A comparative study of apoptosis in reticular and erosive oral lichen planus

The oral lichen planus (OLP) is a chronic inflammatory disease, probably autoimmune, with different clinical forms. The most common types are the reticular and the erosive ones. Apoptosis participates in the destruction of basal keratinocytes, but its role in the perpetuation of the subepithelial lymphocytic infiltrates was not yet investigated. To evaluate the involvement of apoptosis in the epithelium and in subepithelial lymphocytic infiltrates, 15 samples of reticular and erosive OLP and 10 samples of healthy oral mucosa were collected and processed histologically. Apoptosis was quantified in the epithelium and in inflammatory cell infiltrates. TUNEL reaction was used to measure apoptosis in the infiltrates. Erosive OLP showed more intense epithelial apoptosis than reticular OLP and controls. In contrast, apoptosis in the inflammatory cell infiltrates was more frequent in reticular than in erosive OLP. Lymphocytes were the predominant cells within the inflammatory cell infiltrates and were more frequent in erosive OLP than in reticular type. These results suggest that different apoptotic levels are involved in the erosive/reticular switch in OLP, determining different clinical presentations. In conclusion, decreased apoptosis in inflammatory infiltrates may contribute to the persistence of T lymphocytes, worsening the attack to the epithelium in erosive OLP.

O líquen plano oral (LPO) é uma doença crônica inflamatória, provavelmente auto-imune, com diferentes formas clínicas. Os tipos mais comuns são o reticular e o erosivo. A apoptose participa da destruição dos ceratinócitos basais, no entanto o seu papel na perpetuação do infiltrado linfocitário subepitelial ainda não foi investigado. Para avaliar o envolvimento da apoptose no epitélio e no infiltrado linfocitário subepitelial, quinze amostras de LPO reticular, quinze de LPO erosivo e dez amostras de mucosa oral saudável foram coletadas e processadas histologicamente. A apoptose foi quantificada no epitélio e nas células do infiltrado inflamatório. A reação de TUNEL foi usada para mensurar a apoptose no infiltrado. A intensidade da apoptose no epitélio mostrou ser maior no LPO erosivo que no LPO reticular e estes foram maiores que no controle. Em contraste, a apoptose nas células do infiltrado inflamatório foi mais freqüente no LPO reticular que no LPO erosivo. Os linfócitos foram as células predominantes dentro do infiltrado inflamatório e foram mais freqüentes no tipo erosivo de LPO que no tipo reticular. Estes resultados sugerem que diferentes níveis de apoptose estão envolvidos no tipo erosivo e reticular de LPO, determinando as diferenças nas apresentações clínicas. Em conclusão, a diminuição da apoptose no infiltrado inflamatório pode contribuir para a persistência dos linfócitos T, piorando o ataque ao epitélio no LPO erosivo.

A comparative study of apoptosis in reticular and erosive oral lichen planus.

The oral lichen planus (OLP) is a chronic inflammatory disease, probably autoimmune, with different clinical forms. The most common types are the reticular and the erosive ones. Apoptosis participates in the destruction of basal keratinocytes, but its role in the perpetuation of the subepithelial lymphocytic infiltrates was not yet investigated. To evaluate the involvement of apoptosis in the epithelium and in subepithelial lymphocytic infiltrates, 15 samples of reticular and erosive OLP and 10 samples of healthy oral mucosa were collected and processed histologically. Apoptosis was quantified in the epithelium and in inflammatory cell infiltrates. TUNEL reaction was used to measure apoptosis in the infiltrates. Erosive OLP showed more intense epithelial apoptosis than reticular OLP and controls. In contrast, apoptosis in the inflammatory cell infiltrates was more frequent in reticular than in erosive OLP. Lymphocytes were the predominant cells within the inflammatory cell infiltrates and were more frequent in erosive OLP than in reticular type. These results suggest that different apoptotic levels are involved in the erosive/reticular switch in OLP, determining different clinical presentations. In conclusion, decreased apoptosis in inflammatory infiltrates may contribute to the persistence of T lymphocytes, worsening the attack to the epithelium in erosive OLP.

