Cytotoxic and Antimicrobial Effects of Herbal Formulation (Ficus benghalenis, Azadirachta indica and Menthapiperita) Based Mouthwash

Background: F.Benghalensis is known to have medicinal effect and is beneficial in treating mouth ulcers. A.Indicais rich in antioxidants and helps to boost the immune response in gum and tissues of the mouth. Neem offers a good remedy for curing mouth ulcers, tooth decay and acts as a pain reliever.M.Piperita contains antimicrobial and anti- inflammatory properties that promote oral hygiene. Aim: To prepare a mouth wash formulation containing F.benghalensis, A.indica, M.piperita. To determine the cytotoxic and antimicrobial effects of mouthwash formulation containing F.benghalensis, A.indica, M.piperita. Materials and Methods: In the present study, preparation of herbal mouthwash formulation was carried out initially and then the cytoxicity and antimicrobial activity of the mouthwash was assessed. The antimicrobial activity of the mouthwash formula- tion was carried out using agar well diffusion method. Different concentrations of mouthwash were tested against Streptococ- cus mutans, E.faecalis, lactobacillus and Candida albicans. The cytoxicity activity of the mouthwash was assessed using brine shrimp lethality assay method. Results: The cytotoxic activity was found to be better at low concentration. 8 nauplii survived at 5 ?L, 10 ?L, 20 ?L after 24 hours. The antimicrobial activity of the mouthwash formulation showed that the mouthwash formulation had the highest zone of inhibition seen in 100 ?L concentration on C.albicans almost similar to the control antibiotic in comparison to the oral pathogens. Discussion: Ficus benghalensis, Azadirachta indica & Mentha piperita extracts have many medicinal properties. Our study has evaluated the combination of these three extracts in a mouthwash formulation and has found positive results. Conclusion: The mouth wash formulation had better antimicrobial activity and exhibited less cytotoxicity at lower concentrations.

Gene Expression of GSK3, TGF ?-1 and PI3 Kinase in Oral Squamous Cell Carcinoma – A Real Time PCR Based Approach

Background: Oral squamous cell carcinoma (OSCC) is the most predominant type of oral cancer which has a poor prognosis, with 5-year survival rates less than 50%. Clinical characteristics such as tumor position, TNM classification and method of treat- ment, as well as histological grades have all been studied as OSCC prognostic factors but evaluating the genetic expression is the evolving trend in early diagnosis. Aim: To compare the gene expression of TGF-?-1, GSK3, Pi3 kinase in OSCC and normal tissue samples and to correlate the expression levels of these molecules with the pathological grading and survival in OSCC patients. Also to understand the role of GSK3 in Pi3 kinase pathway and TGF-? signaling pathway in OSCC progression thereby attempting targeted therapy in OSCC patients. Materials and Methods: 10 OSCC samples as well as normal healthy samples were collected and RNA isolation was done us- ing RNA easy kit from Qiagen (Valencia, CA), and thensubjected to cDNA synthesis using Human TGF-?1, Human GSK3? and Human Pi3 kinase primers. Real time PCR was performed using gene specific primers at 40 cycles. The results were retrieved, tabulated and analyzed. Results: The current research results revealed that there were up regulation of mRNA expression in GSK3, TGF ?-1 and Pi3 kinase in OSCC patients than in healthy individuals. On comparison, Pi3 kinase showed highest mRNA expression levels than GSK3 and TGF ?-1. Conclusion: The expression of GSK3 and its role in activation of Pi3 kinase pathway plays a crucial role in progression of oral cancer and targeting GSK3?could be a novel and targeted approach for treating OSCC.

Volatile Organic Compounds in Human Breath

A comprehensive analysis of volatile organic compounds (VOCs) from the exhaled breath sample is termed as breathomics. Breath samples are a complex mixture composed of a multitude of VOCs and other molecules. The analysis of total VOCs in exhaled breath provides a promising tool for the diagnosis of many diseases because it enables the observation of biochemical processes in the body in a non?invasive way. VOCs are produced in various physiological and pathophysiological conditions thus making it a potential biomarker for several diseases.

