A importância do uso das células tronco para a saúde pública

Em 1999, as células-tronco foram eleitas "Scientific Breakthrough of the Year" (avanço científico do ano) pela revista Science¹. Naquele ano, foi demonstrado que células-tronco de tecidos adultos mantinham a capacidade de se diferenciar em outros tipos de tecidos. No ano anterior, as primeiras linhagens de células-tronco embrionárias humanas foram estabelecidas. Desde então, o número de artigos científicos sobre células-tronco vem crescendo exponencialmente, onde novos paradigmas são estabelecidos. Neste artigo, farei uma revisão da área de células-tronco com um foco especial em seu uso como agente terapêutico em doenças comuns como diabetes e cardiopatias. As células-tronco serão tratadas em dois grupos distintos: as embrionárias e as adultas. Enquanto o potencial de diferenciação das primeiras está bem caracterizado em camundongos e em humanos, seu uso em terapia celular e em pesquisa tem sido dificultado por questões de histocompatibilidade, segurança e ética. Em contraste, células-tronco adultas não apresentam estes empecilhos, apesar da extensão de sua plasticidade ainda estar sob investigação. Mesmo assim, diversos testes clínicos em humanos estão em andamento utilizando células-tronco adultas, principalmente derivadas da medula óssea. Discutirei ainda a importância de se trabalhar com as duas classes de células-tronco humanas de forma a se cumprir suas promessas terapêuticas.

Avaliação da proliferação celular como indicador prognóstico para mastocitomas cutâneos caninos

Este estudo teve como objetivo avaliar o valor prognóstico de marcadores de proliferação celular em casos de mastocitomas cutâneos caninos. Vinte e três casos foram analisados quanto à expressão imuno-histoquímica de Ki67 e do Antígeno Nuclear de Proliferação Celular (PCNA), sendo subsequentemente acompanhados clinicamente. Observou-se que a expressão de Ki67 mantém relação negativa com a tradicional graduação histopatológica (p= 0,0418; p<0,05 entre os graus I e III), sendo um indicador confiável para o tempo de sobrevida pós-cirúrgica (p=0,0089). A imunoexpressão de PCNA, apesar de estar correlacionada à marcação por Ki67, não apresentou valores estatisticamente significantes na predição da mortalidade em função da doença e do tempo de sobrevida pós-cirúrgico. Os resultados obtidos confirmam que informações sobre a atividade proliferativa tumoral pela detecção imuno-histoquímica de Ki67 podem incrementar a classificação de mastocitomas cutâneos caninos quanto à malignidade.

Modelo de suplementação nutricional com fatores hepatotróficos aumenta proliferação celular em fígado de ratos sadios

Foram avaliados dois protocolos de administração, em ratos sadios, de uma solução de fatores hepatotróficos (FH), composta por aminoácidos, vitaminas, sais minerais, glicose, insulina, glucagon e triiodotironina (T3). A solução foi administrada durante 10 dias, 40mg/kg/dia, i.p., em duas, grupo 2xFH (n=15), ou três doses, grupo 3xFH (n=15), diárias. Foram observados os efeitos na proliferação celular dos hepatócitos, na angiogênese e na matriz extracelular hepática, assim como as possíveis reações adversas. Os animais dos grupos 2xFH e 3xFH apresentaram aumento da massa hepática de 30,1% e 22,5%, respectivamente, em relação ao grupo-controle (CT; n=15). O índice de proliferação hepatocelular foi maior nos grupos 2xFH (1,4%) e 3xFH (1,2%) em relação ao grupo CT (0,53%), e a densitometria relativa do fator de crescimento do endotélio vascular pelo imunoblot não revelou diferença estatística entre os três grupos. Nos grupos 2xFH e 3xFH, houve redução do colágeno intersticial em relação ao grupo CT. A solução de FH estimulou o crescimento hepático e reduziu o volume de colágeno perissinusoidal. A administração em três doses diárias resultou em mortalidade de 26,7%, possivelmente pelo excessivo estresse da manipulação e pela menor adaptação fisiológica dos ratos, o que não ocorreu nos grupos 2xFH e CT. Para esse tipo de abordagem em ratos, o procedimento experimental mais apropriado, seguro, com melhor chance de adaptação dos animais e com resultados significativos é a aplicação dos FH em duas doses diárias.