Caspase-3/caspase-8, bax and bcl2 in pulps of human primary teeth with physiological root resorption.

OBJECTIVE. Physiological root resorption is a programmed event that takes place in primary teeth leading to elimination of all root structures. The mechanism behind pulp elimination indicates apoptosis, but its pathway has not been well characterised yet. To better understand this event, we evaluated the gene expression of bax, bcl-2, caspase-3 and caspase-8 through real-time polymerase chain reaction (PCR) and immunohistochemistry expression of Caspase-8 and Bax in pulps. METHODS. Samples were split into two groups: pulps from primary teeth with physiological root resorption (n = 40) and control (n =40), pulps from permanent teeth. Samples of each group were split into PCR (n = 20) and immunohistochemistry (n = 20). RESULTS. Pulps from primary teeth showed a higher caspase-3 mRNA level than pulps from permanent teeth. The expression of bax gene was more intense than caspase-8 but both did not show difference between groups. The bcl-2 mRNA level was incipient and similar between groups. Histopath slides did not show any evidence of inflammatory infiltration, which implies that extrinsic via is not likely to be involved. Immunohistochemistry reaction to Bax and Caspase-8 supported PCR results. CONCLUSIONS. Pulp apoptosis is likely to occur via caspase-3 activation through the mitochondrial pathway.

Apoptose na maturação placentária de vacas em diferentes estágios de gestação: evidenciação imuno-histoquímica e bioquímica / Apoptosis in placental maturation of cows in different stages of pregnancy: Immunohistochemical and biochemical evidences

Apoptose tem um papel importante na manutenção da homeostase placentária, e o desequilíbrio desse processo pode comprometer a gestação. O objetivo do presente estudo foi avaliar a ocorrencia de apoptose em amostras de placenta de vacas em diferentes fases de gestação. Amostras de placentomos de 15 vacas saudáveis com 4 (n=5), 6 (n=5) e 9 (n=5) meses de gestação foram colhidas e processadas rotineiramente para a histologia, imuno-histoquímica e isolamento de DNA. As lâminas obtidas foram coradas em HE, ou submetidas à análise imuno-histoquímica das proteínas pró-apoptóticas caspase-3 e Bax, e da proteína anti-apoptótica Bcl-2. O DNA isolado foi submetido à eletroforese em gel de agarose para detecção da fragmentação internucleossômica do genoma. Os resultados de histomorfometria revelaram que as células apoptóticas aumentaram progressivamente com o avanço da gestação. Confirmou-se a apoptose pela fragmentação característica do DNA genômico, visualizada pelo clássico "padrão em escada" na eletroforese em gel de agarose. Adcionalmente, a imunoexpressão de caspase-3, Bax e Bcl-2 foram observadas em todas as amostras. Entretanto, a proteína caspase-3 apresentou marcação mais intensa em todos os tempos gestacionais, quando comparada com a marcação das proteínas Bcl-2 e Bax. Esses resultados confirmam e reforçam a importância da apoptose na maturação placentária. Além disto, indica que caspase-3, Bax e Bcl-2 estão envolvidas nos mecanismos de ativação da apoptose pela via intrínseca mitocondrial ao longo da gestação, contribuindo para o equilíbrio fisiológico da celularidade e renovação celular na placenta bovina.(AU)

