RESUMO
The significant expansion in the use of nanoparticles and submicron particles during the last 20 years has led to increasing concern about their potential toxicity to humans and particularly their impact on male fertility. Currently, an insufficient number of studies have focused on the testicular biodistribution of particles. The aim of our study was to assess the distribution of 450 nm fluorescent particles in mouse testes after intramuscular injection. To this end, testes were removed from 5 groups of 3 mice each at 1 h (H1), 4 days (D4), 21 days (D21), 45 days (D45) and 90 days (D90) after the injection of 7.28 × 109 particles in the tibialis anterior muscles of each mouse. We examined histological sections from these samples by epifluorescence microscopy and confocal microscopy and identified testicular biodistribution of a small number of particles in groups H1, D4, D21, D45 and D90. Using CD11b immunostaining, we showed that particles were not carried into the testis by macrophages. The intratesticular repartition of particles mainly followed testicular vascularization. Finally, we found some particles in seminiferous tubules but could not determine if the blood-testis barrier was crossed.
Assuntos
Corantes Fluorescentes/química , Corantes Fluorescentes/farmacocinética , Látex/química , Látex/farmacocinética , Tamanho da Partícula , Testículo/metabolismo , Animais , Corantes Fluorescentes/administração & dosagem , Injeções Intramusculares , Látex/administração & dosagem , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Espectrometria de Fluorescência , Coloração e Rotulagem , Testículo/citologia , Distribuição Tecidual , Preservação de TecidoRESUMO
BACKGROUND: Micrometric and nanometric particles are increasingly used in different fields and may exhibit variable toxicity levels depending on their physicochemical characteristics. The aim of this study was to determine the impact of the size parameter on cellular uptake and biological activity, working with well-characterized fluorescent particles. We focused our attention on macrophages, the main target cells of the respiratory system responsible for the phagocytosis of the particles. METHODS: FITC fluorescent silica particles of variable submicronic sizes (850, 500, 250 and 150 nm) but with similar surface coating (COOH) were tailored and physico-chemically characterized. These particles were then incubated with the RAW 264.7 macrophage cell line. After microscopic observations (SEM, TEM, confocal), a quantitative evaluation of the uptake was carried out. Fluorescence detected after a quenching with trypan blue allows us to distinguish and quantify entirely engulfed fluorescent particles from those just adhering to the cell membrane. Finally, these data were compared to the in vitro toxicity assessed in terms of cell damage, inflammation and oxidative stress (evaluated by LDH release, TNF-α and ROS production respectively). RESULTS AND CONCLUSION: Particles were well characterized (fluorescence, size distribution, zeta potential, agglomeration and surface groups) and easily visualized after cellular uptake using confocal and electron microscopy. The number of internalized particles was precisely evaluated. Size was found to be an important parameter regarding particles uptake and in vitro toxicity but this latter strongly depends on the particles doses employed.
Assuntos
Macrófagos/efeitos dos fármacos , Nanopartículas/toxicidade , Dióxido de Silício/toxicidade , Animais , Linhagem Celular , Fluoresceína-5-Isotiocianato/química , Fluoresceína-5-Isotiocianato/metabolismo , Fluorescência , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , L-Lactato Desidrogenase/metabolismo , Macrófagos/metabolismo , Camundongos , Microscopia Eletrônica de Varredura , Microscopia Eletrônica de Transmissão , Nanopartículas/química , Nanopartículas/ultraestrutura , Tamanho da Partícula , Espécies Reativas de Oxigênio/metabolismo , Dióxido de Silício/química , Dióxido de Silício/metabolismo , Fator de Necrose Tumoral alfa/metabolismoRESUMO
BACKGROUND: The use of micro- or nanometric particles is in full expansion for the development of new technologies. These particles may exhibit variable toxicity levels depending on their physicochemical characteristics. We focused our attention on macrophages (MA), the main target cells of the respiratory system responsible for the phagocytosis of the particles. The quantification of the amount of phagocytosed particles seems to be a major element for a better knowledge of toxicity mechanisms. The aim of this study was to develop a quantitative evaluation of uptake using both flow cytometry (FCM) and confocal microscopy to distinguish entirely engulfed fluorescent microsized particles from those just adherent to the cell membrane and to compare these data to in vitro toxicity assessments. METHODS: Fluorescent particles of variable and well-characterised sizes and surface coatings were incubated with MA (RAW 264.7 cell line). Analyses were performed using confocal microscopy and FCM. The biological toxicity of the particles was evaluated [lactate dehydrogenase (LDH) release, tumor necrosis factor (TNF)-α, and reactive oxygen species (ROS) production]. RESULTS AND CONCLUSION: Confocal imaging allowed visualization of entirely engulfed beads. The amount of phagocytic cells was greater for carboxylate 2-µm beads (49 ± 11%) than for amine 1-µm beads (18 ± 5%). Similarly, side scatter geometric means, reflecting cellular complexity, were 446 ± 7 and 139 ± 12, respectively. These results confirm that the phagocytosis level highly depends on the size and surface chemical groups of the particles. Only TNF-α and global ROS production varied significantly after 24-h incubation. There was no effect on LDH and H(2)O(2) production.
