Made available in DSpace on 2015-12-06T23:47:21Z (GMT). No. of bitstreams: 0 / Objetivo: Analisar as diferenças histopatológicas entre os lobos pulmonares submetidos a biópsia cirúrgica de pacientes com fibrose pulmonar idiopática. Métodos: Foram incluídos 16 pacientes com resultados de biópsias concordantes de dois sítios pulmonares, o lobo médio ou o segmento lingular. Técnicas semiquantitativas foram utilizadas na avaliação histológica pulmonar, aplicando-se um escore com base nas alterações encontradas no padrão histológico de pneumonia intersticial usual (PIU). Resultados: Encontrou-se maior incidência de foco fibroblástico em lobo inferior esquerdo e maior volume médio dos fragmentos pulmonares originários do lobo médio. Nenhuma diferença significante, que viesse a mudar o estadiamento da doença, e independentemente do local de origem da biópsia, foi encontrada. Conclusão: Não há dados que fundamentem a rejeição ao uso dos lobos médios e segmentos lingulares na investigação histológica de doenças intersticiais, em especial a fibrose pulmonar idiopática.. / Objective: To analyze the histopathology differences between the pulmonary lobes submitted to surgical biopsy in patients with idiopathic pulmonary fibrosis. Methods: Sixteen patients with usual interstitial pneumonia and concordant biopsies of two distinct lobes (the middle lobe or the lingula) were included in this study. Semiquantitative techniques were used in the histological evaluation of fragments of different lobes of the lung. A score was applied for the evaluation of usual interstitial pneumonia. Results: A greater incidence of fibroblastic foci was found in the left inferior lobe, and a greater mean volume of pulmonary fragments was obtained from the middle lobe. No significant difference was found in variables related to disease staging, no matter what was the pulmonary lobe. Conclusion: There is no reason to despise the middle lobe and the lingula in the histopathological investigation of interstitial diseases of the lung, especially the idiopathic pulmonary fibrosis. / BV UNIFESP: Teses e dissertações

A atresia das vias biliares é afecção hepática da infância. A etiologia é desconhecida, e o diagnóstico baseia-se na biópsia hepática, cujo achado é a proliferação ductular. A ligadura do ducto biliar comum em ratos é modelo utilizado para estudo das doenças colestáticas. A proposta do trabalho foi estudar, em modelo experimental de obstrução biliar, as alterações histológicas hepáticas em ratos jovens e compará-las com o animal adulto. Avaliou-se a semiquantificação da proliferação ductular e inflamação pelo HE; quantificação da fibrose portal e periportal pelo picrosírius; semiquantificação da expressão de desmina e a-actina de músculo liso pelas células estreladas e miofibroblastos. Apesar das respostas de proliferação ductular e inflamação mais lentas no rato jovem, a fibrose e a expressão de desmina foram mais intensas neste grupo / Biliary atresia is an hepatic disease of infancy. Etiology is unknown, and diagnosis is made by liver biopsy, with ductular proliferation being the main histological feature. Bile duct ligation in rats is an useful experimental model of biliary obstruction. The aim of this study of extra-hepatic cholestasis was analyse hepatic histological alterations in young rats compared to adult animals. The responses were studied by semiquantification of ductular proliferation and inflammatory infiltrated by HE stain; quantification of portal and periportal fibrosis with the sirius-red stain; semiquantification of the expression of desmin and a-smooth muscle actin by the hepatic stellated cells and myofibroblasts. In young animals, despite the very slow response of ductular proliferation and inflammation observed with HE, there were significantly more fibrosis and expression of desmin than in adult group

A atresia das vias biliares é afecção hepática da infância. A etiologia é desconhecida, e o diagnóstico baseia-se na biópsia hepática, cujo achado é a proliferação ductular. A ligadura do ducto biliar comum em ratos é modelo utilizado para estudo das doenças colestáticas. A proposta do trabalho foi estudar, em modelo experimental de obstrução biliar, as alterações histológicas hepáticas em ratos jovens e compará-las com o animal adulto. Avaliou-se a semiquantificação da proliferação ductular e inflamação pelo HE; quantificação da fibrose portal e periportal pelo picrosírius; semiquantificação da expressão de desmina e a-actina de músculo liso pelas células estreladas e miofibroblastos. Apesar das respostas de proliferação ductular e inflamação mais lentas no rato jovem, a fibrose e a expressão de