Detection and consistency of mucosal fluid T lymphocyte phenotypes and their relationship with blood, age and gender.

Innate and adaptive immune responses at mucosal surfaces play a role in protection against most infectious diseases. However, the relative importance either of mucosal versus systemic, or of cellular versus humoral immunity in protection against such infections remains unclear. We aimed to determine the relative percentages and reproducibility of detection of five major T lymphocyte phenotypes in stimulated whole mouth fluid (SWMF); to compare matched mucosal and blood phenotypes; to evaluate the consistency of phenotypes in SWMF over time; and to determine any associations with age or gender. Peripheral blood and SWMF samples were collected from 194 participants and sequential concomitant samples were collected from 27 of those and from 12 subjects living with HIV. CD3, CD4, CD8, Th1 and Th2 T lymphocyte phenotypes were determined by FACS. All the five T lymphocyte phenotypes were detected consistently by FACS in PBMC and SWMF with experimental replicates (N = 10; PBMC CV: 3-30%; SWMF CV: 12-36%). In longitudinal samples detection rates were reproducible in both fluids but variations were higher in SWMF (CV: 23-79.6%) than PBMC (CV: 9.7-75%). Statistically significant correlations of the percentages of all the T lymphocyte phenotypes except CD8 was seen between the two fluids. In PBMCs a negative correlation with age was found with CD3, CD4 and CD8 phenotypes, whilst a positive correlation was found in both SWMF and PBMC with the Th2 phenotype. CD3, CD4 and CD8 phenotypes in SWMF and PBMCs from an HIV-positive cohort were not significantly correlated in contrast with the HIV-negative controls. Our study provides a robust FACS protocol for the detection of the five major T lymphocyte phenotypes in SWMF which should prove useful for research with other mucosal fluids.

Clinical aspects of oral cancer and potentially malignant disorders in South and Southeast Asia.

BACKGROUND: Oral cancer and Oral Potentially Malignant Disorders (OPMD) are major health problems in South and Southeast Asia. AIMS: To describe and discuss the clinical aspects of Oral Cancer and OPMD in South and Southeast Asia. MATERIALS AND METHODS: Literature review of concepts and data over the last four decades. DISCUSSION: Asian countries account for about two-thirds of new cases of oral cancer (OC) globally, with the highest burden in the South and Southeast Asian countries, including Pakistan and India. Habits, dietary patterns, socioeconomic status, and access to routine dental care play a crucial role in defining the demographics and clinical presentation of OC in these regions and significantly influence the morbidity and mortality of the disease. This region sees the use of different types of tobacco with or without areca nut (AN), such as pan masala, gutka, gul, snuff, mawa, and mishri. Tobacco use is high among men in Sri Lanka, Myanmar, Maldives, Bangladesh, Nepal, India and Bhutan. Areca nut is the fourth most common addictive substance globally and is frequently used in South and Southeast Asian countries, including Southeast China, Hainan Island, India, Taiwan, and the Pacific Islands, and immigrants from these regions in Africa, Europe, and North America. The use of these products results in mucosal alterations with varied clinical presentation of Oral Potentially Malignant Disorders (OPMDs) and OC. We discuss here the different types of OPMD and OC, the diagnostic aids and their relevance in clinical practice, and factors that influence their prognosis.

E-cadherin Expression in Premalignant Lesions, Premalignant Conditions, Oral Squamous Cell Carcinoma, and Normal Mucosa: An Immunohistochemical Study.

