DNA methylation pattern of apoptosis-related genes in ameloblastoma.

OBJECTIVES: DNA methylation is an important mechanism of gene control expression, and it has been poorly addressed in odontogenic tumours. On this basis, we aimed to assess the methylation pattern of 22 apoptosis-related genes in solid ameloblastomas. MATERIALS AND METHODS: Ameloblastoma fresh samples (n = 10) and dental follicles (n = 8) were included in the study. The percentage fraction of methylated and unmethylated DNA promoter of 22 apoptosis-related genes was determined using enzymatic restriction digestion and quantitative real-time PCR (qPCR) array. The relative expressions of the genes that showed the most discrepant methylation profile between tumours and controls were analysed by reverse-transcription quantitative PCR (RT-qPCR). RESULTS: Lower methylation percentages of TNFRSF25 (47.2%) and BCL2L11 (33.2%) were observed in ameloblastomas compared with dental follicles (79.3% and 59.5%, respectively). The RT-qPCR analysis showed increased expression of BCL2L11 in ameloblastomas compared with dental follicles, in agreement with the methylation analysis results, while there was no difference between the expression levels of TNFRSF25 between both groups. CONCLUSIONS: On the basis of our results, the transcription of the apoptosis-related gene BCL2L11 is possibly regulated by promoter DNA methylation in ameloblastoma. The biological significance of this finding in ameloblastoma pathobiology remains to be clarified.

Cutaneous loxoscelism caused by Loxosceles similis venom and neutralization capacity of its specific antivenom.

Members of the spider genus Loxosceles pose a marked health risk to humans because of the seriousness of the necrotic and systemic effects of their bite, known as loxoscelism. The recent confirmation of Loxosceles similis in residences of Belo Horizonte in Minas Gerais Province, Brazil increases the local potential risk of loxoscelism at higher levels. The first characterization of the venom from this species showed that its main biological effects had a similar intensity as other species (e.g. Loxosceles intermedia, Loxosceles laeta, and Loxosceles gaucho). Therefore, we wished to further analyse the biological activity of the L. similis venom as well as the capacity of anti-L. similis-venom serum to reduce dermonecrotic effects to rabbit skin. Histological analysis of rabbit skin 2, 4 and 8h after intradermal injection of L. similis venom demonstrated a dense inflammatory infiltrate, edema, degeneration and necrosis of the skin muscle, dissociation of collagen fibers, and disruption of reticular fibers. Importantly, pre-incubation of the venom with anti-L. similis-venom serum significantly decreased all of these effects. Anti-L. similis antivenom generated antibodies that were strongly reactive to L. similis venom and capable of neutralizing the dermonecrotic effects in rabbits caused by this venom. Moreover, the antivenom significantly reduced the sphingomyelinase activity of L. similis crude venom. Venoms produced by male and female spiders were equally reactive towards anti-L. similis and anti-L. intermedia antivenoms, but female venom induced larger lesions on rabbits. In contrast, female venom acted as an immunization enhancer and protected animals from L. similis envenomation to a greater degree than male venom. In conclusion, the results shown in this study for L. similis antivenom merits a more in depth study of its properties, which may become a valuable tool against loxoscelism.

Histopathological characterization of experimentally induced cutaneous loxoscelism in rabbits inoculated with Loxosceles similis venom

Envenomation by Loxosceles bites is characterized by dermonecrotic and/or systemic features that lead to several clinical signs and symptoms called loxoscelism. Dermonecrotic lesions are preceded by thrombosis of the dermal plexus. Recent studies show that atheromatous plaque is prone to thrombosis due to endothelial cell apoptosis. To the best of our knowledge, there are no reports of microscopic dermal lesion and endothelial cell apoptosis induced by Loxosceles similis venom in the literature. Thus, the aim of the present study is to describe histological lesions induced by L. similis venom in rabbit skin and to elucidate whether apoptosis of endothelial cells is involved in the pathogenesis of loxoscelism. Forty male rabbits were split into two groups: the control group (intradermally injected with 50 µL of PBS) and the experimental group (intradermally injected with 0.5 µg of L. similis crude venom diluted in 50 µL of PBS). After 2, 4, 6 and 8 hours of injection, skin fragments were collected and processed for paraffin or methacrylate embedding. Sections of 5 µm thick were stained by HE, PAS or submitted to TUNEL reaction. Microscopically, severe edema, diffuse heterophilic inflammatory infiltrate, perivascular heterophilic infiltrate, thrombosis, fibrinoid necrosis of arteriolar wall and cutaneous muscle necrosis were observed. Two hours after venom injection, endothelial cells with apoptosis morphology were evidenced in the dermal plexus. Apoptosis was confirmed by TUNEL reaction. It seems that endothelial cell apoptosis and its consequent desquamation is an important factor that induces thrombosis and culminates in dermonecrosis, which is characteristic of cutaneous loxoscelism.

Inflammatory response, parasite load and AgNOR expression in ear skin of symptomatic and asymptomatic Leishmania (Leishmania) chagasi infected dogs

The skin has an important role in the transmission of visceral leishmaniasis (VL) as the infection pathway in dogs. To better characterize the inflammatory response of intact skin in VL, sixty infected dogs (30 symptomatic and 30 asymptomatic) and six non-infected controls were studied. Diagnosis of visceral leishmaniasis was confirmed by RIFI and ELISA; direct visualization of the parasite in bone marrow aspirate; imprints of popliteal lymph nodes, spleen, liver and skin; culture in NNN-phase liquid Schneider's medium; and PCR (performed only in the ear skin). Amastigote forms of the parasite in intact skin were found only in symptomatic dogs. Inflammatory infiltrates were observed in all groups, varying from intense and/or moderate in symptomatic to discrete and/or negligible in asymptomatic and control animals. Parasite load was associated with the intensity of the inflammatory response and with clinical manifestations in canine visceral leishmaniasis. AgNOr as active transcription markers were expressed in inflammatory cells and within apoptotic bodies in all groups, including controls, with no statistical difference. Therefore, cell activation and transcription do occur in both symptomatic and asymptomatic canine visceral leishmaniasis and may result in more necrosis and inflammation or in apoptosis and less symptoms, depending on the parasite load.