobin antibodies at different concentrations, and scored their membrane labelling. We found that plakoglobin protein levels were significantly reduced in BMC obtained from diagnosed ACM patients and preclinical variant carriers when compared to controls. This effect was independent from age and sex. Moderate to strong correlations were found with the revised 2010 Task Force Criteria score which is commonly used for ACM diagnosis (rs = −0.67, n = 64, p < 0.0001 and rs = −0.71, n = 64, p < 0.0001). In contrast, plakoglobin scores in PLN p.Arg14del patients were comparable to controls (p > 0.209), which suggests differences in underlying etiology. However, for the individual diagnosis of the ‘classical’ ACM patient, this method might not be discriminative enough to distinguish true patients from variant carriers and controls, because of the high interindividual variability.

Micronuclei and Other Nuclear Anomalies in Exfoliated Buccal Mucosa Cells in Breast Cancer

Objectives: This study designed to assess the genomic instability between healthy women and women with breast cancer by means of buccal cells micronucleus (MN) cytome assay. Methods: The current study comprised 25 healthy women and 30 breast cancer patients. The exfoliated cells of buccal mucosa were taken after each participants rinse their mouths with tap water. The micronucleated cell and nuclear anomalies were analyzed under a total magnification of X1000, 2000 cells per subjects ( patient and control group) were scored and the frequencies of nuclear anomalies including MN, binucleates (BN), Pycnotic cell, karyolysis (KL) and karyorrhexis (KR) were evaluated in exfoliated buccal mucosa cells of women with primary BC and healthy women. Results: The frequencies of micronuclei and all nuclear anomalies in buccal cells of BC patients were significantly increased compared with the controls. (For Binucleates cells only, p<0.001; in all other cases, P < 0.0001). The mean scores of micronuclei and all nuclear anomalies for the breast cancer patients were (10.66±0.3845, 6.20±0.26, 8.40±0. 22, 18.40±0. 34, 19.13 ±0.40) were significantly higher than that of healthy women). Conclusion: Elevated frequency of micronucleated cells and all nuclear anomalies in the buccal mucosa of breast cancer patients reveal the genomic instability. These findings propose that the buccal MN-cytome assay can be used to measure both genotoxic and cytotoxic effects in primary cancer patients.

Viability and DNA Damage of Buccal Mucosa Cells in Patients Exposed to Panoramic X-ray

Panoramic X-ray is well known to cause DNA damage and induces cellular death. The aim of the present study was to evaluate the cytotoxicity of radiation exposure from panoramic radiography on human buccal mucosa cells by assessing the cell viability using the simple-trypan blue exclusion test. The genotoxicity effect was evaluated by assessing comet assay score. This research included a total of 20 healthy patients who had panoramic radiography for a routine dental examination. Buccal mucosa cells were collected from all participants before X-ray exposure and at 30 min or 24 h after exposure in Groups 1 and 2, respectively, and subjected to a comet assay and trypan blue exclusion test to assess cell viability and DNA damage. Cell viability was calculated as the ratio of live (translucent) to total counted cells. Comet assay output images were analysed using OpenComet software and a visual score by measuring the percentages of tail DNA and summing the visual score, respectively. A statistically significant (p < 0.05) reduce in cell viability was observed at 30 min after exposure, furthermore there is no more reduction after 24 h. Both comet assay measurements showed a significant (p < 0.05) increase in the percentage of tail DNA and visual score at 30 min after exposure, then tend to decrease after 24 h of exposure, although it was not significant (p > 0.05). The results showed that panoramic radiography interfered cell viability and induced DNA damage in buccal mucosa cells within 30 min after exposure, but these effects were ceased after 24 h.

Staining criteria, collection and number of cells evaluated to identify micronuclei in buccal mucosa cells of agricultural workers exposed to pesticides.

Although there are several publications that refer to the basic criteria established for the micronucleus (MN) test in exfoliated buccal cells, there is still a difference in the quantity, method of obtention and staining of the cells for the evaluation of the presence of micronuclei. Objective. Identify the criteria for evaluation of MN in oral mucosa cells exposed to pesticide in investigations carried out. Material and methods. A systematic review was carried out on the internet, based on articles published in Crossref, JCR, Scopus, PubMed, Google academic, using keywords such as: micronuclei, buccal mucosa cells, genotoxic damage, pesticides and biomarker. Results. In the six selected articles, four presented statistically significant values ​​with the presence of MN when comparing the exposed groups with respect to the control groups, and the criteria for staining, collection and number of cells evaluated to identify micronuclei were very varied. Conclusions. It is important to follow validated and standardized protocols for the MN oral cytoma assay. Being considered in all the parameters suggested in the protocol will increase the reliability of the studies and will give the possibility of comparing the results obtained.

