Tongue Epithelial Cells Research Articles

Effect of CBD on anti-tumor immune response and interaction with GPR55 on MAPK signaling in head and neck squamous cell carcinoma.

6060 Background: Head and neck squamous cell carcinoma (HNSCC) is a lethal malignancy and comprises two distinct etiology: human papillomavirus (HPV)+ve and -ve. Marijuana use is associated with HPV+ oropharyngeal infection and conflicting data show positive and negative associations of daily marijuana use with HPV+ and tobacco-associated HPV- HNSCC. However, cannabinoid use continues to increase in the US general population for recreational purposes as well as in cancer patients for palliative care. G-protein coupled receptors (GPCRs), including cannabinoid GPCRs, stimulated by specific ligands interact with multiple G proteins creating diversity and complexity of signaling. In this study, we aimed to investigate the CBD-induced GPCR coupling and its effect on anti-tumor activity by modulating immune response in HNSCC by using pre-clinical models. Methods: The anti-cancer effect of CBD was measured by cell proliferation, apoptosis and migration analysis. Next, TGF-α shedding assay was performed to identify the CBD-induced GPCR coupling with different cannabinoid receptors. The cannabinoid receptor GPR55 was subjected to silencing by siRNA and western blot analysis was performed to evaluate its role in activation of p38 MPAK pathway. Next, the anti-tumor immune response of CBD was evaluated by using an immunocompetent syngeneic C57BL/6 mEER RasG12D+/- HPV E6/E7 transformed tongue epithelial cell model that serves as a model for HPV+ HNSCC and the 4MOSC1 orthotopic, syngeneic, 4NQO (4-nitroquinoline-1 oxide)-induced murine oral tongue squamous cell carcinoma C57BL/6 mouse model that serve as HPV- HNSCC model as well as in immune-deficient Rag1 -/- and athymic nude mouse followed by measurement of immune cell infiltration by IHC analysis. Results: We found that 10μM of CBD treatment inhibited cell proliferation and migration in HNSCC cells by promoting apoptosis. Interestingly, we observed that CBD resulted in GPCR coupling for Gαi with GPR55 and Gαq/o and Gαq/s for CNR1, CNR2 and GPR55. Furthermore, we silenced GPR55 and observed that 10μM of CBD treatment inhibits the activation of p38 MAPK pathway whereas it promotes activation in non-silenced HNSCC cells. Next, we observed that treatment with 10μM of CBD significantly inhibited tumor growth compared to control in both HPV- and HPV+ models in immune-intact animals whereas, no significant difference in the tumor growth was observed in immune-deficient (Rag1 -/- and athymic nude) mouse, indicating role of CBD in modulating tumor immune microenvironment. We also observed CBD treatment enhanced CD4+ and CD8+ T cell infiltration into the primary tumors of HPV+ and HPV- mice models, implicating a downstream cytotoxic T cell response. Conclusions: Our study suggests that CBD promotes anti-tumor activity by modulating the immune microenvironment and interacts with GPR55 to activate intrinsic MAPK signaling pathway.

Digital manufacturing techniques and the in vitro biocompatibility of acrylic-based occlusal device materials

ObjectivesMaterial chemistry and workflow variables associated with the fabrication of dental devices may affect the biocompatibility of the dental devices. The purpose of this study was to compare digital and conventional workflow procedures in the manufacturing of acrylic-based occlusal devices by assessing the cytotoxic potential of leakage products.MethodsSpecimens were manufactured by 3D printing (stereolithography and digital light processing), milling, and autopolymerization. Print specimens were also subjected to different post-curing methods. To assess biocompatibility, a human tongue epithelial cell line was exposed to material-based extracts. Cell viability was measured by MTT assay while Western blot assessed the expression level of selected cytoprotective proteins.ResultsExtracts from the Splint 2.0 material printed with DLP technology and post-cured with the Asiga Flash showed the clearest loss of cell viability. The milled and autopolymerized materials also showed a significant reduction in cell viability. However, by storing the autopolymerized material in dH2O for 12 h, no significant viability loss was observed. Increased levels of cytoprotective proteins were seen in cells exposed to extracts from the print materials and the autopolymerized material. Similarly to the effect on viability loss, storing the autopolymerized material in dH2O for 12 h reduced this effect.Conclusions/Clinical relevanceBased on the biocompatibility assessments, clinical outcomes of acrylic-based occlusal device materials may be affected by the choice of manufacturing technique and workflow procedures.