desmina foram mais intensas neste grupo / Biliary atresia is an hepatic disease of infancy. Etiology is unknown, and diagnosis is made by liver biopsy, with ductular proliferation being the main histological feature. Bile duct ligation in rats is an useful experimental model of biliary obstruction. The aim of this study of extra-hepatic cholestasis was analyse hepatic histological alterations in young rats compared to adult animals. The responses were studied by semiquantification of ductular proliferation and inflammatory infiltrated by HE stain; quantification of portal and periportal fibrosis with the sirius-red stain; semiquantification of the expression of desmin and a-smooth muscle actin by the hepatic stellated cells and myofibroblasts. In young animals, despite the very slow response of ductular proliferation and inflammation observed with HE, there were significantly more fibrosis and expression of desmin than in adult group

INTRODUÇÃO: A endomiocardiofibrose (EMF) é uma miocardiopatia de padrão restritivo de etiologia desconhecida, prevalente em regiões tropicais. Caracteriza-se por espessamento fibroso do endocárdio e miocárdio subjacente, comprometendo ponta e via de entrada de um ou de ambos os ventrículos. Sua etiopatogenia é pouco conhecida e muitos autores a associam à doença infecciosa cardíaca ou sistêmica, à eosinofilia prévia e/ou à carência nutricional. O prognóstico em geral é grave e a ressecção cirúrgica da lesão é indicada aos pacientes com insuficiência cardíaca refratária a tratamento clínico, em classe funcional III ou IV (NYHA). Os estudos anatomopatológicos até o momento foram realizados em material de autópsia ou de biópsia endomiocárdica. OBJETIVOS: Este estudo retrospectivo se propõe a (1) descrever os três aspectos morfológicos da lesão endocárdica (fibrose, infiltrado inflamatório e vasos) utilizando microscopia óptica comum e técnicas imunoistoquímicas, assim como correlacioná-los aos dados clínicos, laboratoriais e de imagem dos pacientes (2) comparar os aspectos morfológicos de espécimes de ressecção cirúrgica com os de autópsia a fim de verificar se os primeiros podem ser empregados para diagnóstico histológico da doença; e (3) discutir a patogenia da EMF e realizar pesquisa de agentes infecciosos cardiotrópicos em amostra endomiocárdica incluída em parafina por técnica de biologia molecular. MÉTODOS: Foram utilizadas amostras de ressecção cirúrgica endocárdica incluídas em parafina provenientes de 31 pacientes com diagnóstico clínico e cineangiocardiográfico de EMF, operados no InCor entre 1991 e 2005. As amostras foram coradas por técnicas convencionais (HE, tricômico de Masson, Verhoeff e reticulina) e submetidas a reações imunoistoquímicas para fibras colágenas tipo I, III e IV, para células inflamatórias (CD3, CD20, CD68) e para endotélio de linfáticos (D2-40). Amostras de nove corações de autópsia de pacientes com o mesmo diagnóstico serviram de controle positivo da doença. A pesquisa de agentes infecciosos foi feita por reação de cadeia de polimerase e reação de transcrição reversa de cadeia de polimerase (PCR e RT-PCR) em amostras endomiocárdicas incluídas em parafina, para T. gondii e para vírus cardiotrópicos (enterovirus, adenovirus, influenza A e B, citomegalovirus, parvovirus B19 e herpes simples). Para identificar alterações vasculares intramiocárdicas, procedeu-se à revisão de prontuários clínicos dos pacientes e de 16 cineangiocoronariografias. RESULTADOS: Foi observado intenso espessamento endocárdico ventricular à custa de fibrose hialina superficial escassamente celular, com fibras colágenas tipos I e III, predominando o tipo I sobre o III. O colágeno tipo IV foi identificado na membrana basal de vasos. Na porção profunda da lesão endocárdica observou-se escasso infiltrado inflamatório crônico com macrófagos, linfócitos T e B em menor número. O infiltrado estava distribuído ao redor de vasos proliferados com intensas alterações estruturais na parede e com participação de linfáticos. No miocárdio superficial identificou-se miocardite \"borderline\" (critério de \"Dallas\"). Foram obtidos ácidos nucléicos em quantidade suficiente para a reação de PCR/RT-PCR em 12/36 (33%) amostras. Genomas de agentes infecciosos foram identificados em 6/12 (50%) pacientes. Dois casos foram positivos para enterovirus (EV), dois para citomegalovirus (CMV), um para ambos (CMV e EV) e um para T. gondii. Não foram observadas diferenças histopatológicas entre as amostras cirúrgicas e as de autópsia. Foram detectadas alterações vasculares à cineangiocoronariografia em 9/16 (56%) pacientes. Não houve correlação anatomoclínica definida entre os múltiplos dados