Assuntos
Poeira/análise , Macrófagos/metabolismo , Nanopartículas/toxicidade , Fagocitose , Análise de Variância , Animais , Linhagem Celular , Citometria de Fluxo/métodos , Fluorescência , Peróxido de Hidrogênio/metabolismo , L-Lactato Desidrogenase/análise , L-Lactato Desidrogenase/biossíntese , Camundongos , Microscopia Confocal/métodos , Microesferas , Estresse Oxidativo , Espécies Reativas de Oxigênio/análise , Fator de Necrose Tumoral alfa/análiseRESUMO
Myelodysplastic syndromes (MDS) are characterized by abnormal growth of committed progenitors in clonogenic assay, with reduced number of colonies and decreased colony/cluster ratio. It has been suggested that excessive apoptosis is the cause of marrow failure in MDS. We studied the expression of caspase-1 (interleukin-1beta-converting enzyme, ICE) and caspase-3 (CPP32/apopain) in marrow mononuclear cells, and the growth pattern of committed progenitors in a series of 83 MDS cases. The percentage of apoptotic cells as detected by TUNEL technique, and the percentage of caspase-3-positive cells were significantly higher in refractory anemia (RA) and RA with ringed sideroblasts (RAS) than in chronic myelomonocytic leukemia (CMML), refractory anemia with excess of blasts (RAEB) and RAEB in transformation (RAEB-T). Spontaneous growth of CFU-GM was associated with a higher percentage of blasts, and with a lower expression of caspase-3 and caspase-1. The yield of CFU-E, BFU-E, and CFU-GM (in the presence of growth factors) was decreased by comparison to normal marrow, but large individual differences were observed in all cytological categories. Inhibition of caspase-1 and caspase-3 activities by specific inhibitors resulted in a significant increase of the production of all types of colonies (up to 50-fold of control). In the presence of caspase-3 inhibitor, the number of BFU-E and CFU-E was in the range of normal values in most cases of RA and RAS. In addition, caspase-1 and -3 protease activities were detectable by fluorogenic assay in all cases studied. Western blot analysis confirmed the expression of caspase-3, including the cleaved (activated)-p17 form in most cases of RA/RAS analyzed. It is concluded that caspase-3 is implicated in the increased apoptosis observed in MDS and that inhibition of its activity can restore at least partially the growth of committed progenitors.
Assuntos
Caspase 1/metabolismo , Caspases/metabolismo , Síndromes Mielodisplásicas/enzimologia , Adulto , Antígenos CD34/imunologia , Apoptose , Western Blotting , Caspase 3 , Citometria de Fluxo , Humanos , Síndromes Mielodisplásicas/imunologia , Síndromes Mielodisplásicas/metabolismo , Síndromes Mielodisplásicas/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismoRESUMO
PURPOSE: Investigate the involvement of auditory spatial compensation, which is observed in blind humans, in self-localization processes. METHOD: Sighted and early-blind subjects had to indicate, on a two-dimensional view of the experimental room, the position where they previously sat and had passively listened to auditory spatial cues. Two different environments were distinguished. In a first session, auditory cues (i.e., white broadband sounds) were displayed successively in a dark anechoic room. This condition was defined as a simple acoustic environment. In a second session, four different auditory cues were displayed simultaneously at regular intervals in an experimental room, where echo cues were salient. This condition, which is more reminiscent of the natural situation, was described as a complex acoustic environment. RESULTS: Self-localization capacities were significantly better in early-blind individuals than in sighted subjects, whatever the type of acoustic environment. CONCLUSIONS: Auditory compensation leads to improved self-localization capacities in early-blind humans and indicates that prior visual experience is not essential for the development of spatial competence.