Establishment of a protocol for obtention of neuronal stem cells lineages from the dog olfactory epithelium

A morphological and cell culture study from nasal mucosa of dogs was performed in order to establish a protocol to obtain a cell population committed to neuronal lineage, as a proposal for the treatment of traumatic and degenerative lesions in these animals, so that in the future these results could be applied to the human species. Twelve mongrel dogs of 60-day aged pregnancy were collected from urban pound dogs in São Paulo. Tissue from cribriform ethmoidal lamina of the fetuses was collected at necropsy under sterile conditions around 1h to 2h postmortem by uterine sections and sections from the fetal regions described above. Isolated cells of this tissue were added in DMEM/F-12 medium under standard conditions of incubation (5% CO², >37ºC). Cell culture based on isolated cells from biopsies of the olfactory epithelium showed rapid growth when cultured for 24 hours, showing phase-bright sphere cells found floating around the fragments, attached on culture flasks. After 20 days, a specific type of cells, predominantly ellipsoids or fusiform cells was characterized in vitro. The indirect immunofluorescence examination showed cells expressing markers of neuronal precursors (GFAP, neurofilament, oligodendrocyte, and III â-tubulin). The cell proliferation index showed Ki67 immunostaining with a trend to label cell groups throughout the apical region, while PCNA immunostaining label predominantly cell groups lying above the basal lamina. The transmission electron microscopy from the olfactory epithelium of dogs revealed cells with electron-dense cytoplasm and preserving the same distribution as those of positive cell staining for PCNA. Metabolic activity was confirmed by presence of euchromatin in the greatest part of cells. All these aspects give subsidies to support the hypothesis about resident progenitor cells among the basal cells of the olfactory epithelium, committed to renewal of these cell populations, especially neurons.

Estudo clínico da aplicação de matriz inorgânica de osso associado a peptídeo sintético de adesão celular (MIO/P-15), PepGen P-15®, em lesões periodontais avançadas de cães

Os mecanismos biológicos desenvolvidos para aumentar a qualidade da regeneração óssea e da reparação tecidual de sítios periodontais específicos continuam a ser um desafio e têm sido complementado pela capacidade de adesão celular do colágeno do tipo I, promovida por um peptídeo sintético de adesão celular (P-15), associado a uma matriz inorgânica de osso (MIO) para formar MIO/P-15. O objetivo deste estudo foi avaliar a perda do nível clínico de inserção e a resposta da bolsa periodontal em dentes após 3 e 6 meses da aplicação de enxerto com MIO/P-15. Vinte e um cães do Hospital Veterinário da Universidade de São Paulo foram anestesiados para realização de tratamento periodontal e 132 faces dentais com perda de nível clínico de inserção foram tratadas, sendo que 36,4% (48 faces) receberam o peptídeo de adesão celular e 63,6% (84 faces) compuseram o grupo controle que recebeu tratamento convencional (retalho muco-gengival e aplainamento radicular). O procedimento foi documentado através de radiografia intra-oral e todas as sondagens de bolsas periodontais foram fotografadas. Depois de 3 e de 6 meses, os animais foram re-anestesiados a fim de se obter novas avaliações, radiografias, fotografias e sondagens periodontais. As 48 faces com perda de nível clínico de inserção que receberam material de enxertia apresentaram taxa de 40% de recuperação do nível clínico de inserção após 6 meses. O grupo controle de faces dentais não apresentou alteração do nível clínico de inserção. A face palatina foi a que apresentou melhor taxa de regeneração (40%) e os dentes caninos e molares mostraram as melhores respostas (57,14% e 65%, respectivamente). Não houve sinais de infecção pós-cirúrgica relacionadas à falta de higienização oral dos animais. Pode-se concluir que o MIO/P-15 auxilia na regeneração e re-aderência das estruturas periodontais, incluindo osso alveolar. Sua aplicação mostrou-se fácil e prática e a incidência de complicações pós-cirúrgicas foi baixa. Ainda assim, mais estudos e pesquisas são necessários para que se avalie a quantidade e a qualidade do osso e do ligamento periodontal formados.

Análise proliferativa nas células trofoblásticas em bovinos

The aim of the present study was to evaluate the cell proliferative activity, by AgNORs number, in different regions of bovine placenta throughout gestation. A total of 28 bovine placentas were separated into four groups: group I (60 to 120 days), group II (121 to 170 days), group III (171 to 220 days), and group IV (221 to 290 days). It was found a greater number of AgNORs in giant trophoblastic cells (GTC) when compared with mononuclear trophoblastic cells (MTC) (p<0,001) in all regions and gestational groups analyzed, that confirms their intensive synthesis activity in trophoblast epithelium. The central region of the placentome begins an intense proliferative activity in group II, observed by clusters, while placentomes edges showed a higher number of clusters on group III. These data suggest that the central region of the placentomes began an intense proliferative activity prior to its edge, both declines at the end of pregnancy. Interplacentomal area showed a higher number of AgNORs in the group IV, suggesting a higher proliferative activity of these cells at the end of pregnancy. The results of this study indicate that the proliferative activity, as determined by the amount of intranuclear AgNORs, exhibits patterns that are not only specific to each type of trophoblastic cells, but also for each specific region of bovine placenta throughout pregnancy.