comparados. CONCLUSÕES: Os resultados indicam que na EMF ocorre processo inflamatório crônico mantido por rede vascular anômala rica em linfáticos localizada na profundidade da lesão endocárdica. A rede vascular provavelmente contribui para a manutenção da placa fibrótica e deve ser considerada como fator importante na patogenia da doença. O diagnóstico anatomopatológico pode ser feito com segurança em material de ressecção cirúrgica. A análise molecular do endomiocárdio possibilitou a detecção de alta incidência de genomas de agentes infecciosos cardiotrópicos. Seu significado, contudo, permanece controverso. / BACKGROUND: Endomyocardial fibrosis (EMF) is a restrictive cardiomyopathy of unknown etiology prevalent in tropical regions. The disease involves the inflow tract and apex of either one or both ventricles and is characterized by a fibrous thickening of the endocardium and the underlying myocardium. Although its etiology remains unknown, most authors believe it could be related to systemic or heart infection/parasitism, previous blood eosinophilia or malnutrition. Surgical resection of the thickened endocardium is recommended to patients with advanced heart failure of functional class III or IV, New York Heart Association (NYHA). The gross and histological features of the heart have been comprehensively studied in autopsies and endomyocardial biopsies. Studies in surgical samples, however, are still lacking. AIMS: This study was conducted to evaluated: (1) the histomorphological changes of EMF as seen in surgical specimens by means of routine histological and immunohistochemical methods in an attempt to correlate them with clinical symptoms and coronary angiographic features; (2) to compare histological data between surgical and autopsy samples, and (3) to discuss probable pathogenetic mechanisms of the disease, as well as to investigate cardiotropic infective agents by means of molecular analysis of endomyocardial surgical samples. METHODS: We collected all available clinical records and endomyocardial surgical samples from 31 patients with EMF who had been submitted to surgery between 1991 and 2005 at InCor. The diagnosis was based on clinical, hemodynamic and angiocardiographic findings. The surgical samples were fixed in 10% formalin, submitted to standard processing, and stained with H&E, Masson\'s trichrome, reticulin and elastic stains. Immunohistochemical methods were employed to detect collagen fibers type I, III, and IV, inflammatory cells (CD3, CD20, CD68) and lymphatic vessels\' endothelium (D2-40). Nine samples from autopsied hearts of EMF patients were used as a positive control group. Polymerase chain reaction (PCR) and reverse transcription-PCR were used retrospectively to search for genomes of T. gondii and cardiotropic viruses (enterovirus, adenovirus, influenza A e B, cytomegalovirus, parvovirus B19 and herpes simplex) in the surgical material. All clinical and surgical reports were reviewed, including follow-ups and 16 coronary cineangiocardiographies. RESULTS: Ventricular endocardium was thickened by superficial acellular hyaline collagen fibers type I and III. Type-IV collagen fibers were seen only around vessels. Focal chronic inflammatory infiltrate with T-lymphocytes, macrophages and a few B-lymphocytes was seen around blood vessels with a peculiar pattern of vascular changes and numerous lymphatics within the endocardium. The superficial myocardium showed borderline myocarditis (Dallas criteria). RNA and DNA were successfully extracted from 12/36 samples. Infective agents were detected in 6/12 (50%) patients; two of them were positive for cytomegalovirus (CMV), two for enterovirus (EV), one for both (CMV and EV) and one for T. gondii. No histopathological differences between surgical samples and autopsy fragments were observed. Vascular blush or neovascularity was detected in 9 of the 16 coronary cineangiocardiographies reviewed. Clinicopathologic characteristics are associated neither with infective genomes in the endocardium nor with vascular blush. CONCLUSIONS: Results indicate that there is an non especific chronic inflammatory process maintained by an anomalous vascular net rich in lymphatics situated deep within the endocardium. This angiolymphatic web probably contributes to the maintenance of the fibrotic plaque and might be considered an important pathological finding concerning in the pathogenesis of EMF. Histopathological changes as seen in surgical material are diagnostic of EMF. Molecular analysis of the endomyocardium revealed high incidence of cardiotropic infective agents, but their role in the pathogenesis of the disease is still controversial.