Conditional knockout mouse model reveals a critical role of peroxiredoxin 1 in oral leukoplakia carcinogenesis

Peroxiredoxin 1 (Prx1) is an antioxidant protein that may promote the carcinogenesis in oral leukoplakia (OLK). To investigate the effect of Prx1 on the oral mucosal epithelium of OLK, we generated a Prx1 conditional knockout (cKO) mouse model. The mRNA and gRNA were generated using the clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) technique. An infusion cloning method was used to construct a homologous recombination vector. To obtain the F0 generation mice, fertilized eggs of C57BL/6J mice were microinjected with Cas9 mRNA, gRNA, and a donor vector. Polymerase chain reaction (PCR) amplification and sequencing were used to identify F1 generation mice. Using the cyclization recombination-enzyme-locus of the X-overP1 (Cre-loxP) system, we created a Prx1 cKO mouse model, and the effectiveness of the knockout was confirmed through immunohistochemistry. We examined the influence of Prx1 knockout on the occurrence of OLK in mice by constructing a model of tongue mucosa carcinogenesis induced by 4-nitroquinoline-1-oxide (4NQO). Prx1 modification was present in the F1 generation, as evidenced by PCR amplification and sequencing. Prx1flox/flox: Cre + mice exhibited normal growth and fertility. Immunohistochemical analysis revealed that tongue epithelial cells in Prx1flox/flox: Cre + mice displayed a distinct deletion of Prx1. An examination of the heart, liver, spleen, lung, and kidney tissues revealed no visible histological changes. Histological analysis showed a reduction in the occurrence of the malignant transformation of OLK in the tongue tissues of Prx1flox/flox: Cre + mice. Ki67 immunostaining showed that Prx1 knockout significantly inhibited cell proliferation in the tongue epithelial. Our research developed a conditional knockout mouse model for Prx1. The obtained results provide insights into the function of Prx1 in the development of oral cancer and emphasize its potential as a therapeutic target for precancerous oral lesions.

Pragmatic Return to Effective Dental Infection Control through Triage and Testing (PREDICT): an observational, feasibility study to improve dental office safety

BackgroundDuring the COVID-19 pandemic, there was a substantial interruption of care, with patients and workers fearful to return to the dental office. As dental practice creates a highly aerosolized environment, the potential for spread of airborne illness is magnified. As a means to increase safety and mitigate risk, pre-visit testing for SARS-CoV-2 has the potential to minimize disease transmission in dental offices. The Pragmatic Return to Effective Dental Infection Control through Testing (PREDICT) Feasibility Study examined the logistics and impact of two different testing mechanisms (laboratory-based PCR viral testing and point-of-care antigen testing) in dental offices.MethodsDental healthcare workers (DHCWs) and patients in four dental offices within the National Dental Practice-based Research Network participated in this prospective study. In addition to electronic surveys, participants in two offices completed POC testing, while participants in two offices used lab-based PCR methods to detect SARS-CoV-2 infection. Analysis was limited to descriptive measures, with median and interquartile ranges reported for Likert scale responses and mean and standard deviation for continuous variables.ResultsOf the total 72 enrolled, 28 DHCWs and 41 patients completed the protocol. Two patients (4.9%) tested positive prior to their visit, while 2 DHCWs (12.5%) tested positive for SARS-CoV-2 infection at the start of the study. DHCWs and patients shared similar degree of concern (69% and 63%, respectively) for contracting COVID-19 from patients, while patients feared contracting COVID-19 from DHCWs less (49%). Descriptive statistics calculations revealed that saliva, tongue epithelial cells, and nasal swabs were the most desirable specimen collection method; both testing (LAB and POC) protocols took similar amounts of total time to complete; and DHCWs and patients reported feeling more comfortable when both groups were tested.ConclusionsWhile a larger-scale, network study is necessary for generalizability of results, this feasibility study suggests that SARS-CoV-2 testing can be effectively implemented into dental practice workflows and positively impact perception of safety for DHCWs and patients. As new virulent infectious diseases emerge, preparing dental personnel to employ an entire toolbox of risk mitigation strategies, including testing, may have the potential to decrease dental practice closure time, maintaining continuity of dental care services for patients.Trial registrationClinicalTrials.gov: NCT05123742.