Quantitative analysis of copper levels in areca nut plantation area – A role in increasing prevalence of oral submucous fibrosis: An in vitro study

Background: Oral submucous fibrosis (OSMF) commonly seen in the South Asian countries is mostly associated with the chewing of areca nut (AN). Copper content in the AN has been implicated to play a major role in the pathogenesis of OSMF. It was found that most of the AN plantations in South India commonly use a copper-based fungicide, Bordeaux mixture (BM). Objective: To evaluate the level of copper in the AN, root, and soil of the AN plantation with and without the application of BM and to correlate the role of copper in the pathogenesis of OSMF. Materials and Methods: ANs, roots, and soil were obtained from plantations located in Tanniadi, Kerala. Four areas were selected from the plantation with and without BM application. The samples were collected twice with the interval of 6 months during January 2015 and July 2015, respectively. Statistical Analysis: Arithmetic mean and standard deviation were calculated. The differences between means were calculated by paired sample t-test. Results: There was statistically significant difference in the copper content of ANs, soil, and roots from both groups (P < 0.05). Samples treated with BM showed significantly higher copper levels as compared to their counterparts in January and July 2015. Conclusions: External copper from BM and increased processing for the commercial products could collectively increase the total copper content of the commercial AN products, and this high copper concentration may be implicated to the pathogenesis and the increasing prevalence of OSMF.

Expression of CD 68, CD 45 and human leukocyte antigen‑DR in central and peripheral giant cell granuloma, giant cell tumor of long bones, and tuberculous granuloma: An immunohistochemical study.

Background: Multinucleated giant cells (MNCs) form an integral part of numerous bone and soft tissue tumors, tumor‑like lesions and are often associated with granulomas of immunological and nonimmunological origin. The presence of various types of giant cells depends on the lesions in which they are present which are difficult to be diagnosed under routine histological techniques. Immunohistochemistry can be used for a better diagnosis and understanding of the origin of various giant cells using various markers of immune response like human leukocyte antigen‑DR (HLA‑DR) and those expressed on monocytes and macrophages like CD 68 and leukocyte common antigen (LCA). Materials and Methods: The study group consisted of 10 cases of giant cell tumor (GCT) of long bones, tuberculous granuloma, and giant cell granuloma to evaluate and analyze the expression pattern of LCA, CD 68, and HLA‑DR in various giant cell lesions. Results: Strong expression of CD 68 was observed in 80% of the lesions, strong and moderate expression of CD 45 observed in 70% of the lesions among and within the groups. In contrast, HLA‑DR demonstrated negative expression in 80% of cases except for tuberculous granuloma where all the 10 cases showed moderate to strong immunoreactivity. Conclusion: CD 68 and CD 45 expression was found in central giant cell granuloma, peripheral giant cell granuloma and GCT, suggesting the origin from mononuclear phagocyte system and considering their clinical behavior of osteoclast type. High expressivity of HLA‑DR in tuberculous granulomas which is an essential factor for presentation of the microbial antigen to CD 4 helper cells thus reassuring the fact that they are up‑regulated in response to infection.

Promoter hypermethylation patterns of P16, DAPK and MGMT in Oral Squamous Cell Carcinoma: A systematic review and meta-analysis.

Background: Oral squamous cell carcinoma (OSCC) is a common cancer world‑wide that is highly lethal due to its recurrence and metastasis. Methylation is a common epigenetic mechanism that leads to gene silencing in tumors and could be a useful biomarker in OSCC. The prevalence of P16, death‑associated protein kinase (DAPK) and O6‑methylguanine‑DNA‑methyltransferase (MGMT) promoter hypermethylation in OSCC has been evaluated for several years while the results remain controversial. Objective: The aim of this systematic review is to critically analyze and perform a meta‑analysis on the various studies in the literature that have reported the promoter hypermethylation of P16, DAPK and MGMT genes in OSCC. Search Strategy: Articles were searched and selected through PubMed. Hand search from the relevant journals was also performed. Articles were reviewed and analyzed. Results: The estimated prevalence of P16 methylation was 43%, DAPK methylation was 39.7% and MGMT methylation was 39.8%. Heterogeneity in methylation prevalences and correlations with the clinical outcomes of the disease prevailed in various studies. Conclusion: We can conclude from our systematic review that a higher prevalence of methylation of P16, DAPK and MGMT occur in OSCC. Further studies are required to substantiate the role of methylation of P16, DAPK and MGMT as a marker in OSCC.