Background Oral squamous cell carcinoma (OSCC) is a multi-step process. Epithelial-mesenchymal transition (EMT) is an important step in the progression of OSCC. One of the components that influence EMT is E-cadherin. The aim of this study was to determine the expression of E-cadherin in oral submucous fibrosis (OSMF), various grades of epithelial dysplasia, OSCC, and to compare it with the expression in the normal mucosa. Material and methods E-cadherin immunohistochemical detection was done using a monoclonal antibody of clone EP-6TM and the PolyExcel HRP/DAB chromogen detection system. A total of 100 samples, were divided into four groups, which included epithelial dysplasia (group 2) (30 cases), oral submucous fibrosis (group 3) (OSMF-30 cases), and oral squamous cell carcinoma (group 4) (OSCC-30 cases), which was compared with normal mucosa (group 1) (10 cases). The positive control used for E-cadherin was ductal breast carcinoma. Results All the cases of normal mucosa, epithelial dysplasia, and OSMF showed positivity for E-cadherin expression. In OSCC, 97% of cases expressed E-cadherin except one case. Out of 30 cases of epithelial dysplasia, 53% of mild epithelial dysplasia had a moderate intensity of expression and 75% had a mild intensity of E-cadherin expression. In moderately differentiated OSCC, 82% of cases showed mild intensity. Tissue localization of the E-cadherin stain in the basal layer decreased from normal mucosa to grades of epithelial dysplasia and OSCC. The pattern of E-cadherin staining in all the cases of group I, group II, and group III was membranous. In 97% of OSCC cases, both membranous and cytoplasmic staining were seen. Conclusion E-cadherin expression was reduced in increasing grades of epithelial dysplasia, OSCC, and OSMF compared to that of normal mucosa. E-cadherin expression is reduced as the lesions progress to malignancy. Hence, E-cadherin can be considered a surrogate marker of malignancy.

Expression of CD44 in Head and Neck Squamous Cell Carcinoma-An In-Silico Study.

Introduction CD44, a multistructural and multifunctional transmembrane glycoprotein, is a promising cancer stem cell (CSC) marker that regulates the properties of CSCs, including self-renewal, tumor initiation, and metastasis, and confers resistance to chemotherapy and radiotherapy. The aim of the present study was to evaluate the gene and protein expression of CD44 and explore its prognostic value in head and neck squamous cell carcinoma (HNSCC). Methodology The present observational study employs computational tools for analysis. The Cancer Genome Atlas Head-Neck Squamous Cell Carcinoma dataset (520 primary HNSCC and 44 normal tissues) from the University of Alabama at Birmingham Cancer platform was used to study the association of CD44 mRNA transcript levels with various clinicopathological characteristics of HNSCC including age, gender, tumor grade, tumor stage, human papillomavirus (HPV) status, p53 mutation status, and overall survival. The CD44 protein expression in HNSCC and normal tissues was ascertained using the National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium Head-and-Neck cancer dataset (108 primary HNSCC and 71 normal tissues). Results CD44 mRNA transcript and protein expression levels were significantly higher in HNSCC tissues than in normal tissues, and high CD44 expression was correlated with poor survival. CD44 was upregulated in Stage 1 and Grade 2 HNSCC compared with other stages and grades. Overexpression of CD44 was observed in HPV-negative and TP53-positive mutant status in HNSCC. Conclusion The pleiotropic roles of CD44 in tumorigenesis urge the need to explore its differential expression in HNSCC. The study concludes that CD44 can be a potential diagnostic and prognostic biomarker for HNSCC and offer new molecular targets for CD44-targeted therapy for cancer management.

Do Clear Cell Changes in Oral Squamous Cell Carcinoma Warrant It Being Recognised as a Variant?

Histological variants of oral squamous cell carcinoma (OSCC) include verrucous, basaloid, spindle cell, adenosquamous, papillary, and acantholytic types. Clear-cell changes in OSCC are rare. We report a case of a 65-year-old male Chinese patient who presented with a swelling in the lower-left mandible for three weeks, causing ill-fitting of his lower denture and an ulcer on the floor of the mouth. Histologically, the lesion showed a dense proliferation of malignant tumor cells arranged in islands and sheets consisting of squamoid cells intermixed with signet ring-shaped clear cells. The clear cells were negative for mucicarmine, periodic acid Schiff (PAS), periodic acid Schiff-diastase (PAS-D), and alcian blue (AB). Immunohistochemistry showed the tumor cells were immuno-positive for cytokeratin (CK) and p63, but CK7, CK20, and S100 were immuno-negative. Therefore, a metastatic tumor in the oral cavity was suggested. However, the CT scan did not show any primary tumors in other sites. Histopathologically, the surgical specimen showed signet-ring-shaped clear cells in the stroma with squamoid cells invading the underlying connective tissue from the surface epithelium, suggesting a diagnosis of clear cell changes in OSCC. Follow-up showed recurrent OSCC at the base of the tongue with lymph node metastasis and distant metastasis in the lung. Only a few cases of clear-cell changes have been reported, with most having a poor prognosis. This case report adds one more case of clear cell changes in the OSCC with a poor prognosis. We reviewed the literature to understand their clinical behavior. Due to the rarity of its (clear cell changes) occurrence, further research is required in order to obtain a better understanding of the clinical behavior and prognosis of these clear cell changes seen in OSCC.