Assessment of Genotoxic Biomarker in Tongue and Buccal Mucosal Epithelial Cells of COVID-19 Patients: An Observational Study

Assessment of Genotoxic Biomarker in Tongue and Buccal Mucosal Epithelial Cells of COVID-19 Patients: An Observational Study

Abstract PR06: Cooperative HPV and YAP-mediated oncogenic reprogramming of oral progenitors into cancer stem cells at single cell resolution

Abstract Human papillomavirus-positive head and neck squamous cell carcinoma (HPV+HNSC) constitutes the most common HPV-driven malignancy in the United States, and yet the molecular and cellular mechanisms underlying carcinogenesis in HPV+HNSC remain poorly understood. The decades long latency period and incomplete penetrance linking oral HPV infection to HPV+HNSC suggest that oncogenic events beyond E6-E7 expression are required for carcinogenesis. YAP is a transcriptional co-activator that coordinates cell stemness, growth, proliferation, and survival. We previously identified alterations resulting in YAP activation as a potential oncogenic event in HNSC. We thus hypothesized YAP activation in the context of E6-E7 expression may represent a key tumor initiating event leading to HPV+HNSC. We developed a genetically engineered murine system to investigate the effects of local induction of YAP activation and E6-E7-expression in Keratin14+ (K14+) oral epithelial progenitor cells (OEPCs). In this system, intralingual administration of tamoxifen and dietary administration of doxycycline enables spatiotemporally controlled expression of E6-E7 and/or YAP. Employing this model, we identify combined E6-E7 and YAP activation (E+Y) as sufficient to induce highly penetrant carcinoma formation as early as 10 days after transgene induction. Immunofluorescent analyses showed E+Y resulted in the ectopic expansion of a mitotically active (Ki67+), stem-like (TP63+, SOX2+) epithelial cell population with invasive potential. Bulk RNAseq of microdissected tongue epithelia showed that E+Y uniquely induced transcriptional programs promoting cell cycle progression, epithelial cell plasticity, and acute inflammatory response, which were not observed upon sole activation of either the E6-E7 or YAP transgene. scRNAseq of transgene-induced tongue epithelial cell suspensions identified 8 distinct clusters, including previously defined physiologic oral epithelial cell states and novel transgene-associated clusters. E+Y resulted in the emergence of a unique epithelial cell cluster with transcriptional characteristics consistent with a cancer stem cell population, with enrichment in YAP, mTORC1, E2F, and MYC-driven programs, and pan-cancer cell states including cell cycle, hypoxia, squamous differentiation, and partial EMT. Implantation of E+Y cells resulted in tumor formation in 100% of NSG mice, indicating that E+Y drives the emergence of a cancer stem cell population. We next defined transcriptionally co-expressed modules driving E+Y tumor initiating capacity. Among TCGA-HNSC subjects, 8 of 12 modules were enriched in tumor compared to matched normal tissue. Interestingly, 3 modules (G1/S, cell migration, and focal adhesion) were associated with overall and disease-free survival. Our findings show YAP activation in the context of E6-E7 expression is sufficient for rapid carcinogenesis. We illuminate cellular processes driving malignant conversion at single cell resolution, and show enrichment of these processes in human HNSC exhibiting poor prognosis. Citation Format: Farhoud Faraji, Sydney I. Ramirez, Lauren M. Clubb, Kuniaki Sato, Paola Y. Anguiano Quiroz, William M. G. Galloway, Thomas S. Hoang, Kate Medetgul-Ernar, Pauline Marangoni, Alfredo A. Molinolo, Joseph A. Califano, Quinton Smith, Ophir D. Klein, Pablo Tamayo, J. Silvio Gutkind. Cooperative HPV and YAP-mediated oncogenic reprogramming of oral progenitors into cancer stem cells at single cell resolution [abstract]. In: Proceedings of the AACR-AHNS Head and Neck Cancer Conference: Innovating through Basic, Clinical, and Translational Research; 2023 Jul 7-8; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2023;29(18_Suppl):Abstract nr PR06.