INTRODUÇÃO: A endomiocardiofibrose (EMF) é uma miocardiopatia de padrão restritivo de etiologia desconhecida, prevalente em regiões tropicais. Caracteriza-se por espessamento fibroso do endocárdio e miocárdio subjacente, comprometendo ponta e via de entrada de um ou de ambos os ventrículos. Sua etiopatogenia é pouco conhecida e muitos autores a associam à doença infecciosa cardíaca ou sistêmica, à eosinofilia prévia e/ou à carência nutricional. O prognóstico em geral é grave e a ressecção cirúrgica da lesão é indicada aos pacientes com insuficiência cardíaca refratária a tratamento clínico, em classe funcional III ou IV (NYHA). Os estudos anatomopatológicos até o momento foram realizados em material de autópsia ou de biópsia endomiocárdica. OBJETIVOS: Este estudo retrospectivo se propõe a (1) descrever os três aspectos morfológicos da lesão endocárdica (fibrose, infiltrado inflamatório e vasos) utilizando microscopia óptica comum e técnicas imunoistoquímicas, assim como correlacioná-los aos dados clínicos, laboratoriais e de imagem dos pacientes (2) comparar os aspectos morfológicos de espécimes de ressecção cirúrgica com os de autópsia a fim de verificar se os primeiros podem ser empregados para diagnóstico histológico da doença; e (3) discutir a patogenia da EMF e realizar pesquisa de agentes infecciosos cardiotrópicos em amostra endomiocárdica incluída em parafina por técnica de biologia molecular. MÉTODOS: Foram utilizadas amostras de ressecção cirúrgica endocárdica incluídas em parafina provenientes de 31 pacientes com diagnóstico clínico e cineangiocardiográfico de EMF, operados no InCor entre 1991 e 2005. As amostras foram coradas por técnicas convencionais (HE, tricômico de Masson, Verhoeff e reticulina) e submetidas a reações imunoistoquímicas para fibras colágenas tipo I, III e IV, para células inflamatórias (CD3, CD20, CD68) e para endotélio de linfáticos (D2-40). Amostras de nove corações de autópsia de pacientes com o mesmo diagnóstico serviram de controle positivo da doença. A pesquisa de agentes infecciosos foi feita por reação de cadeia de polimerase e reação de transcrição reversa de cadeia de polimerase (PCR e RT-PCR) em amostras endomiocárdicas incluídas em parafina, para T. gondii e para vírus cardiotrópicos (enterovirus, adenovirus, influenza A e B, citomegalovirus, parvovirus B19 e herpes simples). Para identificar alterações vasculares intramiocárdicas, procedeu-se à revisão de prontuários clínicos dos pacientes e de 16 cineangiocoronariografias. RESULTADOS: Foi observado intenso espessamento endocárdico ventricular à custa de fibrose hialina superficial escassamente celular, com fibras colágenas tipos I e III, predominando o tipo I sobre o III. O colágeno tipo IV foi identificado na membrana basal de vasos. Na porção profunda da lesão endocárdica observou-se escasso infiltrado inflamatório crônico com macrófagos, linfócitos T e B em menor número. O infiltrado estava distribuído ao redor de vasos proliferados com intensas alterações estruturais na parede e com participação de linfáticos. No miocárdio superficial identificou-se miocardite \"borderline\" (critério de \"Dallas\"). Foram obtidos ácidos nucléicos em quantidade suficiente para a reação de PCR/RT-PCR em 12/36 (33%) amostras. Genomas de agentes infecciosos foram identificados em 6/12 (50%) pacientes. Dois casos foram positivos para enterovirus (EV), dois para citomegalovirus (CMV), um para ambos (CMV e EV) e um para T. gondii. Não foram observadas diferenças histopatológicas entre as amostras cirúrgicas e as de autópsia. Foram detectadas alterações vasculares à cineangiocoronariografia em 9/16 (56%) pacientes. Não houve correlação anatomoclínica definida entre os múltiplos dados comparados. CONCLUSÕES: Os resultados indicam que na EMF ocorre processo inflamatório crônico mantido por rede vascular anômala rica em linfáticos localizada na profundidade da lesão endocárdica. A rede vascular provavelmente contribui para a manutenção da placa fibrótica e deve ser considerada como fator importante na patogenia da doença. O diagnóstico anatomopatológico pode ser feito com segurança em material de ressecção cirúrgica. A análise molecular do endomiocárdio