Immunohistochemical biomarkers in oral submucous fibrosis: A scoping review.

INTRODUCTION: This scoping review was done to study the immunohistochemical biomarkers involved in the pathogenesis and malignant transformation of oral submucous fibrosis (OSF), in literature published from 2010 to 2021. METHOD: The protocol was adapted from the Joanna Briggs Institute Reviewer's Manual (2017), and reported according to the PRISMA guidelines for Scoping Reviews. RESULTS: Eighty-six studies included in this review reported 84 immunohistochemical (IHC) biomarkers in OSF: 10 epithelial markers, 28 connective tissue markers, 22 proliferative markers, and 24 other biomarkers that are transcription factors, cancer stem cell markers, cell signaling markers, proteins, and enzymes. The commonly reported IHC biomarkers were alpha-smooth muscle actin (α-SMA) and E-cadherin (seven articles each) followed by vascular endothelial growth factor (VEGF) and CD34 (six articles each), p53, p63, and Ki67 (five articles each). α-SMA, Ki67, CD105, and hTERT were significantly increased in oral squamous cell carcinoma arising in a background of OSF (OSCC-OSF) compared with OSF and normal subjects. CONCLUSION: The identified surrogate IHC biomarkers reported in OSF in this scoping review require validation with long-term prospective studies to facilitate early diagnosis, for use in risk assessment, and plan appropriate treatment for OSF in clinical practice. Open Science Framework ID: osf.io/epwra.

Decoding the link associated with areca nut chewing and cardiovascular disease using hsCRP as a biomarker.

Background: Chewing of areca nut is associated with various oral and systemic ill effects. The deleterious oral effects of areca nut have been widely documented to range from reactive lesions, periodontal health compromise to oral pre-cancer and cancerous states and have been described widely in the literature. The reported systemic effects of areca nut chewing are relatively less documented but reported in literature. Areca nut chewing may predispose to cardiovascular disease due to a systemic inflammatory response from the elevated levels of circulating inflammatory mediators. High-sensitivity C-reactive protein (hsCRP) is a systemic inflammatory biomarker to assess the risk of cardiovascular disease. Materials and Methods: This cross-sectional, observational study was conducted among areca nut chewers (n = 50) and non-chewers (n = 50). Areca nut chewers were considered as chewers/cases. Blood samples were collected from the participants and hsCRP levels were studied. The data were analysed using SPSS software, version 21 for statistical significance. Chi-square test was used to compare categorical variables. Mann-Whitney U test was done to analyse continuous variables. The level of statistical significance was set at P value < 0.05. Results: hsCRP mean was higher among the chewers (2.3 ± 3.7) compared to non-chewers (0.9 ± 1.3). The difference in hsCRP levels between the study group was statistically significant (P = 0.002). Conclusion: This study demonstrates the link between areca nut and cardiovascular disease using hsCRP as biomarker.

Evaluation of stress among dental undergraduates and postgraduates using salivary cortisol as a biomarker.

Background: Stress is a non-specific response of the body to any demand. Professional education has a certain level of stress inherent in it. Dental students have been reported in the literature to experience more stress than medical students. Objective: To evaluate the stress levels of dental students: first-year undergraduates, house surgeons, and final-year postgraduates, using salivary cortisol as a stress biomarker. Materials and Methods: Dental students above 18 years of age and without any systemic illness were included in the study. Those students with a drug history of corticosteroids, anti-depressants, and oral contraceptives were excluded from the study. Female participants experiencing pre-menstrual symptoms, pregnancy, and familial stressor issues were excluded from the study. First-year undergraduates, house surgeons, and final-year postgraduates were enrolled in the study. Unstimulated whole saliva was collected in the morning and evening between 9:00 and 10:00 am and between 3:00 and 4:00 pm. Salivary cortisol levels were assessed by chemiluminescence immuno-assay (CLIA). Results: Paired sample t-test was used to compare the diurnal variation among morning and afternoon samples within the groups. The salivary cortisol levels were found to be significant within the groups. One-way ANOVA was used to compare the salivary cortisol levels between the groups. The salivary cortisol levels within the groups were not statistically significant. Conclusion: Salivary cortisol is a non-invasive biomarker of stress. Postgraduates had higher salivary cortisol levels than other study groups.