Apoptosis is important for placental homeostasis maintenance, and a misbalance in this process may compromise the success of pregnancy. The aim of this study is to evaluate apoptosis in bovine placental samples at different stages of pregnancy. Placentome samples from 15 healthy cows, at gestational ages of 4 (n=5), 6 (n=5) and 9 (n=5) months were collected and routinely processed for histological and immunohistochemical analysis, and for DNA isolation. Histopathology sections were stained with HE. Others were submitted to immunohistochemistry, using pro-apoptotic caspase-3 and Bax protein, and anti-apoptotic Bcl-2 specific antibodies. Isolated DNAs were processed for electrophoresis in agarose gel, for detection of internucleosomal fragmentation. Histomorphometry results demonstrated that apoptotic cells gradually increase with the advance of the gestation. Also, the DNA ladder pattern was observed in all groups. In addition, caspase-3, Bax and Bcl-2 were all expressed in the three different gestation periods. However, caspase-3 presented a higher expression in all groups, in comparison to Bcl-2 and Bax. These results confirm the importance of the apoptosis in the placental maturation. Also, these results indicated that caspase-3, Bcl-2 and Bax are involved in apoptotic activation mechanisms by mitochondrial intrinsic pathway during placental maturation, contributing for physiological cellularity and cellular turn over balance in bovine placenta.(AU)

Apoptose na maturação placentária de vacas em diferentes estágios de gestação: evidenciação imuno-histoquímica e bioquímica / Apoptosis in placental maturation of cows in different stages of pregnancy: Immunohistochemical and biochemical evidences

Apoptose tem um papel importante na manutenção da homeostase placentária, e o desequilíbrio desse processo pode comprometer a gestação. O objetivo do presente estudo foi avaliar a ocorrencia de apoptose em amostras de placenta de vacas em diferentes fases de gestação. Amostras de placentomos de 15 vacas saudáveis com 4 (n=5), 6 (n=5) e 9 (n=5) meses de gestação foram colhidas e processadas rotineiramente para a histologia, imuno-histoquímica e isolamento de DNA. As lâminas obtidas foram coradas em HE, ou submetidas à análise imuno-histoquímica das proteínas pró-apoptóticas caspase-3 e Bax, e da proteína anti-apoptótica Bcl-2. O DNA isolado foi submetido à eletroforese em gel de agarose para detecção da fragmentação internucleossômica do genoma. Os resultados de histomorfometria revelaram que as células apoptóticas aumentaram progressivamente com o avanço da gestação. Confirmou-se a apoptose pela fragmentação característica do DNA genômico, visualizada pelo clássico "padrão em escada" na eletroforese em gel de agarose. Adcionalmente, a imunoexpressão de caspase-3, Bax e Bcl-2 foram observadas em todas as amostras. Entretanto, a proteína caspase-3 apresentou marcação mais intensa em todos os tempos gestacionais, quando comparada com a marcação das proteínas Bcl-2 e Bax. Esses resultados confirmam e reforçam a importância da apoptose na maturação placentária. Além disto, indica que caspase-3, Bax e Bcl-2 estão envolvidas nos mecanismos de ativação da apoptose pela via intrínseca mitocondrial ao longo da gestação, contribuindo para o equilíbrio fisiológico da celularidade e renovação celular na placenta bovina.

Apoptosis is important for placental homeostasis maintenance, and a misbalance in this process may compromise the success of pregnancy. The aim of this study is to evaluate apoptosis in bovine placental samples at different stages of pregnancy. Placentome samples from 15 healthy cows, at gestational ages of 4 (n=5), 6 (n=5) and 9 (n=5) months were collected and routinely processed for histological and immunohistochemical analysis, and for DNA isolation. Histopathology sections were stained with HE. Others were submitted to immunohistochemistry, using pro-apoptotic caspase-3 and Bax protein, and anti-apoptotic Bcl-2 specific antibodies. Isolated DNAs were processed for electrophoresis in agarose gel, for detection of internucleosomal fragmentation. Histomorphometry results demonstrated that apoptotic cells gradually increase with the advance of the gestation. Also, the DNA ladder pattern was observed in all groups. In addition, caspase-3, Bax and Bcl-2 were all expressed in the three different gestation periods. However, caspase-3 presented a higher expression in all groups, in comparison to Bcl-2 and Bax. These results confirm the importance of the apoptosis in the placental maturation. Also, these results indicated that caspase-3, Bcl-2 and Bax are involved in apoptotic activation mechanisms by mitochondrial intrinsic pathway during placental maturation, contributing for physiological cellularity and cellular turn over balance in bovine placenta.

