RESUMEN
Magnetic hyperthermia is an oncological therapy where magnetic nanostructures, under a radiofrequency field, act as heat transducers increasing tumour temperature and killing cancerous cells. Nanostructure heating efficiency depends both on the field conditions and on the nanostructure properties and mobility inside the tumour. Such nanostructures are often incorrectly bench-marketed in the colloidal state and using field settings far off from the recommended therapeutic values. Here, we prepared nanoclusters composed of iron oxide magnetite nanoparticles crystallographically aligned and their specific absorption rate (SAR) values were calorimetrically determined in physiological fluids, agarose-gel-phantoms and ex vivo tumours extracted from mice challenged with B16-F0 melanoma cells. A portable, multipurpose applicator using medical field settings; 100 kHz and 9.3 kA m-1, was developed and the results were fully analysed in terms of nanoclusters' structural and magnetic properties. A careful evaluation of the nanoclusters' heating capacity in the three milieus clearly indicates that the SAR values of fluid suspensions or agarose-gel-phantoms are not adequate to predict the real tissue temperature increase or the dosage needed to heat a tumour. Our results show that besides nanostructure mobility, perfusion and local thermoregulation, the nanostructure distribution inside the tumour plays a key role in effective heating. A suppression of the magnetic material effective heating efficiency appears in tumour tissue. In fact, dosage had to be increased considerably, from the SAR values predicted from fluid or agarose, to achieve the desired temperature increase. These results represent an important contribution towards the design of more efficient nanostructures and towards the clinical translation of hyperthermia.
Asunto(s)
Óxido Ferrosoférrico/química , Hipertermia Inducida , Melanoma Experimental/terapia , Nanopartículas/química , Sefarosa/química , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Coloides/química , Microscopía por Crioelectrón , Femenino , Magnetismo , Melanoma Experimental/diagnóstico , Melanoma Experimental/diagnóstico por imagen , Ratones , Ratones Endogámicos C57BL , Método de Montecarlo , Nanopartículas/metabolismo , Nanopartículas/toxicidad , Fantasmas de Imagen , TemperaturaRESUMEN
INTRODUCTION: Although the expression of the granulocyte colony-stimulating factor (G-CSF) and its receptor (G-CSFR) in placental tissues suggests that the cytokine could play a role in placental development, the relevance of G-CSF:G-CSFR interaction in trophoblast cells remains to be studied. Thus, the possible functional role of G-CSF was examined in a human trophoblast cell line (Swan 71 cells). METHODS AND RESULTS: The expression of G-CSFR was detected by immunocytochemistry and Western blot assays. G-CSF treatment exerted neither a proliferative nor a protective effect on H2O2-mediated cell death in trophoblast cells. Gelatin zymography of supernatants collected from G-CSF-treated cells showed an increment of metalloproteinase-2 (MMP-2) activity. We also found higher MMP-2 and VEGF expression levels in conditioned medium from cells exposed to G-CSF. In addition, it was demonstrated that G-CSF induced the activation of PI3K/Akt and Erk1/2 pathways, which in turn activated NF-kB. By using selective pharmacological inhibitors, it was showed that these pathways are mediating the biological effects produced by G-CSF in Swan 71 cells. DISCUSSION AND CONCLUSION: We have demonstrated for the first time that G-CSF increases MMP-2 activity and VEGF secretion in Swan 71 cells through activation of PI3K/Akt and Erk signaling pathways, both contributing to the translocation of NF-kB to the nucleus. These data suggest that G-CSF is involved in the regulation of trophoblast function, and should be considered as a locally produced cytokine probably contributing to embryo implantation and the development of a functional placenta.