Antioxidant and pro-oxidant activity of Vitamin C in oral environment.

Context: Antioxidant properties and Vitamin C. Background: Vitamin C is a naturally occurring organic compound and a potent antioxidant preventing oxidative damage to lipids and other macromolecules. It can also exhibit bimodal activity as a pro‑oxidant at a higher concentration. Vitamin C has a switch over role from being an antioxidant in physiologic conditions to a pro‑oxidant under pathologic conditions. A systematic review of this role would help to elucidate whether it is an antioxidant or a pro‑oxidant in the oral environment. Objective: To review studies reported in the literature elucidating the activity of Vitamin C and determine whether it is an antioxidant or a pro‑oxidant. Materials and Methods: Articles were searched in PubMed, MEDLINE using appropriate key words like “Vitamin C,” “antioxidant activity,” “pro‑oxidant activity,” “oral health” “oral disease.” Hand search of journals was also performed. Articles were reviewed and analyzed. Results: Search strategy reviewed 10 relevant articles which studied the dual role of Vitamin C. 65% of authors analyzed antioxidant action of ascorbic acid compared to 35% of the pro‑oxidant potential. Vitamin C acts as an antioxidant and a pro‑oxidant by a plethora of mechanisms. Factors determining its bimodal activity were studied, and the frequencies of their occurrence in the literature were depicted in percentage. Conclusion: The data validates the role of Vitamin C as an antioxidant under physiologic conditions exhibiting a cross over role as a pro‑oxidant in pathological conditions. Further studies are required to substantiate its pro‑oxidant activity to draw concrete conclusions.

Salivary characteristics of diabetic children

The objectives of this study were to evaluate the levels of glucose, cholesterol, protein and albumin in saliva, and to correlate the levels of glucose of the saliva to oral health and blood glucose of diabetic and non-diabetic children. Methods: 32 children with type 1 diabetes mellitus formed the study group (DC) and 32 non-diabetic children formed the control group (ND). Thepatients had their saliva collected and evaluated for glucose, cholesterol, total protein and albumin. Blood glucose analysis was also performed. The dental health status of the subjects was measured by DMFT index and def index. Independent Student’s t-test was performed to compare metabolic status values in DC and ND groups. Correlation test was applied between blood glucose and salivary glucose (Spearman’s correlation), and salivary glucose and DMFT/def (Spearman’stest). Results: A statistically significant difference was observed between DC and ND considering salivary glucose (p=0.000). Elevated levels of cholesterol were evident in DC in correlation with ND. Total protein and albumin had increased values in DC (nonsignificant p value). The dental health status was not statistically different. Conclusions: Salivary parameters can act as adjuncts in assessing the overall metabolic status of the patient.

Stromal changes in apparently normal mucosa of smokers and pan chewers: a multi-parametric approach