Reduction of oral pathogens and oxidative damage in the CAL 27 cell line by Rosmarinus officinalis L. and Taraxacum officinale Web. extracts

Ethnopharmacological relevanceDandelion (Taraxacum officinale Web.) and rosemary (Rosmarinus officinalis L.) are treasured botanicals with a long usage history in traditional herbal practices worldwide. Dandelion was used to treat kidney, spleen, and liver disease, as well as cardiovascular disease, diabetes, and bacterial infections, whereas rosemary was used to treat pain, spasms, and to improve blood circulation. Aim of the studyThe aim of this study was to determine the influence of rosemary and dandelion leaves aqueous extracts on the human tongue epithelial carcinoma cell line (CAL 27) at the level of interaction between oral microbiota and tongue epithelial cells, genomic damage, and H2O2 - induced oxidative damage protection. Materials and methodsThe polyphenolic composition of the extracts was determined by spectrophotometric and HPLC analyses. After extract treatment, cytotoxic impact and ROS generation in CAL 27 cells were measured using the MTT assay and the 2',7'-dichlorofluorescein-diacetate (DCFH-DA) assay, respectively. Microdilutions were applied to investigate the antimicrobial and adhesive properties against representatives of the oral microbiota. The single-cell gel electrophoresis (comet assay) and cytokinesis-blocked micronucleus cytome assay (CBMN cyt) were used to detect induced genomic damages. ResultsBoth extracts increased the adhesion of the lactic acid bacteria L. plantarum but decreased the adhesion of the bacterial pathogens S. enterica serovar Typhimurium LT21 and E. coli K-12 MG1655 adhesion onto CAL 27 cells. 1 h treatment with 5x concentrated dandelion extract and 1x, 2.5x, and 5x of rosemary extract caused an increase in comet tail intensity. CBMN cyt results demonstrated a significant increase in micronucleus formation even at concentrations several times lower than the usual bioactive compound concentrations found in a cup of beverage, with higher concentrations also inducing cell apoptosis and necrosis. Rosemary extract showed a protective effect against H2O2 - induced oxidative damage by decreasing the apoptotic cell number, probably preventing mutations leading to tumor aggressiveness, invasion, and metastasis. ConclusionsBoth tested extracts demonstrated their usefulness in maintaining good oral bacteria balance and their protective capability as powerful antitumor agents by causing a protective apoptotic effect in tumor cell line already at the dosage of an average daily cup.

Oral Problems and Related Factors in Elderly COVID-19 Patients in an Intensive Care Unit: A Cross-Sectional Study

Objective: Elderly individuals were more affected by the COVID-19 pandemic. Many complications, changes in tongue epithelial cells, and salivary gland dysfunction are associated with COVID-19. This study aimed to explore oral problems and related factors in elderly COVID-19 patients receiving treatment in an intensive care unit. Material and Methods: This cross-sectional study was conducted with 86 individuals aged ≥ 65 years who received treatment for COVID-19 in the intensive care unit of a state hospital in western Turkey between May and November 2021. Patient’s demographic characteristics, chronic diseases, nutritional status, fluid intake, vital signs, and presence of secretion, intubation status, oxygen therapy, and blood biochemistry tests were evaluated. Eilers’ Oral Assessment Guide was used for oral assessment. The data were analysed using t-tests, one-way ANOVAs, Cox regression analysis, and Kaplan−Meier test. Results: The mean age of the participants was 72.93±7.91 years, and 60.5% of them were male. The mean length of stay in the intensive care unit was 8.55±6.54 days. Of the participants, 41.9% were intubated and 80.2% had at least one chronic disease. The mean Oral Assessment Guide score of the participants was 11.43±2.03, and 31.4% of them showed limited salivary secretion. There were significant differences between the mean Oral Assessment Guide score and length of stay, body mass index, intubation, continuous positive airway pressure treatment, presence of secretion, aspiration status, type of nutrition, and activated partial thromboplastin time value. Conclusion: Our results indicated that elderly COVID-19 patients, who were intubated, received continuous positive airway pressure treatment, had a body mass index of ≥ 35 kg/m2, received total parenteral nutrition, and had blood coagulation problems demonstrated an increased risk of deterioration of the oral mucous membrane as their length of stay in the intensive care unit increased.

