Avaliação da incorporação de enxerto osseo homogeno fresco congelado em humanos: análise histologica e imuno-histoquímica

Allogeneic, fresh-frozen bone has been used in order to replace bone autografts. However, its osteoinduction and osteoconduction properties are not well-defined in the scientific literature. This work aimed to evaluate samples of homogenous bone grafts in humans by qualitative histological and immunohistochemical analysis. For this, ten pre-selected patients underwent surgical augmentation of bone defects. The homogenous fresh frozen block bone graft was stabilized and fixed by bicortical screws. After six months, the reopening procedure was performed for installation of osseointegrated implants. At this time surgical bone graft samples were removed by means of drill trephine. The samples were fixed in 10% formalin, processed with decalcified paraffin, and stained with hematoxylin and eosin. Immunohistochemistry was performed for the expression of Caspase 3 enzyme. The slides were brought to light microscopy for qualitative histology and immunohistochemistry. The results showed non-vital bone tissue, with few areas of deposition of new bone formation on the amorphous matrix, presence of chronic inflammatory infiltrate with areas of osteomyelitis, and expressive immunolabeling of Caspase 3. Given the methods employed and the results it was concluded that the allograft fresh-frozen block is not incorporated into the recipient bed after a healing period of six months.

Análise histológica e imunoistoquímica do tecido pulpar de ratos Wistar submetidos a procedimento clareador com duas concentrações de peróxido de hidrogênio

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Perfil fenotípico de potenciais células iniciadoras tumorais no tumor venéreo transmissível canino ex vivo

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Influência da suplementação de açaí (Euterpe oleracea Mart.) na ração sobre remodelação cardíaca em ratos expostos à fumaça do cigarro

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Modulação da atividade das interleucinas 17B e 25 associadas à melatonina como indutores de apoptose em cultivo celular de neoplasias mamárias

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Correlation between immunoexpression of BCL-2 protein family with apoptosis index, cellular proliferation and survival in colorectal carcinomas

Objectives: To evaluate the Bcl-2, Bax, Bad and Bak immunoexpression in tumor and nontumorous tissue of 130 patients with colorectal carcinoma submitted to surgery at São Paulo Hospital, EPM/ UNIFESP, from 2002 to 2005, and to correlate the immunoexpression data with the apoptotic index (AI, obtained by anti-cleaved caspase 3 and M30 labeling), cell proliferation score (CPS, obtained by Ki-67), immunoexpression of p53 and patient’s clinical prognosis. Results: Positive correlation was verified between Bcl-2 protein family in tumor and nontumor tissue. Only Bcl-2 protein correlated with IA and CPS in the tumor. Positive correlation was observed between pro- -apoptotic proteins and Bcl-2 protein. In the adjacent mucosa, Bcl-2 correlated with Ki-67 and p53, but not with IA. Carcinomas exhibited higher immunoexpression of CPS and IA markers. No correlation occurred between immunoexpression data and patient survival. Conclusion: Positive correlation was observed between the pro-apoptotic proteins of the Bcl-2 family and the anti-apoptotic protein Bcl-2. In the adjacent nontumor mucosa, Bcl-2 correlated with Ki-67 and p53, but not with AI. Carcinomas presented greater immunoexpression for CPS and AI markers; however immunoexpression of these markers was not correlated with patient survival.

Metabolismo de proteínas na musculatura esquelética de animais diabéticos tratados com iogurte enriquecido com curcumina

A perda de massa muscular observada no diabetes mellitus (DM) tipo 1 é consequência da combinação entre redução na velocidade de síntese proteica e aumento na velocidade de proteólise. A curcumina, pigmento amarelo extraído dos rizomas de Curcuma longa L., promove diversos benefícios no metabolismo de carboidratos e lipídeos no DM. Deste modo, buscamos avaliar o efeito do tratamento de ratos diabéticos com curcumina incorporada em iogurte sobre o metabolismo proteico muscular. Ratos Wistar machos (150±10 g) receberam estreptozotocina (40 mg/kg, i.v.) para indução do DM e foram divididos nos grupos (n=8): diabético tratado com iogurte (DIOG), 90 mg/kg de curcumina (DC90), 4U de insulina (DINS) e ratos normais, não diabéticos, tratados com iogurte (NIOG). Após 35 dias de tratamento, os animais foram eutanasiados e os músculos esqueléticos soleus e extensor digitorium longus (EDL) foram retirados e utilizados para a determinação das atividades proteolíticas de caspase-3, calpaína e proteassoma (atividade quimiotripsina-like). O tratamento de animais diabéticos com curcumina incorporada em iogurte reduziu a glicemia, os níveis de ureia urinária e promoveu um maior ganho de peso corporal em relação aos animais diabéticos tratados somente com iogurte (DIOG). Animais DIOG apresentaram um aumento nas atividades de calpaína e proteassoma em músculos soleus e EDL em relação aos valores encontrados em músculos de animais NIOG; já o tratamento com curcumina reduziu as atividades de calpaína e proteassoma em EDL de ratos diabéticos, o que explica, pelo menos em parte, a menor perda de massa deste músculo em ratos DC90. Houve uma redução na atividade de caspase-3 em músculos de animais DIOG em comparação aos grupos...

