RESUMO
CD59 is one of the key molecules involved in cell protection against autologus complement. The fact that complement regulatory proteins are able to prevent hyperacute rejection of organs in pig to primate model, raises the question of possible complement regulatory protein (CRP) involvement in the maturation of immunological system. We report here that in foetal and postnatal human thymus, CD59 and CD55 are primarily located on Hassall's corpuscles and medullary epithelial cells. This localization highly correlates with the expression of CD30L, which is the member of the tumour necrosis factor superfamily. Additionally, TUNEL technique was used to visualize distribution of apoptotic cells in the thymus, which revealed the presence of apoptotic cells closely associated with the Hassall's corpuscles. The observed co-localization of CD59, CD55 and CD30L might suggest an involvement of the complement system in thymic selection in humans.
Assuntos
Antígenos CD55/biossíntese , Antígenos CD59/biossíntese , Células Epiteliais/metabolismo , Feto/metabolismo , Regulação da Expressão Gênica/fisiologia , Timo/metabolismo , Adolescente , Apoptose/fisiologia , Antígenos CD55/imunologia , Antígenos CD59/imunologia , Criança , Pré-Escolar , Células Epiteliais/citologia , Feminino , Feto/citologia , Feto/imunologia , Humanos , Imuno-Histoquímica , Lactente , Recém-Nascido , Masculino , Timo/citologia , Timo/imunologiaRESUMO
Detection and characterisation of chemical-induced toxic effects in the central and peripheral nervous system represent a major challenge for employing newly developed technologies in the field of neurotoxicology. Precise cellular predictive test batteries for chemical-induced neurotoxicity are increasingly important for regulatory decision making, but also the most efficient way to keep costs and time of testing within a reasonable margin. Current in vivo test methods are based on behavioural and sensory perturbations coupled with routine histopathological investigations. In spite of the empirical usefulness of these tests, they are not always sensitive enough and often, they do not provide information that facilitates a detailed understanding of potential mechanisms of toxicity, thus enabling predictions. In general, such in vivo tests are unsuitable for screening large number of agents. One way to meet the need for more powerful and comprehensive tests via an extended scientific basis is to study neurotoxicity in specific cell types of the brain and to derive generalised mechanisms of action of the toxicants from such series of experiments. Additionally, toxicokinetic models are to be developed in order to give a rough account for the whole absorption, distribution, metabolism, excretion (ADME) process including the blood-brain barrier (BBB). Therefore, an intensive search for the development of alternative methods using animal and human-based in vitro and in silico models for neurotoxic hazard assessment is appropriate. In particular, neurotoxicology represents one of the major challenges to the development of in vitro systems, as it has to account also for heterogeneous cell interactions of the brain which require new biochemical, biotechnological and electrophysiological profiling methods for reliable alternative ways with a high throughput.
RESUMO
The heterotopically induced ossicles are used in our research on bone tissue. The ossicles are formed in the thigh muscle of BALB/c mice under the influence of injected suspension of 3 x 10(6) HeLa cells. We postulate that the mechanism of bone induction is based on the secretion of bone morphogenetic proteins BMP-4 and BMP-6 by the grafted HeLa cells. This was proved by the use of specific immunohistochemical reaction and Western blots of conditioned culture medium. It seems that HeLa cells secrete BMPs continuously into the culture medium, even without contact with the mice muscle tissue, were induction of bone tissue is observed.
Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Diferenciação Celular , Células HeLa/metabolismo , Células HeLa/patologia , Osteogênese , Animais , Proteína Morfogenética Óssea 4 , Proteína Morfogenética Óssea 6 , Meios de Cultivo Condicionados , Células HeLa/transplante , Humanos , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos BALB CRESUMO
Until now, no in vitro methods for determining neurotoxic effects, on Phase I and Phase II biotransformation-driven metabolite formation or for the evaluation of the metabolism-mediated hazard of a chemical, have been validated. The current test guidelines are based on studies in vivo, involving animals exposed to the test substance. Novel in vitro testing instead of animal testing is required by Directive 86/609/EEC. In the EU White Paper on a Strategy for a Future Chemicals Policy, which may result in up to 20,000 chemicals being screened for toxicity, the use of non-animal test methods is seen as essential and is encouraged. The aim of the present work was to demonstrate the significance of novel technologies, including the use of genetically engineered cell lines and three-dimensional cell culture techniques for direct application in the regulatory hazard-assessment process. Furthermore, attempts were made to make in vitro toxicity tests for specific applications more-readily available for inclusion in the chemical hazard-assessment process, by exploiting advances made in the life sciences.