Ascl1-expressing cell differentiation in initially developed taste buds and taste organoids.

Mammalian taste bud cells are composed of several distinct cell types and differentiated from surrounding tongue epithelial cells. However, the detailed mechanisms underlying their differentiation have yet to be elucidated. In the present study, we examined an Ascl1-expressing cell lineage using circumvallate papillae (CVP) of newborn mice and taste organoids (three-dimensional self-organized tissue cultures), which allow studying the differentiation of taste bud cells in fine detail ex vivo. Using lineage-tracing analysis, we observed that Ascl1 lineage cells expressed type II and III taste cell markers both CVP of newborn mice and taste organoids. However, the coexpression rate in type II cells was lower than that in type III cells. Furthermore, we found that the generation of the cells which express type II and III cell markers was suppressed in taste organoids lacking Ascl1-expressing cells. These findings suggest that Ascl1-expressing precursor cells can differentiate into both type III and a subset of type II taste cells.

Acanthus ilicifolius Methanolic Extract for Oral Candidiasis Treatment through Tongue Epithelial STAT3 and Cell Death Evaluation.

This study aimed to evaluate the effects of topical application of Acanthus ilicifolius methanolic extract on signal transducer and activator of transcription 3 (STAT3) expression and tongue epithelial cell death caused by oral candidiasis under immunosuppressive conditions. To obtain the oral candidiasis model, 20 healthy male Rattus norvegicus (Wistar) rats were administered oral dexamethasone and tetracycline for 14 days and oral candidiasis was induced with Candida albicans (ATCC-10231) 1 McFarland. They were then randomized into four groups-immunosuppression (K-), oral candidiasis (K + ), nystatin treatment (P1), and 20% A. ilicifolius methanolic extract treatment (P2) and were treated for 14 days. Histological analyses of cell death and candida invasion and immunohistochemical analysis of STAT3 in epithelial cells were performed. Epithelial cell death data were analyzed using one-way analysis of variance (ANOVA) and the post hoc Games-Howell test (p < 0.05) and STAT3 expression with one-way ANOVA and the post hoc least significant difference test (p < 0.05). Cell death was significantly different between K- and K+ and between K+ and P1 and P2 (p < 0.05); there were no significant differences between K- and P1 and P2 and between P1 and P2 (p > 0.05). STAT3 expression was significantly different between K- and P1 and P2 and between K+ and P1 and P2 (p < 0.05), but there were no significant differences between K+ and K- and between P1 and P2 (p > 0.05). Topical administration of A. ilicifolius methanol extract increased STAT3 expression and decreased tongue epithelial cell death caused by oral candidiasis.

Preparation, Characterization, and Evaluation of Cytotoxicity of Fast Dissolving Hydrogel Based Oral Thin Films Containing Pregabalin and Methylcobalamin

The oral availability of many drugs is problematic due to the pH of the stomach, enzymes, and first-pass effects through the liver. However, especially geriatric, pediatric, bedridden, or mentally handicapped patients and those with dysphagia have difficulty swallowing or chewing solid dosage forms. Oral Thin Films (OTFs) are one of the new drug delivery systems that can solve these problems. Pregabalin (PG) and Methylcobalamin (MC), which are frequently preferred for pain originating in the central nervous system, were brought together for the first time using OTF technology in this study. In this study, a quantification method for PG and MC was developed and validated simultaneously. Optimum formulations were selected with organoleptic and morphological controls, moisture absorption capacity, swelling capacity, percent elongation, foldability, pH, weight variability, thickness, disintegration time, and transparency tests on OTFs prepared by the solvent pouring method. Content uniformity, dissolution rate, determination of release kinetics, SEM, XRD, FT-IR, DSC, long-term stability, and cytotoxicity studies on the tongue epithelial cell line (SCC-9) were performed on selected OTFs. As a result, OTFs containing PG-MC, which are non-toxic, highly flexible, transparent, compatible with intraoral pH, with fast disintegration time (<30 s), and acceptable in taste and appearance, have been developed successfully.

