Malathi Narasimhan

Immunolocalization of Ki-67 in different periodontal conditions

Background: Ki-67 which is a non-histone nuclear protein which is expressed in proliferating cells, during all the active phases of the cell cycle. Increased Ki-67 expression has been seen in several inflammatory and malignant conditions like diabetes, rheumatoid arthritis, atherosclerosis, pancreatitis and squamous cell carcinoma. Aim: The aim of the present study is to analyze the expression of Ki-67 in gingival tissues by immunohistochemistry in smokers and non-smokers with healthy gingiva and chronic periodontitis. Materials and Methods: Gingival biopsies (n = 32) were obtained from smokers who had clinically healthy gingiva (n = 8), smokers with periodontitis (n = 8), chronic periodontitis (n = 8) and healthy gingiva (n = 8). The expression of Ki-67 was evaluated immunohistochemically. Statistical analysis used: Mean and standard deviation were estimated for the gingival tissue extract sample for each study group. Mean values were compared between different study groups by, one way ANOVA, post hoc analysis. In this study P < 0.05 was considered as the level of significance. Results: The mean number of Ki-67 positive cells/field was higher in the smokers with periodontitis group. When the mean Ki-67 positive cells were compared between different groups, statistical significant difference was observed between healthy and both the periodontitis groups (P = 0.000) and between smokers group (P = 0.001). Conclusions: Ki-67 was maximally expressed in smoker with periodontitis followed by chronic periodontitis patients, healthy smokers and healthy control patients which shed light on the toxic effects of tobacco in dysregulating the cell cycle and cellular proliferation. The findings of this study also help us to understand the role of the cell cycle in resolution of periodontal inflammation which is a salient feature in the pathogenesis of chronic periodontitis.

Invitro Anti-mycotic Activity of Hydro Alcoholic Extracts of Some Indian Medicinal Plants against Fluconazole Resistant Candida albicans.

BACKGROUND Candidiasis is one of the most common opportunistic infections caused by Candida albicans. Fluconazole is the drug of choice for prevention and management of this condition. However, the emergence of fluconazole resistant candidal strains has become a major concern. Many herbs like fenugreek, cinnamon, papaya, oregano, garlic are rich in phytochemical constituents known to express antimycotic activity. With the available information, the present research study was carried out to assess the invitro anti-mycotic activity of hydro alcoholic extracts of Trigonella foenum-graecum seeds, Cinnamomum verum bark and Carica papaya leaves and seeds against fluconazole resistant Candida albicans. MATERIALS AND METHODS Hydro alcoholic extracts of Trigonella foenum-graecum (seeds), Cinnamomum verum (bark), Carica papaya CO.2 strain (male and female leaves) and Carica papaya CO.2 strain (seeds) were prepared by maceration. The anti-mycotic activity of the prepared extracts against Candida albicans was assessed by agar well diffusion method. Three independent experiments were performed in triplicates and the mean and standard deviation were calculated. Minimum inhibitory concentration was determined. RESULTS The results of the present study revealed that all the extracts exhibited anti-mycotic activity in a dose dependent manner and minimum inhibitory concentration of all the extracts was found to be 15.62 μg/ml. CONCLUSION The results of the present study shed light on the fact that plant extracts could be used not only as an alternate drug for management of fluconazole resistant candidiasis but also explored further for oral cancer prevention as a therapeutic adjunct.

A comparison of oral and dental manifestations in diabetic and non-diabetic uremic patients receiving hemodialysis

Background: The purpose of the study was to evaluate the oral and dental findings of uremic patients receiving hemodialysis and to compare the Results between diabetic and non-diabetic groups. Materials and Methods: A total of 100 patients undergoing hemodialysis were classified into diabetic and non-diabetic groups and examined for uremic oral manifestations, dental caries (DMFT), and periodontal status (CPITN). Mann-Whitney test of significance has been applied for analyzing DMFT score and chi-square test is used for analyzing CPITN score. Results: Of the study group, 46% were diabetic and only 11% of them did not have any oral manifestation. Oral manifestations observed were xerostomia and uremic odor, which contributed to 47 (23%) and 37 (17%), respectively. Hyperpigmentation was present in 26 (12%), macroglossia in 23 (11%), and uremic tongue coating in 24 (11%). Mucosal petechiae were seen in 17 patients contributing to 8% of total patients. Eleven patients had tongue pallor (5%), 9 patients had glossitis with depapillation (4%), and 7 patients had dysgeusia (3%). Angular cheilitis and gingival swelling were seen in 5 patients (2%). Conclusion: The oral and dental manifestations were higher in prevalence in the study group. However, there was no significant difference between the two groups.

Antifibrotic effect of Centella asiatica Linn and asiatic acid on arecoline-induced fibrosis in human buccal fibroblasts.