Implications in the quantification of SARS-CoV2 copies in concurrent nasopharyngeal swabs, whole mouth fluid and respiratory droplets.

OBJECTIVE: Association of SARS-CoV2 burden in the aerodigestive tract with the disease is sparsely understood. We propose to elucidate the implications of SARS-CoV2 copies in concurrent nasopharyngeal swab (NPS), whole mouth fluid (WMF) and respiratory droplet (RD) samples on disease pathogenesis/transmission. METHODS: SARS-CoV2 copies quantified by RT-PCR in concurrent NPS, WMF and RD samples from 80 suspected COVID-19 patients were analysed with demographics, immune response and disease severity. RESULTS: Among the 55/80 (69 %) NPS-positive patients, SARS-CoV2 was detected in 44/55 (80 %) WMF (concordance with NPS-84 %; p = 0.02) and 17/55 (31 %) RD samples. SARS-CoV2 copies were similar in NPS (median:8.74 × 10^5) and WMF (median:3.07 × 10^4), but lower in RD (median:3.60 × 10^2). The 25-75 % interquartile range of SARS-CoV2 copies in the NPS was significantly higher in patients who shed the virus in WMF (p = 0.0001) and RD (p = 0.01). Multivariate analyses showed that hospitalized patients shed significantly higher virus copies in the WMF (p = 0.01). Hospitalized patients with more severe disease (p = 0.03) and higher IL-6 values (p = 0.001) shed more SARS-CoV2 virus in the RD. CONCLUSIONS: WMF may be used reliably as a surrogate for diagnosis. High copy numbers in the NPS probably imply early disease onset, while in the WMF and RD may imply more severe disease and increased inflammation.

Study of expression of endoglin (CD105) in oral squamous cell carcinoma.

Context: Oral cancer is the 8th most common cancer in the world. An important feature of carcinogenesis is angiogenesis. Endoglin is a powerful marker of neovascularization in solid malignancies. This study was done to ascertain its role as an indicator of metastasis and prognosis. Aim: This study aimed to evaluate and compare the expression of endoglin (CD105) in metastatic primary tumor, lymph node of the metastasized tumor, nonmetastatic primary tumor and in normal buccal mucosa immunohistochemically. Settings and Design: The total sample size comprised 45 formalin-fixed paraffin-embedded tissue blocks, n = 10 metastasized primary tumor, n = 10 lymph nodes of metastasized primary tumor, n = 20 nonmetastasized oral squamous cell carcinoma and n = 5 normal buccal mucosa were studied. Subjects and Methods: Immunohistochemistry for endoglin was performed and microvessel density (MVD) was determined by hot spot method. Microvessel density was compared between the groups. Statistical Analysis: Statistical analysis was used using one-way ANOVA. P < 0.05 was statistically significant. Results: Endoglin expression in metastatic cases (0.68 + 0.10) was higher than nonmetastatic cases (0.45 + 0.20) and the difference was statistically significant (P = 0.002). Conclusion: This study shows that presence of endoglin determines the metastatic potential of the tumor and its prognosis, thus, could be considered as a potential target of therapy.

Study of Hypoxia-inducible factor-2α expression in the malignant transformation of Oral submucous fibrosis.