possibilitou a detecção de alta incidência de genomas de agentes infecciosos cardiotrópicos. Seu significado, contudo, permanece controverso. / BACKGROUND: Endomyocardial fibrosis (EMF) is a restrictive cardiomyopathy of unknown etiology prevalent in tropical regions. The disease involves the inflow tract and apex of either one or both ventricles and is characterized by a fibrous thickening of the endocardium and the underlying myocardium. Although its etiology remains unknown, most authors believe it could be related to systemic or heart infection/parasitism, previous blood eosinophilia or malnutrition. Surgical resection of the thickened endocardium is recommended to patients with advanced heart failure of functional class III or IV, New York Heart Association (NYHA). The gross and histological features of the heart have been comprehensively studied in autopsies and endomyocardial biopsies. Studies in surgical samples, however, are still lacking. AIMS: This study was conducted to evaluated: (1) the histomorphological changes of EMF as seen in surgical specimens by means of routine histological and immunohistochemical methods in an attempt to correlate them with clinical symptoms and coronary angiographic features; (2) to compare histological data between surgical and autopsy samples, and (3) to discuss probable pathogenetic mechanisms of the disease, as well as to investigate cardiotropic infective agents by means of molecular analysis of endomyocardial surgical samples. METHODS: We collected all available clinical records and endomyocardial surgical samples from 31 patients with EMF who had been submitted to surgery between 1991 and 2005 at InCor. The diagnosis was based on clinical, hemodynamic and angiocardiographic findings. The surgical samples were fixed in 10% formalin, submitted to standard processing, and stained with H&E, Masson\'s trichrome, reticulin and elastic stains. Immunohistochemical methods were employed to detect collagen fibers type I, III, and IV, inflammatory cells (CD3, CD20, CD68) and lymphatic vessels\' endothelium (D2-40). Nine samples from autopsied hearts of EMF patients were used as a positive control group. Polymerase chain reaction (PCR) and reverse transcription-PCR were used retrospectively to search for genomes of T. gondii and cardiotropic viruses (enterovirus, adenovirus, influenza A e B, cytomegalovirus, parvovirus B19 and herpes simplex) in the surgical material. All clinical and surgical reports were reviewed, including follow-ups and 16 coronary cineangiocardiographies. RESULTS: Ventricular endocardium was thickened by superficial acellular hyaline collagen fibers type I and III. Type-IV collagen fibers were seen only around vessels. Focal chronic inflammatory infiltrate with T-lymphocytes, macrophages and a few B-lymphocytes was seen around blood vessels with a peculiar pattern of vascular changes and numerous lymphatics within the endocardium. The superficial myocardium showed borderline myocarditis (Dallas criteria). RNA and DNA were successfully extracted from 12/36 samples. Infective agents were detected in 6/12 (50%) patients; two of them were positive for cytomegalovirus (CMV), two for enterovirus (EV), one for both (CMV and EV) and one for T. gondii. No histopathological differences between surgical samples and autopsy fragments were observed. Vascular blush or neovascularity was detected in 9 of the 16 coronary cineangiocardiographies reviewed. Clinicopathologic characteristics are associated neither with infective genomes in the endocardium nor with vascular blush. CONCLUSIONS: Results indicate that there is an non especific chronic inflammatory process maintained by an anomalous vascular net rich in lymphatics situated deep within the endocardium. This angiolymphatic web probably contributes to the maintenance of the fibrotic plaque and might be considered an important pathological finding concerning in the pathogenesis of EMF. Histopathological changes as seen in surgical material are diagnostic of EMF. Molecular analysis of the endomyocardium revealed high incidence of cardiotropic infective agents, but their role in the pathogenesis of the disease is still controversial.