Influência da suplementação de alecrim (Rosmarinus oficinallis L.) na ração sobre a remodelação cardíaca em ratos submetidos ao infarto do miocádio

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

AÇÃO REGULATÓRIA DO GnRH NO DESENVOLVIMENTO EMBRIONÁRIO PRECOCE E VITRIFICAÇÃO DE EMBRIÕES SUÍNOS PELO MÉTODO DE MICROGOTA (ROLE OF GnRH DURING EARLY EMBRYONIC DEVELOPMENT AND DEVELOPMENT OF SWINE EMBRYOS FOLLOWING VITRIFICATION BY MICRODROPLET METHOD)

The objective of this study was to investigate the role of GnRH on the preimplantation development of mouse embryos in vitro. GnRH-I, GnRH-II, and GnRH agonists: Des-Gly, Des-Trp and histrelin did not improve embryo development. However, treatment with the specific GnRH antagonist SB-75 blocked embryo development at morula stage. The inhibition of embryo development by SB-75 could be rescued by the addition of histrelin. To determine which intracellular signaling cascade is involved following binding of GnRH to the GnRHR, embryos were cultured in the presence of specific PKC (GFX) or PKA (SQ22536) inhibitors. The PKC inhibitor blocked embryo development at a similar stage as SB-75, whereas SQ22536 had an inhibitory effect, diminishing blastocyst formation and hatched rates. There are evidences that GnRH has an essential autocrine effect on mouse embryonic development via GnRHR, probably by activating PKC signaling cascade while the inhibition of the GnRH signaling does not activate apoptotic mechanisms involving caspase-3. In another experiment, development in vitro of embryos from Chinese Meishan (M) and occidental white crossbred (WC) females were investigated after improving the vitrification protocol for pig embryos. Efficient cryopreservation of zona pellucida-intact porcine embryos and studies of the difference among breeds could greatly impact the swine industry. The percentage of embryos surviving 24 h after cryopreservation without lysis or degeneration was higher for M (72%) than WC (44%). However, in vitro development of embryos that survived cryopreservation was not different between M and WC at the expanded (64%) or hatched (22%) blastocyst stages. Developmental rates were significantly higher for control embryos than frozen embryos from both breeds at expanded blastocyst stage, but not at hatched blastocyst stage. Rates of expanded blastocyst formation did not differ between M and WC control embryos (98 and 95%, respectively). With a new procedure to warm vitrified pig embryos, the survival rates may be improved. The optimal stages to vitrify pig embryos using the microdroplet method ranges from late compact morula to early expanded blastocyst. The results suggest that M embryos have a higher capacity to survive the vitrification process than WC embryos. O objetivo do presente estudo foi investigar a importância do GnRH no desenvolvimento embrionário precoce em camundongos. GnRH-I, GnRH-II e os GnRH agonistas: Des-Gly, Des-Trp e histrelina não incrementaram o desenvolvimento embrionário. Entretanto, o tratamento com SB-75, um antagonista específico do GnRH, bloqueou o desenvolvimento embrionário no estádio de mórula. A inibição do desenvolvimento embrionário pelo SB-75 pôde ser revertida com a adição de histrelina. Para determinar a cascata do sinal intracelular desencadeada pela ligação do GnRH com o seu receptor, embriões foram cultivados na presença de inibidores específicos da PKC (GFX) e da PKA (SQ22536). O inibidor da PKC bloqueou o desenvolvimento embrionário em estádio similar ao bloqueio mediado pelo SB- 75, enquanto o SQ22536 teve efeito inibitório diminuindo a formação de blastocisto e taxas de eclosão. Os resultados sugerem que o GnRH tem um efeito autócrino essencial no desenvolvimento embrionário através do GnRHR, provavelmente, ativando a cascata da PKC. Por outro lado, a inibição do sinal do GnRH não ativa mecanismos apoptóticos que involvam caspase-3. Em outro experimento, foi investigado o desenvolvimento in vitro de embriões da raça Meishan (M) e branco cruzado (WC) após vitrificação pelo método microgota. O desenvolvimento de protocolos eficientes para criopreservação de embriões suínos com a zona pelúcida intacta e a avaliação das diferenças entre raças pode ter um significativo impacto na suinocultura. A percentagem de embriões que sobreviveram à criopreservação depois de 24 h foi maior na M (72%) do que na WC (44%). No entanto, o desenvolvimento in vitro dos embriões que sobreviveram à criopreservação não foi diferente entre M e WC nos estádios de blastocisto expandido (64%) ou eclodido (22%). Os índices de desenvolvimento foram significativamente mais altos para os embriões controle do que para os embriões vitrificados nas duas raças no estádio de blastocisto expandido, porém não foram diferentes para o estádio de blastocisto eclodido. A formação de blastocisto expandido não diferiu entre os embriões controle M e WC (98 e 95%, respectivamente). Com o novo procedimento (“hot warm”) para descongelar embriões vitrificados pelo método de microgota, pode-se aumentar dos índices de sobrevivência. Os melhores estádios embrionários para a vitrificação de embriões suínos variam de mórula compacta tardia até blastocisto expandido inicial. Os resultados sugerem que embriões M têm mais capacidade de sobreviver ao processo de vitrificação do que embriões WC.