Role of apoptosis in erosive and reticular oral lichen planus exhibiting variable epithelial thickness.

Oral lichen planus (OLP) is a chronic inflammatory disease with different clinical types. Reticular and erosive forms are the most common. Although the cause of OLP remains speculative, many findings suggest auto-immune involvement, mediated by T lymphocytes against the basal keratinocytes. Inflammation, mechanical trauma or toxic agents can affect the epithelial homeostasia. Increased apoptosis may cause a decrease in epithelial thickness reflecting in the activity of the lesion. The objective of this study was to evaluate the occurrence of apoptosis and epithelial thickness in reticular and erosive forms of OLP. 15 samples of OLP each type (reticular and erosive) plus 10 of healthy mucosa were collected and processed. After morphometry, the apoptotic index and epithelial thickness were obtained. TUNEL and M30 CytoDEATH immunohistochemical assay were used to validate the morphologic criteria used. Apoptosis in the erosive OLP was significantly more intense than in the reticular type and both forms of OLP presented more apoptosis than the healthy oral mucosa. Healthy oral mucosa was thicker than both OLP forms and thicker in OLP reticular form than in the erosive one. The clinical differences between reticular and erosive forms of OLP are related to variations in epithelial thickness and in intensity of apoptosis.

Role of apoptosis in erosive and reticular oral lichen planus exhibiting variable epithelial thickness

Oral lichen planus (OLP) is a chronic inflammatory disease with different clinical types. Reticular and erosive forms are the most common. Although the cause of OLP remains speculative, many findings suggest auto-immune involvement, mediated by T lymphocytes against the basal keratinocytes. Inflammation, mechanical trauma or toxic agents can affect the epithelial homeostasia. Increased apoptosis may cause a decrease in epithelial thickness reflecting in the activity of the lesion. The objective of this study was to evaluate the occurrence of apoptosis and epithelial thickness in reticular and erosive forms of OLP. 15 samples of OLP each type (reticular and erosive) plus 10 of healthy mucosa were collected and processed. After morphometry, the apoptotic index and epitelial thickness were obtained. TUNEL and M30 CytoDEATH immunohistochemical assay were used to validate the morphologic criteria used. Apoptosis in the erosive OLP was significantly more intense than in the reticular type and both forms of OLP presented more apoptosis than the healthy oral mucosa. Healthy oral mucosa was thicker than both OLP forms and thicker in OLP reticular form than in the erosive one. The clinical differences between reticular and erosive forms of OLP are related to variations in epithelial thickness and in intensity of apoptosis.

O líquen plano oral (LPO) é uma doença inflamatória crônica com diferentes tipos clínicos. As mais comuns são as formas reticular e erosiva. Embora a causa do LPO permaneça no campo especulativo, muitos achados sugerem tratar-se de uma doença auto-imune, mediada por linfócitos T que têm como alvo os ceratinócitos basais. Inflamação, trauma mecânico ou agentes tóxicos podem afetar a homeostasia epitelial. O aumento da apoptose pode levar a uma diminuição da espessura epitelial e isto refletir na atividade da doença. O objetivo deste estudo foi avaliar a ocorrência de apoptose e a espessura epitelial nas formas reticular e erosiva de LPO. 15 amostras de LPO de cada tipo reticular e erosivo, além de 10 amostras de mucosa saudável foram coletadas e processadas. Depois da morfometria, o índice apoptótico (IA) e a espessura do epitélio foram obtidas. Reação de TUNEL e imunohistoquímica do M30 CytoDeath foram usadas para validação dos critérios morfológicos. A apoptose no LPO erosivo foi significativamente maior que no tipo reticular e ambas as formas de LPO apresentaram mais apoptose que a mucosa oral normal. A mucosa oral normal foi mais espessa que ambas as formas de LPO, sendo que, a forma reticular foi mais espessa que o tipo erosivo. As diferenças clínicas entre as formas reticular e erosiva de LPO têm relação com as variações na espessura epitelial e na intensidade da apoptose.