Buccal mucosa cells as a diagnostic tool in patients with arrhythmogenic cardiomyopathy

Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Netherlands Cardio Vascular Research Initiative (CVON): the Dutch heart foundation Background Arrhythmogenic cardiomyopathy (ACM) is predominantly caused by mutations in genes encoding desmosomal proteins (most often multiple different mutations in plakophilin-2), however also mutations in non-desmosomal proteins like phospholamban (PLN, PLNR14Del) are found. Previous investigations showed that plakoglobin protein levels and localization in cardiac tissue of ACM patients is disturbed and this could be a valuable additional tool to discriminate between non-affected family members and those being at risk during progression of the disease. Information about cardiac plakoglobin status can only be obtained via endocardial biopsies, however, this technique has major draw-backs and risks, and therefore an alternative is needed. A promising new non-invasive tool is the use of buccal mucosa cells (BMC), as it has been reported that effects of mutations in desmosomal proteins are additionally reflected in non-cardiac tissues like buccal mucosa smears that do also express those desmosomal genes. Purpose To investigate the clinical usability of BMC as tool to classify patients at risk of developing ACM by comparing healthy controls with asymptomatic and symptomatic classical ACM patients, and patients carrying a PLNR14Del mutation. Methods and results BMC of 84 human subjects, 33 ACM patients (19 males, 28 with a PKP2 mutation), 17 PLN patients (6 males) and 34 healthy controls (14 males), were collected on glass slides, fixed and labelled with anti-plakoglobin antibodies diluted 1:5.000, 1:10.000, 1:20.000 and 1:40.000, and scored for their membrane labelling. Data revealed that for each applied dilution, plakoglobin protein levels at the membrane were significantly reduced in BMC obtained from ACM patients compared to healthy controls. This effect appeared independent from age and sex of the patients. Furthermore, dilutions 1:5.000 and 1:10.000 showed a moderate correlation with the revised 2010 Task Force Criteria Score (TFC) which are commonly used for ACM diagnosis (Rs -0.67, n = 64, p &lt;0.0001 and Rs -0.71, n = 64, p &lt; 0.0001 resp.) In contrast, plakoglobin scores of PLN patients were similar to controls. Conclusion On the population level, there is a significant reduction of plakoglobin protein in BMC membranes of ACM patients but not for patients bearing the PLNR14Del mutation when compared to healthy controls. However, for clinical diagnosis of the individual patient, this method is most likely not discriminative enough to distinguish patients from healthy controls, because of the high interindividual variability.

Confirmation of differentiation clusters’ and endoglin markers preset in porcine buccal mucosa cells

Several genes, namely CD44, CD90, CD105 and PCNA may be important in differentiation of porcine mucosa cell cultures. These genes are, inter alia, responsible for cell adhesion to extracellular matrix and its constituent secretion, cytoskeleton organization, epithelial to mesenchymal transition or proper course of DNA replication. A total of 20 pubertal crossbred Landrace gilts bred on commercial farms were used to produce buccal mucosa cultures, which were harvested on the 7th, 15th and 30th day after initiation of the culture. Expression levels of CD44, CD90, CD105 and PCNA were evaluated employing Real-Time Quantitative Polymerase Chain Reaction. CD44, CD90 and PCNA showed an unchanged expression pattern. Expression of CD44 on day 7 was the highest of all factors measured. The greatest difference between the measurement on 7th and 30th day was found in the PCNA gene. These results broaden the understanding of the transcriptome changes in porcine buccal mucosa cells for the duration of in vitro cultivation. Nevertheless, it is very important to consider that the in vitro conditions do not fully reflect the changes taking place in the living organism. It appears that tissues of the oral cavity possess high regenerative potential, and constitute suitable model for wound healing investigation.Running title: Confirmation of differentiation clusters’ and endoglin markers preset in porcine buccal mucosa cells