Oral Submucous Fibrosis (OSF) is a chronic inflammatory disease resulting in progressive fibrosis of the oral tissues that can cause difficulty in chewing, swallowing, speaking, and mouth opening. Epidemiological studies have shown that OSF is a precancerous condition and 2-8% of the OSF patients develop squamous cell carcinoma. This disease affects 0.5% of the population in the Indian subcontinent and is now a growing public health issue in many parts of the world. Habit of chewing betel quid has been proposed as an important etiological factor in the development of this disease and is coline, a principle alkaloid of areca nut is considered as major causative factor for OSF development. But the exact molecular mechanism of OSF pathogenesis is not known. Therefore, we set the following objectives for this study: 1) Gene expression profiling of OSF using microarray. 2) Role of areca nut constituents in OSF pathogenesis. 3) Effect of areca nut on epithelial and fibroblast cells. In order to delineate the possible molecular mechanism of OSF pathogenesis, we took microarray approach and identified differentially regulated genes in ten OSF tissues against eight pooled normals using whole human genome oligonucleotide arrays. Microarray results revealed differential expression of 5288 genes (p≤0.05 and Fold change≥1.5), among them 2884 were up-regulated and 2404 were down-regulated. Validation employing quantitative real-time PCR and immunohistochemistry confirmed up-regulation of transforming growth factor-β1 (TGF-β1), TGFBI, THBS1, SPP1, TIG1 and down-regulation of bone morphogenic protein 7 (BMP7), C4orf7 and ALOX12 in OSF tissues. Furthermore, activation of TGF-β pathway was evident in OSF tissues as demonstrated by p-SMAD2 strong immunoreactivity. Analysis of IHC data showed that in all the normal tissues and in 70% of the OSF tissues the expression of TGF-β and BMP7 are inversely correlated. In good correlation, treatment of keratinocytes (HaCaT) by TGF-βdown-regulated BMP7, while BMP7 expression could not be detected in fibroblast cells. Hence, the imbalance between TGF-βand BMP7 signalling, which are positive and negative modulators of extracellular matrix production, respectively may trigger the manifestation of OSF. We also studied the regulation few genes (CTGF, TGM2 and THBS1) identified in OSF microarray in response to TGF-βand arecoline. TGF-βwas able to induce all the above genes in both HaCaT and hGF cells but arecoline could only induce TGM2 in hGF and THBS1 in HaCaT. Therefore TGF-βpathway came out to be the most important pathway in OSF microarray and subsequent validations. But areca nut constituents responsible for TGF-βpathway activation and the source (epithelial or fibroblast cells) through which it activates TGF-βare not known. In an attempt to understand the role of areca nut and its constituents in inducing TGF-βsignalling in epithelial cells, we performed microarray on epithelial cells (HaCaT) treated with areca nut water extract. Surprisingly, 64% of the differentially regulated genes by areca nut water extract matched with TGF-βinduced gene expression profile. To find out areca nut induced genes through TGF-β, epithelial cells were treated with areca nut in presence of ALK5 (TβRI) inhibitor. Out of 64% differentially induced genes, 57% genes induced by areca nut got compromised in presence of ALK5 and 7% were independently induced by areca nut, highlighting the effect of areca nut via TGF-β. Accordingly, areca nut treatment induced both p-SMAD2 and TGF-βdownstream targets TGFBI, TGM2, TMEPAI and THBS1 in HaCaT cells. One possible mechanism of TGF-βsignalling induction by areca nut could be via induced ligand (TGF-β2) and its activator (THBS1). Induction of TGF-β2 ligand by areca nut was shown at both RNA (Real Time) and protein (ELISA) levels. To find out areca nut components responsible for inducing TGF-β signalling, areca nut fractionation was performed which gave three fractions namely, Ethyl acetate (polyphenol), water supernatant (alkaloids) and Dichloromethane (impurity). Out of these; polyphenol and alkaloid fractions