Aim: To compare and contrast the various changes in the connective tissue among patients with the habits of smoking, pan chewing and controls by using fluorescence spectroscopy, histopathology and transmission electron microscopy. Method: Thirty subjects were categorized into three groups: pan chewers, smokers and controls without any oral lesions. Fluorescence spectroscopy was carried out using FLUOROMAX-2. Excitation spectroscopy was performed at 280 and 320nm respectively and Emission spectroscopy was performed at 340 and 390nm excitation. Subsequently, histopathological evaluation and transmission electron microscopy was done for biopsies taken from test groups and controls. Results: The mean, standard deviation and test of significance of mean values between different groups for intensity-380nm, intensity–420nm and intensity – 460nm at 320 nm excitations, showed that the Mean values in group I and group II were significantly higher than the mean value in group III with a p-value of less than 0.001. Considering the histopathological parameters, the pattern of the sub-epithelial connective tissue, the presence of chronic inflammatory cells and lysis of connective tissue was significant. The ultrastructural analysis revealed the presence of epithelioid, spindle or elongated and stellate shaped fibroblasts in the connective tissue. Conclusion: In this pilot study, we could find variations in emission characteristics of various amino acids which correlated with histopathology and electron microscopy. Our study suggests that there are connective tissue changes in oral mucosa among smokers and pan chewers, though it is apparently normal in clinical presentation. These initial connective tissue changes could determine the progression of altered mucosa to a pre-cancer or cancer, which is further related to other complex interactions. Elaborate studies are required to evaluate the significance of our hypothesis.

Langerhans cell histiocytosis of the mandible in a six-year-old child.

Histiocytosis is a term applied to a group of rare disorders of the reticuloendothelial system. Eosinophilic granuloma, the most benign and localized of the three Langerhans cell histiocytosis entities, may be solitary or multiple. Eosinophilic granuloma can affect almost any bone, but commonly involves the mandible when the jaws are affected. Conventional treatment of LCH is with surgery, radiotherapy, chemotherapy and steroid injections, alone or in combination. Spontaneous regression of localized disease has also been reported. We report a six and a half-year-old patient with Langerhans cell histiocytosis--solitary eosinophilic granuloma of the mandible that initially regressed but rapidly recurred even after radical treatment and had a fatal outcome.

Use of Rt-PCR in detecting disseminated cancer cells after incisional biopsy among oral squamous cell carcinoma patients.

PURPOSE: Tumour metastasis is the most clinically significant and enigmatic aspect of tumour behavior and is an unequivocal hallmark of malignancy. Until recent years little has been known about the transportation phase of vascular dissemination during biopsy, because of the technical difficulties in demonstrating circulating cancer cells. AIMS: This study examined whether cancer cell dissemination results from incisional biopsy in the peripheral blood by using Cytokeratin 19(CK-19) as the marker for Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR). In-house recipes without utilizing kits were employed to extract genomic and total RNA to make the procedure user friendly. MATERIALS AND METHODS: The study population consisted of n=10 patients who were clinically diagnosed for oral squamous cell carcinoma and who had not undergone any previous biopsies. 5 patients who were to undergo incisional biopsies for benign conditions served as controls. 5 ml of blood aspirates were collected before and within 15 minutes after incisional biopsy. CK-19 gene and a positive control gene beta actin were isolated to confirm the primers. Using the total RNA, RT-PCR was performed for beta actin and Ck 19 gene expression. RESULTS: Rt-PCR did show any expression for the CK-19 gene. CONCLUSION: In conclusion there was no evidence of dissemination of cancer cells in our study and the patients are on a regular follow up for the past one and half years. But larger sample size should be examined to make the procedure a diagnostic tool for cancer metastasis.

Primary extranodal non-Hodgkin's lymphoma of the oral cavity.

Non-Hodgkin's lymphoma (NHL) belongs to a group of lymphoid neoplasms that is diverse in manner of presentation, response to therapy and prognosis. Usually the oral manifestations of NHL are secondary to a more widespread involvement throughout the body. Though primary intraoral lesions in NHL are uncommon, it is important to be aware of them, since intraoral manifestations are the presenting symptom in these patients. A 55 year old woman presented with a swelling in the maxillary right alveolus and buccal vestibule. There were no palpable lymph nodes. A CT scan revealed a mass on the labial aspect of the right maxilla with no involvement of the maxillary sinus. Incisional biopsy of the lesion showed a dense lymphocytic infiltrate with formation of variably sized lymphoid follicles. The infiltrate showed a mixed neoplastic lymphocyte composition which was suggestive of follicular mixed type of malignant B cell primary extranodal lymphoma. The patient was treated with chemotherapy followed by radiation.