Bisphosphonate affects the behavioral responses to HCl by disrupting farnesyl diphosphate synthase in mouse taste bud and tongue epithelial cells.

Little is known about the molecular mechanisms underlying drug-induced taste disorders, which can cause malnutrition and reduce quality of life. One of taste disorders is known adverse effects of bisphosphonates, which are administered as anti-osteoporotic drugs. Therefore, the present study evaluated the effects of risedronate (a bisphosphonate) on taste bud cells. Expression analyses revealed that farnesyl diphosphate synthase (FDPS, a key enzyme in the mevalonate pathway) was present in a subset of mouse taste bud and tongue epithelial cells, especially type III sour-sensitive taste cells. Other mevalonate pathway-associated molecules were also detected in mouse taste buds. Behavioral analyses revealed that mice administered risedronate exhibited a significantly enhanced aversion to HCl but not for other basic taste solutions, whereas the taste nerve responses were not affected by risedronate. Additionally, the taste buds of mice administered risedronate exhibited significantly lower mRNA expression of desmoglein-2, an integral component of desmosomes. Taken together, these findings suggest that risedronate may interact directly with FDPS to inhibit the mevalonate pathway in taste bud and tongue epithelial cells, thereby affecting the expression of desmoglein-2 related with epithelial barrier function, which may lead to alterations in behavioral responses to HCl via somatosensory nerves.

Establishment of a chemical tongue injury-recovery mouse model

Tongue epithelium is one of the most proliferative and regenerative epithelia in our body. However, tongue stem cell research is hampered partly by the lack of optimal animal models to study tongue injury, repair, and regeneration. Here, we establish a novel chemically induced tongue injury-recovery mouse model. Focal application of sodium hydroxide for a limited time led to the denudation of suprabasal layers, leaving the basal layer. Time course study revealed that tongue epithelial cells robustly proliferate over one week after the tongue injury. Importantly, we demonstrated that our novel mouse model could be employed in the lineage tracing of the tongue stem cells under the injury and repair process and further showed that tongue stem cells proliferate faster and generate larger clones in the injury condition than in the steady state condition. Our data indicate the development of a novel chemically induced tongue injury-recovery mouse model for tongue stem cell research, which will significantly facilitate the preclinical study for the pathogenesis and treatment of caustic ingestion.

M6A modification-mediated BATF2 suppresses metastasis and angiogenesis of tongue squamous cell carcinoma through inhibiting VEGFA

ABSTRACT The aim is to explore the underlying mechanism of basic leucine zipper ATF-like transcription factor 2 (BATF2) in tongue squamous cell carcinoma (TSCC). The expression of BATF2 in TSCC tissues and corresponding adjacent normal TSCC tissues, human TSCC cell lines (SCC-15 and CAL-27) and human normal tongue epithelial cells NTEC was detected. Then, SCC-15 cells with stable BATF2 knockdown and CAL-27 cells with BATF2 overexpression were established to investigate the functional effect of BATF2 on TSCC. Thereafter, the effect of BATF2 on TSCC angiogenesis and BATF2 m6A methylation was also examined. BATF2 was significantly downregulated in TSCC tissues and cell lines, and BATF2 overexpression could suppress growth, metastasis and angiogenesis of TSCC. Mechanistically, vascular endothelial growth factor A (VEGFA) was identified as a downstream gene of BATF2, and it was confirmed that BATF2 suppressed growth, metastasis and angiogenesis of TSCC via inhibiting VEGFA. In addition, the N6-methyladenosine (m6A) modification of BATF2 mRNA mediated by METTL14 suppressed its expression in TSCC. METTL14/BATF2 axis could serve as a novel promising therapeutic candidate against angiogenesis for TSCC.

Cytotoxic activity of strawberry tree (Arbutus unedo L.) honey, its extract, and homogentisic acid on CAL 27, HepG2, and Caco-2 cell lines.