were found to be responsible for the induction of TGF-β signalling and its downstream targets. Upon treatment with purified components, catechin and tannin of polyphenol fraction and arecoline, arecaidine and guvacine of alkaloid fraction were found to be responsible for inducing TGF-β signalling, as seen by increased appearance of phopho-SMAD2 in HaCaT cells. Areca nut treatment on human gingival fibroblast cells (hGF) did not induce TGF-β signalling, highlighting that the source of TGF-β induction by areca nut could possibly be the epithelium. Further treatment of areca nut along with TGF-β on hGF cells potentiated TGF-β effect both in terms of TGF-β downstream targets like TGFBI, TGM2, TMEPAI, COL1A1 etc and activation of fibroblast by inducing α-SMA. Increasing concentration of areca nut is cytotoxic on HaCaT cells and pro-proliferative on hGF cells. This could provide a possible explanation for epithelial atrophy and proliferating fibroblast cells in connective tissue of OSF patients. Further exploration on HaCaT cell cytotoxicity by areca nut suggests the involvement of Reactive Oxygen Species (ROS) as a key molecule induced by areca nut. Compromising ROS generation by NAC (N-Acetyl-L-Cysteine) led to reversal of Sub-G1 peak induced by areca nut in HaCaT cells. This highlighted that cell death caused by areca nut could be ROS mediated. Areca nut treatment on hGF cells did not induce ROS generation, leading to no cytotoxicity on these cells. A possible explanation of this differential ROS generation can be due to dose dependent suppression of Catalase activity by areca nut in HaCaT cells but not in hGF cells. We also compared cytotoxicity of areca nut with all the alkaloids and found a good match with arecoline as both of them induce ROS, apoptotic ladder formation, annexin V positivity, suppression of Catalase activity and the cell death induced by them was compromised by NAC. The above results indicated that arecoline could be a mediator of areca nut water extract cytotoxicity on HaCaT cells. Betel nut chewer’s oral epithelium gets regularly exposed to areca nut and hence this exposure could be cytotoxic to oral epithelial cells too. We performed Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) in normal and OSF tissues. Our data showed 62.5% of OSF patients having significant percentage of epithelial cells with TUNEL positivity (Labeling index = 2-60%) compared to all normal tissues that were TUNEL negative. TUNEL positivity was predominantly seen in the upper keratin and supra basal layer of the epithelium. We also studied proliferation status of OSF epithelium and observed that 3-17% (LI) of epithelial cells in all normal tissues showed Ki-67 positivity in the germinal layer of epithelium. However, 65% of the OSF patients showed staining for Ki-67 (LI=.2-58%) in their epithelium. Also analysis of TUNEL positive and Ki-67 positive sections indicated that OSF patients with high TUNEL positivity have high Ki-67 labeling index, but stains in the supra basal or keratin layer (TUNEL) and basal layer (Ki-67) of epithelium respectively. This induced proliferation of epithelial cells could be the result of heavy apoptosis in the outer epithelium. But as these patients are regularly exposed to areca nut, this increased proliferation may not be able to cope up with the heavy apoptosis induced by areca nut, leading to atrophied epithelium. To understand the germinal status of OSF atrophied epithelium we performed staining for OCT4 in OSF tissues. To our surprise there were no OCT4 positive nuclei in the epithelium of 53% of OSF patients but a regular spread of OCT4 positivity has been seen in the epithelium of normal subjects. In conclusion, this thesis highlights the involvement of TGF-β pathway in OSF patho-physiology. In addition, activation of TGF-β pathway by areca nut constituents has been demonstrated. Moreover, the atrophied epithelium of OSF appears to be a consequence of apoptosis and stem cell deprivation. Taken together, areca nut perhaps causes atrophy of the epithelium and activates TGF-β pathway that may lead to manifestation of OSF.