AIM The aim of the present study was to investigate the in vitro antifibrogenic effects of Centella asiatica Linn (CA) and its bioactive triterpene aglycone asiatic acid (AA) on arecoline-induced fibrosis in primary human buccal fibroblasts (HBF). METHODS An ethanolic extract of CA was prepared, and AA was purchased commercially. High-performance thin-layer chromatography (HPTLC) was performed to quantify AA in the CA extract; colorimetric assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) was performed to determine an half-maximal inhibitory concentration. HBF were cultured and stimulated with arecoline. The inhibitory effects of CA and AA at different concentrations were assessed using gene-expression studies on fibrosis-related markers: transforming growth factor-β1, collagen 1 type 2, and collagen 3 type 1. The stimulatory effect of arecoline and the inhibitory effect of AA on fibroblast morphology and extracellular matrix were assessed qualitatively using Masson trichrome stain. RESULTS The HPTLC analysis determined 1.2% AA per 100 g of CA extract. Arecoline produced a concentration-dependent increase in the fibrotic markers, treatment with CA significantly downregulated fibrotic markers at higher concentrations, and AA downregulated at lower concentrations. Arecoline altered fibroblast morphology and stained strongly positive for collagen, and AA treatment regained fibroblast morphology with faint collagen staining. CONCLUSION CA and AA can be used as antifibrotic agents.

Qualitative Analysis of Collagen Fibers in Oral Submucous Fibrosis using Picrosirius Red Stain and Polarising Microscope

INTRODUCTION Oral Submucous Fibrosis (OSMF) is an oral potentially malignant condition caused predominantly by areca nut chewing. Early recognition with accurate staging of the disease and appropriate treatment planning is of utmost importance to prevent the malignant transformation and to improve the quality of life of the patient. Picrosirius red stain is specific for collagen and enhances its birefringence under polarising light producing different colours in different stages of the disease. AIM To compare the clinical and functional staging with histopathologic staging methods used to assess the severity of OSMF and to perform a qualitative analysis of the collagen fibres in various histopathologic stages of OSMF using picrosirius red stain and polarising microscope. MATERIALS AND METHODS It is a retrospective study done on archival samples. The study sample included a total of 30 cases which was divided into two groups. Group I comprised of 20 OSMF samples and Group II comprised of 10 normal tissue samples. Clinical, functional and histopathological staging was performed for all OSMF samples. Comparative analysis between clinical and functional stages with the histopathological staging was done using chi square test. Picrosirius red- stained sections of OSMF were analysed using polarising microscopy to evaluate the qualitative changes in the collagen fibers. STATISTICAL ANALYSIS USED Descriptive data which includes frequency and percentages were calculated for each group. Categorical data were analysed by chi-square test. A p-value of 0.05 or less was considered statistically significant. RESULTS Comparative analysis between clinical and functional stages with the histopathological staging revealed a significant correlation (p < 0.05) between the functional and histopathological stage. Enhanced birefringence of the collagen fibers due to picrosirius red stain yielded characteristic prominent polarising colours in different stages of OSMF. CONCLUSION Comparison of functional staging with histopathologic grading is more reliable in determining the severity of the disease than the clinical staging. Production of characteristic polarising colours by various stages of picrosirius red stained sections of OSMF substantiates the use of picrosirius red stain and polarising microscope as a tool in assessing the severity of this condition thereby aiding in better treatment planning.

Immunohistochemical Localization of Epithelial Mesenchymal Transition Markers in Cyclosporine A Induced Gingival Overgrowth

INTRODUCTION Cyclosporine, an immunosuppressive agent used in the management of renal transplant patients is known to produce Drug Induced Gingival Overgrowth (DIGO) as a side effect. Several mechanisms have been elucidated to understand the pathogenesis of DIGO. Recently, epithelial mesenchymal transition has been proposed as a mechanism underlying fibrosis of various organs. AIM The aim of the study was to investigate if Epithelial Mesenchymal Transition (EMT) operates in Cyclosporine induced gingival overgrowth. MATERIALS AND METHODS The study involved obtaining gingival tissue samples from healthy individuals (n=17) and subjects who exhibited cyclosporine induced gingival overgrowth (n=18). Presence and distribution of E-Cadherin, S100 A4 and alpha smooth muscle actin (α-SMA) was assessed using immunohistochemistry and cell types involved in their expression were determined. The number of α- SMA positive fibroblasts were counted in the samples. RESULTS In control group, there was no loss of E-Cadherin and a pronounced staining was seen in the all layers of the epithelium in all the samples analysed (100%). S100 A4 staining was noted in langerhans cells, fibroblasts, endothelial cells and endothelial lined blood capillaries in Connective Tissue (CT) of all the samples (100%) while α - SMA staining was seen only on the endothelial lined blood capillaries in all the samples (100%). However in DIGO, there was positive staining of E-Cadherin only in the basal and suprabasal layers of the epithelium in all the samples (100%). Moreover there was focal loss of E-Cadherin in the epithelium in eight out of 18 samples (44%). A break in the continuity of the basement membrane was noted in three out of 18 samples (16%) on H & E staining. CONCLUSION Based on the analysis of differential staining of the markers, it can be concluded that EMT could be one of the mechanistic pathways underlying the pathogenesis of DIGO.