Strawberry tree (Arbutus unedo L.) honey (STH), also known as “bitter honey”, is a traditional medicine widely used in the Mediterranean area. Regardless of geographical origin, it usually has a very high content of phenolic compounds and strong antioxidant capacity. Yet, little is still known about the effects of STH, its phenolic extract (STHE), and its main bioactive compound – homogentisic acid (HGA) – at the cell level. The aim of this study was to estimate total phenolic content, DPPH radical scavenging activity, and ferric reducing antioxidant power of STH made in Croatia and investigate cytotoxic and pro-oxidative effects of STH, STHE and HGA on three human cell lines: tongue squamous cell carcinoma (CAL 27), hepatocellular carcinoma (HepG2), and epithelial colorectal adenocarcinoma cells (Caco-2) cells. These substances were tested at four concentrations (0.5–5× average human daily intake of STH) and over 30 min and 1 and 2 h. Croatian STH had a total phenolic content of 1.67 g gallic acid equivalents (GAE) per kg of honey, DPPH radical scavenging activity of 2.96 mmol Trolox equivalents (TE) per kg of honey, and ferric reducing antioxidant power (FRAP) of 13.5 mmol Fe2+ per kg of honey. Our results show no clear and consistent time- or concentration-dependent cytotoxicity in any of the cell lines. ROS levels in all the three cell types at almost all exposure times were not significantly higher than control. The most important observation is that the tested substances have low cytotoxicity and high biocompatibility, regardless of concentration, which is a good starting point for further research of their biological effects in other models.

SARS-COV2 Associated Oral Lesions- A Review

Introduction: COVID-19 disease is caused by SARS-CoV-2 virus and it was declared pandemic by World Health Organization (WHO) on March 11,2020. The coronavirus infection has an affinity for ACE2 receptors and by attaching to them, the virus enters the host cells. Along with many body organs like lungs, kidney, liver, upper respiratory tract, nervous system, skeletal muscles, ACE2 concentration is also found in abundance in epithelial cells of tongue and salivary glands.&#x0D; Materials and Methods: Recent studies, researches, documents and case reports published in the world medical literature in the year 2020-2021 were searched and documented in our study. The search engines used were PUBMED, google scholar, WEB OF SCIENCE etc.&#x0D; Results: Dysgeusia, xerostomia, sore throat, aphthous and herpetiform ulcers, candidiasis, enanthema, Kawasaki like lesions were the most common among various oral manifestations. Others includes plaque like changes, gingival inflammation, necrotizing gingivitis, erythema -multiforme, angina-bullosa like lesions, Melkersson-Rosenthal Syndrome, Oral mucormycosis etc.The sites of infection mainly include tongue, gingiva, hard and soft palate, buccal and labial mucosa etc.&#x0D; Conclusion: The etiopathogenesis of such lesions cannot be directly corelated with COVID-19 and factors such as stress, immunosuppression, co-infections, secondary lesions, opportunistic infections, systemic diseases, poor oral hygiene etc. must be considered. Management of stress is an important factor. In this review article various oral lesions are discussed in COVID-19 infection states in detail. The importance of earliest diagnosis of oral lesions is to be kept in mind to prevent further complications.

The Repurposed ACE2 Inhibitors: SARS-CoV-2 Entry Blockers of Covid-19.

The highly infectious disease COVID-19 is induced by SARS-coronavirus 2 (SARS-CoV-2), which has spread rapidly around the globe and was announced as a pandemic by the World Health Organization (WHO) in March 2020. SARS-CoV-2 binds to the host cell’s angiotensin converting enzyme 2 (ACE2) receptor through the viral surface spike glycoprotein (S-protein). ACE2 is expressed in the oral mucosa and can therefore constitute an essential route for entry of SARS-CoV-2 into hosts through the tongue and lung epithelial cells. At present, no effective treatments for SARS-CoV-2 are yet in place. Blocking entry of the virus by inhibiting ACE2 is more advantageous than inhibiting the subsequent stages of the SARS-CoV-2 life cycle. Based on current published evidence, we have summarized the different in silico based studies and repurposing of anti-viral drugs to target ACE2, SARS-CoV-2 S-Protein: ACE2 and SARS-CoV-2 S-RBD: ACE2. This review will be useful to researchers looking to effectively recognize and deal with SARS-CoV-2, and in the development of repurposed ACE2 inhibitors against COVID-19.

EZH2 knockout in oral cavity basal epithelia causes more invasive squamous cell carcinomas.