Metabolismo de proteínas na musculatura esquelética de animais diabéticos tratados com iogurte enriquecido com curcumina

A perda de massa muscular observada no diabetes mellitus (DM) tipo 1 é consequência da combinação entre redução na velocidade de síntese proteica e aumento na velocidade de proteólise. A curcumina, pigmento amarelo extraído dos rizomas de Curcuma longa L., promove diversos benefícios no metabolismo de carboidratos e lipídeos no DM. Deste modo, buscamos avaliar o efeito do tratamento de ratos diabéticos com curcumina incorporada em iogurte sobre o metabolismo proteico muscular. Ratos Wistar machos (150±10 g) receberam estreptozotocina (40 mg/kg, i.v.) para indução do DM e foram divididos nos grupos (n=8): diabético tratado com iogurte (DIOG), 90 mg/kg de curcumina (DC90), 4U de insulina (DINS) e ratos normais, não diabéticos, tratados com iogurte (NIOG). Após 35 dias de tratamento, os animais foram eutanasiados e os músculos esqueléticos soleus e extensor digitorium longus (EDL) foram retirados e utilizados para a determinação das atividades proteolíticas de caspase-3, calpaína e proteassoma (atividade quimiotripsina-like). O tratamento de animais diabéticos com curcumina incorporada em iogurte reduziu a glicemia, os níveis de ureia urinária e promoveu um maior ganho de peso corporal em relação aos animais diabéticos tratados somente com iogurte (DIOG). Animais DIOG apresentaram um aumento nas atividades de calpaína e proteassoma em músculos soleus e EDL em relação aos valores encontrados em músculos de animais NIOG; já o tratamento com curcumina reduziu as atividades de calpaína e proteassoma em EDL de ratos diabéticos, o que explica, pelo menos em parte, a menor perda de massa deste músculo em ratos DC90. Houve uma redução na atividade de caspase-3 em músculos de animais DIOG em comparação aos grupos...

Influência da suplementação de alecrim (Rosmarinus oficinallis L.) na ração sobre a remodelação cardíaca em ratos submetidos ao infarto do miocádio

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Effect of N-acetylcysteine on pulmonary cell death in a controlled hemorrhagic shock model in rats

PURPOSE: To evaluate the effect of N-acetylcysteine (NAC) combined with fluid resuscitation on pulmonary cell death in rats induced with controlled hemorrhagic shock (HS). METHODS: Two arteries (MAP calculation and exsanguination) and one vein (treatments) were catheterized in 22 anesthetized rats. Two groups of male albino rats were induced with controlled HS at 35mmHg MAP for 60 min. After this period, the RL group was resuscitated with Ringer's lactate and the RL+NAC group was resuscitated with Ringer's lactate combined with 150mg/Kg NAC. The control group animals were cannulated only. The animals were euthanized after 120 min of fluid resuscitation. Lung tissue samples were collected to evaluate the following: histopathology, TUNEL and imunohistochemical expression of caspase 3. RESULTS: RL showed a greater number of cells stained by TUNEL than RL + NAC, but there was no change in caspase 3 expression in any group. CONCLUSION: N-acetylcysteine associate to fluid resuscitation, after hemorrhagic shock, decreased cell death attenuating lung injury.