Expression of Osteopontin in Oral Squamous Cell Carcinoma and its Surgical Margins-An Immunohistochemical Study.

INTRODUCTION Despite the advances in the treatment modalities offered for oral squamous cell carcinoma. The recurrence rate of it still remains quite high. Early detection of recurrence will improve the outcome and the survival of the patient. Osteopontin, a transformation-related phosphorylated protein in epithelial cells has been closely related with tumourigenesis. This study was undertaken to explore the potential of OPN as a tumour marker of recurrence in OSCC. AIM To analyse the expression of Osteopontin (OPN) in Oral Squamous Cell Carcinoma (OSCC), patient matched tumour free surgical margins and normal oral mucosa and to correlate with local & loco regional recurrence. MATERIALS AND METHODS Twenty cases each of formalin fixed paraffin embedded blocks of histopathologically diagnosed cases of OSCC, patient matched tumour free surgical margins and normal oral mucosal tissues were obtained from the archives of the Oral Pathology & Microbiology Department, Faculty of Dental Sciences, SRU and Govt. Arignar Anna Memorial Cancer Hospital, Kancheepuram. Immunohistochemical analysis was performed with an antibody to Osteopontin protein. Patients with secondary tumours and those treated with chemotherapy and radiotherapy were excluded from this study. RESULTS The expression of OPN was elevated in 95% of tumours & 55% of histologically tumour free margin samples. There was negative OPN expression in normal mucosal samples. The result of the study was statistically analysed using Pearson chi-square test and was found to be statistically significant. CONCLUSION OPN can be used as a diagnostic marker in Oral Squamous Cell Carcinoma. In the tumour free surgical margins, elevated levels of OPN may predict a significantly increased risk of recurrence.

Spindle cell carcinoma of the gingiva: A rare occurrence

Of the many neoplasms known, squamous cell carcinoma (SCC) is the most common to affect the oral cavity. Spindle cell carcinoma (SpCC) is considered a rare high malignant variant of SCC occurring predominantly in the upper aerodigestive tract. Soft-tissue spindle cell neoplasms are quite uncommon in the oral cavity reportedly accounting for lesser than 1% of all tumors in the oral region. Our case shows an unusual presentation of SpCC involving the mandibular gingiva in a 46-year-old smoker patient, which presented as a firm, erythematous swelling with surface necrosis. An incisional biopsy was performed for microscopic evaluation to confirm the clinical diagnosis and for treatment planning.

Immunohistochemical Evaluation of Mast Cells in Leukoplakia and Oral Squamous Cell Carcinoma

INTRODUCTION More than 90% of oral cancers are squamous cell carcinomas with oral leukoplakia being the most common potentially malignant disorder. Among the cell types in the stroma, mast cells play an important role in tumourigenesis through various mechanisms. AIM The present study was aimed at comparing the mast cell count among normal oral mucosa, leukoplakia and Oral squamous cell carcinoma (OSSC) and to evaluate the possible role of mast cells in carcinogenesis. MATERIALS AND METHODS Mast cell count was assessed immunohistochemically using anti-mast cell tryptase amongst 20 cases of leukoplakia and OSSC each and 10 normal gingival samples. Overall comparison was done using Kruskal Wallis test and intergroup comparison was done using Mann-Whitney U test. RESULTS The results of the present study showed an increase in mast cell count from normal oral mucosa (Mean: 7.73) to leukoplakia (Mean: 15.11) to squamous cell carcinoma (Mean: 22.73). Comparison of mean number of mast cells amongst three groups (p-value: 0.001) and intergroup comparisons showed statistical significance. CONCLUSION Mast cells favour malignant transformation and can be used as indicators of disease progression.

Langerhans Cell Expression in Oral Submucous Fibrosis: An Immunohistochemical Analysis.

INTRODUCTION Langerhans cells (LCs), are dendritic cells of the epithelium which play a role in an array of oral lesions from gingivitis to oral cancer. Oral Submucous Fibrosis (OSMF), a potentially malignant disorder (PMD), is an insidious chronic disease with juxta-epithelial inflammatory changes leading to fibrosis. Langerhans cells (LCs) may play a part in the ongoing inflammatory dysregulation of OSMF. OBJECTIVE The study was aimed at elucidating the distribution of LCs in varying grades of OSMF. MATERIALS AND METHODS A retrospective study using 39 cases of OSMF, graded using Haematoxylin and Eosin (H&E) stained section. Immunohistochemistry was performed using polyclonal anti- CD1a antibodies to identify LCs in 5 cases of normal tissue and 39 samples of OSMF. The distribution of LCs among the various grades and normal mucosa analysed using Mann-Whitney U test. RESULTS LC population in the OSMF was significantly higher when compared to the normal epithelium (p<0.001). Within the grades the advanced stage had more LCs than the other stages. CONCLUSION The increase in LCs might indicate the role of antigenic exposure in turn leading to cell mediated immunity in OSMF. Thus the fibrosis in OSMF might have a direct link to LCs.