CONTEXT: Hypoxia-inducible factor (HIF)-2α is overexpressed in primary and metastatic human cancers, whose expression is correlated with tumor angiogenesis and patient mortality. HIF plays a role in the progression of fibrosis in oral submucous fibrosis (OSF). AIM AND OBJECTIVE: The aim is to study and compare the expression of HIF-2α in OSF (a), oral squamous cell carcinoma (OSCC) with areca nut usage (b), OSCC without areca nut usage (c) and normal mucosa (d) by immunohistochemistry. SUBJECTS AND METHODS: Immunohistochemical detection of HIF-2α was done on 51 samples, which included 11 cases (a), 15 cases (b), 15 cases (c) and the expression was compared with that of (d). STATISTICAL ANALYSIS: Data were analyzed using the SPSS™ software (ver. 21.0). Chi-square test and kappa analysis were performed to compare the intensity of staining between the groups and for inter-observer agreement, respectively. Value of P ≤ 0.05 was considered statistically significant. The mean labeling index between the groups was studied by the Kruskal-Wallis test. RESULTS: All the cases of (d), (a), (b) and (c) showed HIF-2α expression (P = 0.329). About 13% cases of (c) showed intense expression (P = 0.406) and 27% of (a) cases showed expression only in the connective tissue (P = 0.023). The number of positively stained nuclei in both (b and c) cases reduced as the tumor progression was from well to poorly differentiated. CONCLUSION: Areca nut initiates fibrosis and subsequent hypoxia in OSF which triggers HIF-2α expression in the epithelium. HIF-2α could be a surrogate marker for cancer initiation and progression.

Intra-observer and inter-observer variability in two grading systems for oral epithelial dysplasia: A multi-centre study in India.

BACKGROUND: The presence and grading of oral epithelial dysplasia (OED) are considered the gold standard for predicting the malignant risk of oral potentially malignant disorders. However, inter-observer and intra-observer agreement in the context of reporting on OED grading has been reputedly considered unreliable. METHODS: We undertook a multi-centre study of six Indian oral pathologists to assess variations in reporting OED using the World Health Organization (WHO; 2005) system and also the recently introduced binary system. The observer variability was assessed with the use of kappa statistics. RESULTS: The weighted kappa intra-observer agreement scores improved (κw  = 0.5012) on grouping by two grades as no and mild dysplasia versus moderate and severe dysplasia compared to binary grading system (κ = 0.1563) and WHO grading system (κw  = 0.4297). Poor to fair inter-observer agreement scores were seen between the principal investigator (PI) and the other five observers using the WHO grading system (κ = 0.051-0.231; κw  = 0.145 to 0.361; 35% to 46%) and binary grading system (κ = 0.049 to 0.326; 50 to 65%). CONCLUSIONS: There is considerable room for improvement in the assessment of OED using either system to help in standardised reporting. The professional pathology organisations in India should take steps to provide external quality assessment in reporting OED among oral and general pathologists who are engaged in routine reporting of head and neck specimens.

Areca nut use disorder: A dynamic model map.

PROBLEM: Areca nut (AN) chewing is common among Southeast Asian population. Use of AN products (with or without tobacco) have a multifaceted effect on physical health, especially on cardiovascular, nervous, gastrointestinal, metabolic, respiratory, and reproductive systems. AN is a known group 1 carcinogen and carries addictive potential. Varying degrees of AN-related substance use disorder (SUD) have been reported among AN chewers. There is a lacuna in awareness of the health risk of AN use, prevention, and cessation programs among AN users, particularly in those who have developed SUD. EXISTING LACUNAE: The dynamic interaction of factors that promote AN use and later the risk of developing SUD at individual and community level has not been studied in depth. Understanding of the bio-psycho-socio-economic-cultural factors is necessary to identify the factors that prelude, promote, and reinforce AN usage. For managing AN-related conditions, including the several systemic disorders, there is a knowledge lacunae, among health care providers with respect to the pathophysiology of AN-related health issues, SUD, and nonavailability of structured, evidence-based cessation protocols. SOLUTIONS/RECOMMENDATIONS: This manuscript presents a model-map to study the dynamics of AN use and the impact of AN on health and health care system at individual as well as community level. The model proposed can help the health policymakers to create evidence-based awareness and cessation protocols for AN.

Characterization of oral fibroblasts: An in vitro model for oral fibrosis.