Assuntos
Cegueira/fisiopatologia , Localização de Som/fisiologia , Percepção Espacial/fisiologia , Adolescente , Adulto , Análise de Variância , Estudos de Casos e Controles , Sinais (Psicologia) , Feminino , Movimentos da Cabeça , Humanos , Masculino , Pessoa de Meia-Idade , Atividade Motora/fisiologiaRESUMO
BACKGROUND: Apoptosis very probably plays a key part in endothelial cell loss during corneal storage in organ culture as well as hypothermic storage. However, the mechanisms underlying endothelial apoptosis are poorly understood. The response of a human corneal endothelial cell (HCEC) line to staurosporine, a known inducer of apoptosis, was investigated to gain insights into the intracellular modulators that participate in endothelial cell death. METHODS: Immortalised HCECs were studied after 3, 6, 12, and 24 hours of incubation with 0.2 micro M staurosporine. Cell shedding was monitored. Hoechst 33342 fluorescent DNA staining combined with propidium iodide was used for apoptosis/necrosis quantification and morphological examination. The caspase-3 active form was assessed using western blot, proteolytic activity detection, and immunocytochemistry. The cleaved form of poly(ADP-ribose) polymerase (PARP) was assessed using immunocytochemistry and western blot. The ultrastructural features of cells were screened after 12 hours with staurosporine or vehicle. RESULTS: The specific apoptotic nature of staurosporine induced HCEC death was confirmed. The ultrastructural features of staurosporine treated cells were typical of apoptosis. HCEC shedding and DNA condensation increased with time. Caspase-3 activity was detected as early as 3 hours after exposure with staurosporine, peaking at 12 hours of incubation. The presence of cleaved PARP after 3 hours confirmed caspase-3 activation. CONCLUSIONS: These data suggest strongly that HCEC cell death induced by staurosporine is apoptosis. The main consequence of HCEC apoptosis is shedding. Staurosporine induced apoptosis of endothelial cells involves activation of caspase-3, and could be a useful model to study strategies of cell death inhibition.
Assuntos
Apoptose/efeitos dos fármacos , Endotélio Corneano/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Estaurosporina/farmacologia , Western Blotting , Caspase 3 , Caspases/metabolismo , Linhagem Celular , Endotélio Corneano/enzimologia , Endotélio Corneano/fisiologia , Ativação Enzimática , Humanos , Imuno-Histoquímica/métodos , Microscopia Eletrônica , Poli(ADP-Ribose) Polimerases/metabolismoRESUMO
Viral inactivation is one of the possibilities to reduce the residual risk of blood products. It is now applied to all plasma derived products (PDP). Application of such techniques to labile blood products (LBP) is difficult for two main reasons: any method should inactivate cell-associated viruses and should avoid any injury of the cells constituting the active ingredient. Physical techniques may reduce the viral content of cellular BPL (leucodepletion, washing, gamma irradiation), but none of them is active enough to comply with the present requirements for efficacy. An important work has been dedicated to the development of virus photoinactivation techniques. They consist of the addition of a photoreagent followed by illumination at an appropriate wavelength which results in a photochemical reaction responsible for the viral inactivation. Treatment of platelet concentrates by psoralen derivatives and UV-A illumination significantly inactivate in vitro enveloped and naked viruses, free and cell-associated viruses and also sequences integrated in the viral genome. Recent progresses have led to these results without detectable functional alteration of platelets and mutagenicity. Viral inactivation of red blood cells yet did not reach the same level because hemoglobin does not allow the use of the photoreagent compounds applicable to platelet concentrates. Viral decontamination of fresh frozen plasma by solvent and detergent, active on enveloped viruses, has been used in France since 1992. Other techniques of comparable efficacy, have received an agreement in other countries. The research on viral inactivation of LBP could prove to be of great importance in the near future in bringing additional safety to patients not only for the residual viral risk but maybe also for the residual bacterial risk of LBP.