Transforming growth factor beta and apoptosis in leprosy skin lesions: possible relationship with the control of the tissue immune response in the Mycobacterium leprae infection

The course of leprosy depends of the host immune response which ranges from the lepromatous pole (LL) to the tuberculoid pole (TT). A comparative study was conducted in 60 patients with the LL and TT The results showed a mean expression of TGF-beta of 339 +/- 99.4 cells/field for TT and of 519.2 +/- 68.2 cells/field for LL. Frequency of apoptosis was 6.3 +/- 1.8 in TT and 14.0 +/- 6.1 in LL. A correlation (p = 0.0251) between TGF-beta and caspase-3 in the LL was found. This finding indicates a role of TGF-beta and apoptosis in the immune response in leprosy. (C) 2012 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Neurotoxicity of Anhydroecgonine Methyl Ester, a Crack Cocaine Pyrolysis Product

Smoking crack cocaine involves the inhalation of cocaine and its pyrolysis product, anhydroecgonine methyl ester (AEME). Although there is evidence that cocaine is neurotoxic, the neurotoxicity of AEME has never been evaluated. AEME seems to have cholinergic agonist properties in the cardiovascular system; however, there are no reports on its effects in the central nervous system. The aim of this study was to investigate the neurotoxicity of AEME and its possible cholinergic effects in rat primary hippocampal cell cultures that were exposed to different concentrations of AEME, cocaine, and a cocaineAEME combination. We also evaluated the involvement of muscarinic cholinergic receptors in the neuronal death induced by these treatments using concomitant incubation of the cells with atropine. Neuronal injury was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. The results of the viability assays showed that AEME is a neurotoxic agent that has greater neurotoxic potential than cocaine after 24 and 48 h of exposure. We also showed that incubation for 48 h with a combination of both compounds in equipotent concentrations had an additive neurotoxic effect. Although both substances decreased cell viability in the MTT assay, only cocaine increased LDH release. Caspase-3 activity was increased after 3 and 6 h of incubation with 1mM cocaine and after 6 h of 0.1 and 1.0mM AEME exposure. Atropine prevented the AEME-induced neurotoxicity, which suggests that muscarinic cholinergic receptors are involved in AEME's effects. In addition, binding experiments confirmed that AEME has an affinity for muscarinic cholinergic receptors. Nevertheless, atropine was not able to prevent the neurotoxicity produced by cocaine and the cocaineAEME combination, suggesting that these treatments activated other neuronal death pathways. Our results suggest a higher risk for neurotoxicity after smoking crack cocaine than after cocaine use alone.

Effects of different tidal volumes in pulmonary and extrapulmonary lung injury with or without intraabdominal hypertension

We hypothesized that: (1) intraabdominal hypertension increases pulmonary inflammatory and fibrogenic responses in acute lung injury (ALI); (2) in the presence of intraabdominal hypertension, higher tidal volume reduces lung damage in extrapulmonary ALI, but not in pulmonary ALI. Wistar rats were randomly allocated to receive Escherichia coli lipopolysaccharide intratracheally (pulmonary ALI) or intraperitoneally (extrapulmonary ALI). After 24 h, animals were randomized into subgroups without or with intraabdominal hypertension (15 mmHg) and ventilated with positive end expiratory pressure = 5 cmH(2)O and tidal volume of 6 or 10 ml/kg during 1 h. Lung and chest wall mechanics, arterial blood gases, lung and distal organ histology, and interleukin (IL)-1 beta, IL-6, caspase-3 and type III procollagen (PCIII) mRNA expressions in lung tissue were analyzed. With intraabdominal hypertension, (1) chest-wall static elastance increased, and PCIII, IL-1 beta, IL-6, and caspase-3 expressions were more pronounced than in animals with normal intraabdominal pressure in both ALI groups; (2) in extrapulmonary ALI, higher tidal volume was associated with decreased atelectasis, and lower IL-6 and caspase-3 expressions; (3) in pulmonary ALI, higher tidal volume led to higher IL-6 expression; and (4) in pulmonary ALI, liver, kidney, and villi cell apoptosis was increased, but not affected by tidal volume. Intraabdominal hypertension increased inflammation and fibrogenesis in the lung independent of ALI etiology. In extrapulmonary ALI associated with intraabdominal hypertension, higher tidal volume improved lung morphometry with lower inflammation in lung tissue. Conversely, in pulmonary ALI associated with intraabdominal hypertension, higher tidal volume increased IL-6 expression.