BACKGROUND: Oral submucous fibrosis (OSMF) is a chronic debilitating condition of the oral mucosa that has been classified as a potentially malignant disorder with a malignant transformation rate of 2%-8%. Several in vitro and in vivo experiments have been performed to formulate a treatment modality for OSMF, yet no ideal in vitro primary oral fibroblast model has been developed. AIM: To establish an in vitro primary oral fibroblast model. SETTING AND DESIGN: In vitro laboratory setting. MATERIALS AND METHODOLOGY: Primary cell culture protocol was performed after obtaining normal oral tissue. Karyotyping was performed to rule out chromosomal abnormalities. Immunofluorescence staining was carried with a panel of fibroblast-specific markers (vimentin, phalloidin, transforming growth factor-ß receptor 1 [TGFßR1] and s100a4) and Masson trichrome staining (MTS) to demonstrate the presence of extracellular matrix (ECM) qualitatively. RESULTS: A monolayer of oral fibroblasts was observed on the 9th-day postseeding. No chromosomal abnormality was observed in the patient samples. Positive staining was observed with vimentin, phalloidin, TGFßR1 and s100a4, thereby confirming the cell type. MTS revealed fibroblasts with spindle morphology and scanty ECM. CONCLUSION: The present study lays down a protocol to design and characterize primary buccal fibroblast cell culture model that would aid researchers in performing in vitro preliminary experiments in areas concerning fibrosis.

Oral epithelial dysplasia: Classifications and clinical relevance in risk assessment of oral potentially malignant disorders.

After more than a decade, the World Health Organization (WHO) published the revised grading system for oral epithelial dysplasia in 2017. The revised classification has changes reflecting our evolution of understanding of the dysplastic process. Although the WHO 2017 three-tier grading system is the gold standard for histological diagnosis of oral potentially malignant disorders, it has certain limitations. Suggestions to overcome these limitations include the use of clinical determinants and molecular markers to supplement the grading system. It has also been suggested that a two-tier system may be more reproducible and clinically translatable for better management. These advances in the understanding of epithelial dysplasia are very important globally and for us in the Indian subcontinent, given the prevalence of habits (tobacco/areca nut) and burden of oral cancer in this part of the world. The following review traces the evolution of the grading system of dysplasia, its relevance and clinical utility.

Common oral opportunistic infections in Human Immunodeficiency Virus infection/Acquired Immunodeficiency Syndrome: Changing epidemiology; diagnostic criteria and methods; management protocols.

There were 36.9 million in the world living with human immunodeficiency virus (HIV) infection/acquired immunodeficiency syndrome (AIDS) as of 2017, and new infections have seen a reduction by 18% since 2010. But this rate of decline is not sufficient for the goal of eradication of AIDS by 2030. Only 21.7  million people infected with HIV have accesses to antiretroviral therapy, with the rest at risk of the potential complications of HIV infection. It has been shown that oral lesions are diagnostic and prognostic of HIV infection, and many oral opportunistic infections continue to be a major problem, particularly in developing countries. It is therefore important that dental surgeons be aware and updated to recognize and manage the oral effects of HIV infection/AIDS. This chapter describes the classification, diagnosis, and management of oral lesions in these patients, based on our current understanding of the infection. This review also discusses the standardization of diagnosis of oral lesions in HIV infection/AIDS patients, immune reconstitution inflammatory syndrome case definition, and the research priorities formulated at the 7th World Workshop on Oral Health and Disease in AIDS.

"Burden of the Triumph:" Burden of peri-implantitis in Indian population - A mathematical model.

CONTEXT: India suffers from a heavy burden of oral diseases. Dental implants (DIs) are prescribed widely by the dental practitioners to replace lost natural teeth. There is no estimate, however, to determine the number of DIs or the number of people with peri-implantitis or the failure of implants after placement. In this modeling study, we attempted to estimate the prevalence of adult Indians who would choose DI in the near future and to calculate the peri-implantitis and failure of DI. MATERIALS AND METHODS: Using the Global Burden of Disease database (2016), the number of dental caries in permanent dentition, periodontal diseases, and edentulism was obtained. Empirical assumptions of patients with anodontia in urban and rural areas who opted for DI, percentage of implants placed, the affordability factors, and mathematical models for DI were formed and executed. Peri-implantitis and survival data from literary evidence were collated. RESULTS: Based on assumptions, 909,643 Indians, (830,231-858,703) would choose DI. Estimated number of peri-implantitis would be 145,543-254,700 and estimated number of failures should be 50,940-79,412 in the near future. CONCLUSIONS: In spite of the high economic challenge and the risks or complications of peri-implantitis, DIs are gaining prominence. It is the dentists' burden to face the renewed challenges due to emerge and provide remedial measures.