Histopathological evaluation of root canal filling materials for primary teeth

This study aimed to assess the response of apical and periapical tissues of dogs' teeth after root canal filling with different materials. Forty roots from dogs' premolars were prepared biomechanically and assigned to 4 groups filled with: Group I: commercial calcium hydroxide and polyethylene glycol-based paste (Calen®) thickened with zinc oxide; Group II: paste composed of iodoform, Rifocort® and camphorated paramonochlorophenol; Group III: zinc oxide-eugenol cement; Group IV: sterile saline. After 30 days, the samples were subjected to histological processing. The histopathological findings revealed that in Groups I and IV the apical and periapical regions exhibited normal appearance, with large number of fibers and cells and no resorption of mineralized tissues. In Group II, mild inflammatory infiltrate and mild edema were observed, with discrete fibrogenesis and bone resorption. Group III showed altered periapical region and thickened periodontal ligament with presence of inflammatory cells and edema. It may be concluded that the Calen paste thickened with zinc oxide yielded the best tissue response, being the most indicated material for root canal filling of primary teeth with pulp vitality.

Alveolar osseous defect in rat for cell therapy: preliminary report

PURPOSE: To study were to reproduce an alveolar bone defect model in Wistar rats to be used for testing the efficacy of stem cell therapies. Additionally, we also aimed to determine the osteogenesis process of this osseous defect in the 1 month period post-surgery. METHODS: The animals were randomly divided into two groups of 7 animals each. A gingivobuccal incision was made, and a bone defect of 28 mm² of area was performed in the alveolar region. Animals were killed at 2 weeks after surgery (n=7) and 4 weeks after surgery (n=7). RESULTS: The average area of the alveolar defect at time point of 2 weeks was 22.27 ± 1.31 mm² and the average area of alveolar defect at time point of 4 weeks was 9.03 ± 1.17 mm². The average amount of bone formation at time point of 2 weeks was 5.73 ± 1.31 mm² and the average amount of bone formation at time point of 4 weeks was 19 ± 1.17 mm². Statistically significant differences between the amount of bone formation at 2 weeks and 4 weeks after surgery were seen (p=0.003). CONCLUSION: The highest rate of ossification occurred mostly from 2 to 4 weeks after surgery. This observation suggests that 4 weeks after the bone defect creation should be a satisfactory timing to assess the potential of bone inductive stem cells to accelerate bone regeneration in Wistar rats.

Bleaching of melanin in the epidermis of South American fur seal and its application on enzyme immunohistochemistry

The South American fur seal (Arctocephalus australis) is an amphibious marine mammal distributed along the Atlantic and Pacific coasts of South America. The species is well adjusted to different habitats due to the morphology of its fin-like members and due to some adaptations in their integumentary system. Immunohistochemical studies are very important to evaluate the mechanisms of skin adaptation due the differential expression of the antigens present in the tissue depending of the region of the body surface. However, its strongly pigmented (melanin) epidermis prevents the visualization of the immuno-histochemical chromogens markers. In this study a melanin bleaching method was developed aimed to allow the visualization of the chromogens without interfering in the antigen-antibody affinity for immunohistochemistry. The analysis of PCNA (proliferating cell nuclear antigen) index in the epidermis of A. australis by immunohistochemistry with diaminobenzidine (DAB) as chromogen was used to test the method. The bleaching of the melanin allowed to obtain the cell proliferation index in epidermis and to avoid false positive results without affecting the immunohistochemical results.

Protocols for obtainment and isolation of two mesenchymal stem cell sources in sheep

PURPOSE: To evaluate different protocols to isolate stem cells from ovine umbilical cord blood and adipose tissue. METHODS: There were used 5 samples of umbilical blood and 5 samples of perirenal adipose tissue from 10 female sheep. All the samples were obtained through surgery, to harvest aseptic samples. There were used 3 protocols for obtainment and culture of umbilical cord blood stem cells and 4 protocols for ovine adipose tissue stem cells. RESULTS: It was possible to observe only one successful protocol for the obtainment of umbilical cord blood stem cells. When analyzing the techniques used to obtain adipose tissue stem cells, only one of the methods was effective as well. Through colony forming unit assay, there were obtained 58 colonies of cells after seven days in culture. Flow citometry tests revealed the cells were positive to CD44 and exhibited negative reaction to CD38, CD45, CD41/61. These cells showed a growth curve with very well defined phases LOG, LAG and PLATEAU. This phases are typically seem in mesenchymal stem cells growth curves. CONCLUSIONS: The isolation and culture of mesenchymal stem cells from ovine umbilical cord blood are complex and request more detailed assays. Stem cells from fat tissue sheep showed mesenchymal characteristics, according to their cell growth curve, ability to origin colonies of fibroblastoid cells and positive reactivity with the antibody CD44 by flow citometry.