Assuntos
Antivirais/farmacologia , Transfusão de Componentes Sanguíneos/normas , Plaquetas/virologia , Eritrócitos/virologia , Leucócitos/virologia , Plasma/virologia , Humanos , Fotoquímica , Controle de QualidadeRESUMO
Digital plethysmography allows investigation of maximum digital pulse (M.D.P.) after immersion of hands at 45 degrees C over three minutes. It is correlated with digital arterial blood flow. Reactivity to cold is determined from the ratio M.D.P./digital pulse after local and body cooling over 3 minutes. Tests were performed on 65 controls, 69 patients with idiopathic Raynaud's syndrome, 12 with scleroderma, 10 with digital arteritis and 15 with Raynaud's phenomenon secondary to hemorheologic affections. Maximum digital pulse was significantly decreased in patients with digital arteritis and scleroderma. The M.D.P. was normal in controls and patients with idiopathic Raynaud's syndrome, and was significantly increased in patients with a rheologic Raynaud's phenomenon. Digital artery reactivity differentiates the populations studied: it was maximum in patients with scleroderma, moderate in controls and patients with isolated digital arteritis and marked in patients with idiopathic Raynaud's disease and those with rheologic Raynaud's phenomena.
Assuntos
Viscosidade Sanguínea , Dedos/irrigação sanguínea , Doença de Raynaud/diagnóstico , Adulto , Artérias , Pressão Sanguínea , Temperatura Baixa , Cianose/sangue , Cianose/diagnóstico , Cianose/fisiopatologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Pletismografia , Pulso Arterial , Doença de Raynaud/sangue , Doença de Raynaud/fisiopatologia , Escleroderma Sistêmico/diagnóstico , Trombose/diagnósticoAssuntos
Caspases/metabolismo , Precursores Enzimáticos/metabolismo , Proteínas de Neoplasias/metabolismo , Transporte Proteico/efeitos dos fármacos , Frações Subcelulares/enzimologia , Acetato de Tetradecanoilforbol/farmacologia , Células U937/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Núcleo Celular/enzimologia , Citosol/enzimologia , Humanos , Microssomos/enzimologia , Mitocôndrias/enzimologia , Células U937/citologia , Células U937/enzimologiaRESUMO
Cone photoreceptor breakdown underlies functional vision loss in many blinding diseases. Cone loss is often secondary to that of rods, but little experimental data are available on the relationship between the two populations. Because of its high cone numbers, we used the diurnal rodent Arvicanthis ansorgei to explore changes in rod and cone survival and function during chemically-induced retinal degeneration. Adult animals received intraperitoneal injections of N-methyl-N-nitrosourea (MNU), and changes in retinal fundus appearance, histology, phenotype, apoptosis (TUNEL staining) and functionality (scotopic and photopic electroretinography) were monitored as a function of post-treatment time and retinal topography. Relative to control animals injected with vehicle only, MNU-injected animals showed time-, region- and population-specific changes as measured by morphological and immunochemical criteria. Histological (gradual thinning of photoreceptor layer) and phenotypical (reduced immunostaining of rhodopsin and rod transducin, and mid wavelength cone opsin and cone arrestin) modifications were first observed in superior central retina at 11 days post-injection. These degenerative changes spread into the superior peripheral and inferior hemisphere during the following 10 days. Rod loss preceded that of cones as visualized by differential immunolabelling and presence of apoptotic cells in rod but not cone cells. By 3 months post-injection, degeneration of the photoreceptor layer was complete in the superior hemisphere, but only partial in the inferior hemisphere. Despite the persistence of cone photoreceptors, scotopic and photopic electroretinography performed at 90 days post-treatment showed that both rod and cone function were severely compromised. In conclusion, MNU-induced retinal degeneration in Arvicanthis follows a predictable spatial and temporal pattern allowing clear separation of rod- and cone-specific pathogenic mechanisms. Compared to other rodents in which MNU has been used, Arvicanthis ansorgei demonstrates pronounced resistance to photoreceptor cell loss.