Assuntos
Alternativas aos Testes com Animais , Engenharia Genética , Doenças do Sistema Nervoso , Sistema Nervoso/efeitos dos fármacos , Sistema Nervoso/metabolismo , Organoides , Xenobióticos/metabolismo , Xenobióticos/toxicidade , Animais , Cricetinae , Cricetulus , União Europeia , Humanos , Doenças do Sistema Nervoso/induzido quimicamente , Doenças do Sistema Nervoso/metabolismo , Doenças do Sistema Nervoso/patologia , Organoides/efeitos dos fármacos , Organoides/metabolismo , Organoides/patologia , Células PC12 , Ratos , Reprodutibilidade dos Testes , Xenobióticos/classificaçãoRESUMO
In contrast to the role of lipopolysaccharide from Gram-negative bacteria, the role of Gram-positive bacterial components in inducing inflammation in the CNS remains controversial. We studied the potency of highly purified lipoteichoic acid and muramyl dipeptide isolated from Staphylococcus aureus to activate primary cultures of rat microglia. Exposure of pure microglial cultures to lipoteichoic acid triggered a significant time- and dose-dependent production of pro-inflammatory cytokines (tumour-necrosis factor-alpha, interleukin-1beta, interleukin-6) and nitric oxide. Muramyl dipeptide strongly and selectively potentiated lipoteichoic acid-induced inducible nitric oxide synthase expression and nitric oxide production. However, it did not have any significant influence on the production of pro-inflammatory cytokines. As bacterial components are recognised by the innate immunity through Toll-like receptors (TLRs) we showed that lipoteichoic acid was recognised in microglia by the TLR2 and lipopolysaccharide by the TLR4, as cells isolated from mice lacking TLR2 or TLR4 did not produce pro-inflammatory cytokines and nitric oxide upon lipoteichoic acid or lipopolysaccharide stimulation, respectively. Lipoteichoic acid-induced glia activation was mediated by p38 and ERK1/2 MAP kinases, as pretreatment with inhibitor of p38 or ERK1/2 decreased lipoteichoic acid-induced cytokine release, iNOS mRNA expression and nitric oxide production. The observed pro-inflammatory response induced by lipoteichoic acid-activated microglia could play a major role in the inflammatory response of CNS induced by Gram-positive bacteria.
Assuntos
Acetilmuramil-Alanil-Isoglutamina/farmacologia , Infecções Bacterianas do Sistema Nervoso Central/imunologia , Lipopolissacarídeos/farmacologia , Microglia/imunologia , Óxido Nítrico/metabolismo , Ácidos Teicoicos/farmacologia , Receptor 2 Toll-Like/metabolismo , Acetilmuramil-Alanil-Isoglutamina/imunologia , Acetilmuramil-Alanil-Isoglutamina/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Infecções Bacterianas do Sistema Nervoso Central/metabolismo , Citocinas/imunologia , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Encefalite/imunologia , Encefalite/metabolismo , Encefalite/fisiopatologia , Inibidores Enzimáticos/farmacologia , MAP Quinases Reguladas por Sinal Extracelular/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Mediadores da Inflamação/farmacologia , Lipopolissacarídeos/imunologia , Microglia/efeitos dos fármacos , Microglia/metabolismo , Óxido Nítrico Sintase Tipo II/efeitos dos fármacos , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/imunologia , Ratos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/imunologia , Ácidos Teicoicos/imunologia , Fatores de Tempo , Receptor 2 Toll-Like/efeitos dos fármacos , Receptor 4 Toll-Like/efeitos dos fármacos , Receptor 4 Toll-Like/imunologia , Receptor 4 Toll-Like/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
In this study we investigated the mechanisms of neuronal cell death induced by lipoteichoic acid (LTA) and muramyl dipeptide (MDP) from Gram-positive bacterial cell walls using primary cultures of rat cerebellum granule cells (CGCs) and rat cortical glial cells (astrocytes and microglia). LTA (+/- MDP) from Staphylococcus aureus induced a strong inflammatory response of both types of glial cells (release of interleukin-1beta, tumour necrosis factor-alpha and nitric oxide). The death of CGCs was caused by activated glia because in the absence of glia (treatment with 7.5 microm cytosine-d-arabinoside to inhibit non-neuronal cell proliferation) LTA + MDP did not cause significant cell death (less than 20%). In addition, staining with rhodamine-labelled LTA confirmed that LTA was bound only to microglia and astrocytes (not neurones). Neuronal cell death induced by LTA (+/- MDP)-activated glia was partially blocked by an inducible nitric oxide synthase inhibitor (1400 W; 100 microm), and completely blocked by a superoxide dismutase mimetic [manganese (III) tetrakis (4-benzoic acid)porphyrin chloride; 50 microm] and a peroxynitrite scavenger [5,10,15,20-tetrakis (4-sulfonatophenyl) porphyrinato iron (III); 100 microm] suggesting that nitric oxide and peroxynitrite contributed to LTA-induced cell death. Moreover, neuronal cell death was inhibited by selective inhibitors of caspase-3 (z-DEVD-fmk; 50 microm) and caspase-8 (z-Ile-Glu(O-Me)-Thr-Asp(O-Me) fluoromethyl ketone; 50 microm) indicating that they were involved in LTA-induced neuronal cell death.