Successful transplant of mesenchymal stem cells in induced osteonecrosis of the ovine femoral head: preliminary results

PURPOSE: Evaluate the bone tissue recovery following transplantation of ovine mesenchymal stem cells (MSC) from bone marrow and human immature dental-pulp stem cells (hIDPSC) in ovine model of induced osteonecrosis of femoral head (ONFH). METHODS: Eight sheep were divided in three experimental groups. First group was composed by four animals with ONFH induced by ethanol through central decompression (CD), for control group without any treatment. The second and third group were compose by two animals, six weeks after ONFH induction received transplantation of heterologous ovine MSC (CD + oMSC), and hIDPSC (CD + hIDPSC), respectively. In both experiments the cells were transplanted without application of any type of immunosupression protocol. RESULTS: Our data indicate that both cell types used in experiments were able to proliferate within injured site providing bone tissue recovery. The histological results obtained from CD+hIDPSC suggested that the bone regeneration in such animals was better than that observed in CD animals. CONCLUSION: Mesenchymal stem cell transplant in induced ovine osteonecrosis of femoral head by central decompression technique is safe, and apparently favors bone regeneration of damaged tissues.

Controle da adipogênese por ácidos graxos

A obesidade é um dos principais problemas de saúde pública. Indivíduos obesos são mais suscetíveis a desenvolver doenças cardiovasculares e diabetes melito tipo 2. A obesidade resulta do aumento no tamanho e no número de adipócitos. O balanço entre adipogênese e adiposidade determina o grau de obesidade do indivíduo. Adipócitos maduros secretam adipocinas, tais como TNF&#945;, IL-6, leptina e adiponectina, e lipocina, o ácido palmitoleico &#969;-7. A produção de adipocinas é maior na obesidade, o que contribui para o estabelecimento de resistência periférica à insulina. O conhecimento dos eventos moleculares que regulam a diferenciação dos pré-adipócitos e de células-tronco mesenquimais em adipócitos (adipogênese) é importante para o entendimento da gênese da obesidade. A ativação do fator de transcrição PPAR&#947; é essencial na adipogênese. Certos ácidos graxos são ligantes de PPAR&#947; e podem, assim, controlar a adipogênese. Além disso, alguns ácidos graxos atuam como moléculas sinalizadoras em adipócitos, regulando sua diferenciação ou morte. Dessa forma, a composição lipídica da dieta e os agonistas de PPAR&#947; podem regular o balanço entre adipogênese e morte de adipócitos e, portanto, a obesidade.

HLA-G 14-bp Insertion/Deletion Polymorphism Is a Risk Factor for HTLV-1 Infection

About 95% of HTLV-1 infected patients remain asymptomatic throughout life, and the risk factors associated with the development of related diseases, such as HAM/TSP and ATL, are not fully understood. The human leukocyte antigen-G molecule (HLA-G), a nonclassical HLA class I molecule encoded by MHC, is expressed in several pathological conditions, including viral infection, and is related to immunosuppressive effects that allow the virus-infected cells to escape the antiviral defense of the host. The 14-bp insertion/deletion polymorphism of exon 8 HLA-G gene influences the stability of the transcripts and could be related to HTLV-1-infected cell protection and to the increase of proviral load. The present study analyzed by conventional PCR the 14-bp insertion/deletion polymorphism of exon 8 HLA-G gene in 150 unrelated healthy subjects, 82 HTLV-1 infected patients with symptoms (33 ATL and 49 HAM), and 56 asymptomatic HTLV-1 infected patients (HAC). In addition, the proviral load was determined by quantitative real-time PCR in all infected groups and correlated with 14-bp insertion/deletion genotypes. The heterozygote genotype frequencies were significantly higher in HAM, in the symptomatic group, and in infected patients compared to control (p < 0.05). The proviral load was higher in the symptomatic group than the HAC group (p < 0.0005). The comparison of proviral load and genotypes showed that -14-bp/-14-bp genotype had a higher proviral load than +14-bp/-14-bp and +14-bp/+14-bp genotypes. Although HLA-G 14-bp polymorphism does not appear to be associated