Oral squamous cell carcinoma (oral SCC) is an aggressive disease and despite intensive treatments, 5-year survival rates for patients have remained low in the last 20 years. Enhancer of zeste homolog 2 (EZH2), part of polycomb repressive complex 2 (PRC2), is highly expressed in human oral SCC samples and cell lines and has been associated with greater epithelia-to-mesenchymal transition (EMT), invasion and metastasis. Here, we developed a tamoxifen-regulated, transgenic mouse line (KcEZH2) in which EZH2 is selectively knocked out (KO) in some tongue epithelial basal stem cells (SCs) in adult mice. EZH2 KO SCs do not show the H3K27me3 mark, as assessed by double-label immunofluorescence. We used this mouse line to assess EZH2 actions during oral tumorigenesis with our immunocompetent 4-nitroquinoline 1-oxide model of oral SCC. We report that higher percentages of mice with invasive SCCs and high-grade neoplastic lesions are observed in mice containing EZH2 KO SCs (KcEZH2-2TΔ and KcEZH2-5TΔ mice). Moreover, EZH2 expression does not correlate with the expression of markers of invasive SCCs. Finally, EZH2 KO cells that are E-cadherin+ are present at invasion fronts infiltrating underlying muscle tissue. Our findings indicate that the knockout of EZH2 in basal SCs of tongue epithelia results in more aggressive carcinomas, and this should be considered when targeting EZH2 as a therapeutic strategy.

Morphological alterations in tongue epithelial cells infected by SARS‐CoV‐2: A case–control study

ObjectiveThe aim of the present case–control study was to evaluate the morphological aspects of the epithelial cells from the dorsum of the tongue and the expression of the SARS‐CoV‐2 Spike protein in these cells, in patients with and without COVID‐19 infection.Methods24 individuals with at least one symptom of COVID‐19 were recruited among inpatients from Hospital Universitário Pedro Ernesto (Rio de Janeiro, Brazil). 14 patients who tested positive for COVID‐19 by RT‐PCR were included in the case group, and 10 patients who tested negative were included in the control group. Cytological smears from the dorsum of the tongue were obtained from all patients and analyzed using immunohistochemistry directed against SARS‐CoV‐2‐Spike protein. Morphological changes in epithelial cells were analyzed using light microscopy.ResultsImmunohistochemistry showed that 71% of the COVID‐19 patients presented epithelial cells positive for the presence of the SARS‐CoV‐2 Spike protein, and all cells coming from patients in the control group were negative. Cytological analysis showed significant differences when comparing epithelial cells from COVID‐19‐positive and COVID‐19‐negative patients.ConclusionCOVID‐19 may generate dimensional changes in tongue epithelial cells; however, further studies are necessary to understand how this happens.

Effect of Sonic hedgehog on the proliferation in mouse tongue epithelial cells.

To investigate Sonic hedgehog (Shh) effects on proliferation and apoptosis of tongue epithelial cells in embryonic and ageing mice. Embryonic day 13.5 (E13.5), E14.5, E16.5 and postnatal day 0.5 (PN0.5) K14-Cre;Shhfl/fl mice, and E14.5, E16.5, PN0.5, PN90.5 and postnatal 1.5years (PN1.5Y) wild-type (Wt) mice were employed. Scanning electron microscopy, haematoxylin-eosin and immunohistochemistry were performed. Gel beads containing exogenous Shh protein were embedded in the tongue of PN90.5 and PN1.5Y Wt mice. Three days later, 5-bromodeoxyuridine (BrdU), proliferating cell nuclear antigen (PCNA) immunohistochemical and TUNEL staining were performed. The number of fungiform papillae was decreased with age. The numbers of BrdU- and PCNA-positive cells were the highest at PN0.5 and the lowest at PN1.5Y. Compared with Wt mice, K14-Cre;Shhfl/fl mice had decreased PCNA-positive cells in the epithelium, a smaller tongue volume, and fewer papillae at PN0.5. At E14.5, the number of BrdU-positive cells was decreased in K14-Cre;Shh fl/fl mice. At PN1.5Y, the number of apoptotic cells in tongue tissue exposed to Shh protein was less than that in the BSA group and the numbers of BrdU- and PCNA-positive proliferating cells were increased. Shh maintains cell proliferation and reduces apoptosis during tongue development and ageing.