Asunto(s)
Factor Estimulante de Colonias de Granulocitos/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Trofoblastos/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Línea Celular , Proliferación Celular , Supervivencia Celular , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismoRESUMEN
We have previously demonstrated that tyrosine phosphorylation of STAT1/3 and p38 mitogen-activated protein kinase (p38 MAPK) activation are involved in the apoptotic response triggered by a chimeric cyclic peptide of the interferon-α2b (IFN-α2b) in WISH cells. Since the peptide also induced serine phosphorylation of STAT proteins, in the present study we examined the kinase involved in serine STAT1 phosphorylation and the signaling effectors acting upstream such activation. We first found that p38 MAPK is involved in serine STAT1 phosphorylation, since a reduction of phophoserine-STAT1 levels was evident after incubating WISH cells with cyclic peptide in the presence of a p38 pharmacological inhibitor or a dominant-negative p38 mutant. Next, we demonstrated that the peptide induced activation of protein kinase Cδ (PKCδ). Based on this finding, the role of this kinase was then evaluated. After incubating WISH cells with a PKCδ inhibitor or after decreasing PKCδ expression levels by RNA interference, both peptide-induced serine STAT1 and p38 phosphorylation levels were significantly decreased, indicating that PKCδ functions as an upstream regulator of p38. We also showed that PKCδ and p38 activation stimulated by the peptide was inhibited by a specific pharmacological inhibitor of phosphatidylinositol 3-kinase (PI3K) or by a dominant-negative p85 PI3K-regulatory subunit, suggesting that PI3K is upstream in the signaling cascade. In addition, the role of PI3K and PKCδ in cyclic peptide-induced apoptosis was examined. Both signaling effectors were found to regulate the antiproliferative activity and the apoptotic response triggered by the cyclic peptide in WISH cells. In conclusion, we herein demonstrated that STAT1 serine phosphorylation is mediated by the sequential activation of PI3K, PKCδ and p38 MAPK. This signaling cascade contributes to the antitumor effect induced by the chimeric IFN-α2b cyclic peptide in WISH cells.
Asunto(s)
Apoptosis , Interferón-alfa/farmacología , Proteína Quinasa C-delta/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Antineoplásicos/farmacología , Línea Celular Tumoral/efectos de los fármacos , Proliferación Celular , Activación Enzimática , Humanos , Imidazoles/farmacología , Interferón alfa-2 , Péptidos Cíclicos/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Proteína Quinasa C-delta/genética , Piridinas/farmacología , Interferencia de ARN , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes/farmacología , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo , Serina/metabolismo , Transducción de Señal , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/genéticaRESUMEN
A monoclonal antibody termed MAb R7B4, directed to an epitope present in prolactin receptors (PRLRs), was used as a tool to map the receptor binding sites for human growth hormone (hGH), ovine prolactin (oPRL) and human placental lactogen (hPL). Although the three hormones completely inhibited the binding of each other to Nb2 cells or rat liver receptors, MAb R7B4 behaviour was different depending on the hormone tested and the receptor source. According to the MAb effects, PRLR from Nb2 cells would locate both hGH and oPRL close to R7B4 epitope, whereas hPL would bind far from the MAb binding site. On the other hand, PRLR from rat liver should bind hGH close to the R7B4 epitope but oPRL and hPL would be recognized by a separate region of the same receptor. Thus, results presented in this paper suggest that PRLR binding sites for hGH, oPRL and hPL do not exactly overlap in spite of full competition between ligands.
Asunto(s)
Hígado/metabolismo , Receptores de Prolactina/metabolismo , Animales , Anticuerpos Monoclonales , Sitios de Unión , Bovinos , Línea Celular , Membrana Celular/metabolismo , Femenino , Hormona de Crecimiento Humana/metabolismo , Humanos , Cinética , Lactógeno Placentario/metabolismo , Embarazo , Prolactina/metabolismo , Ratas , Ratas Wistar , OvinosRESUMEN
The binding and antiproliferative activities of synthetic peptides 29-35 and 122-139 of interferon-alpha2b, both of which contain a cysteine residue in their sequences, were studied in the presence or absence of a dissociation medium containing mainly urea, dithiothreitol and 2-mercaptoethanol. Although interferon-alpha2b peptides either did not modify or slightly increased 125I-labelled interferon-alpha2b specific binding to WISH cell-membrane receptors in the absence of dissociation medium, significant binding inhibition was obtained when both peptides were assayed in dissociation medium. Furthermore, also in the presence of dissociating agents, the two fragments inhibited cell growth in a concentration-dependent manner, the 122-139 sequence being more effective than the 29-35 sequence. No additive effect on interferon binding and cell proliferation was observed when both peptides were added simultaneously. Results obtained after submitting peptide 122-139 to gel filtration or PAGE under different experimental conditions showed the presence of dimers and/or noncovalent aggregates arising from intermolecular disulfide bridges or hydrophobic interactions. Thus, our results indicated that peptide effects on 125I-labelled interferon-alpha2b binding and WISH cell proliferation were clearly manifested when the amount of monomeric species increased, showing that suitable experimental conditions should be used to study peptide behavior. The ability of both peptides to effectively trigger an interferon-specific biological action, such as cell growth inhibition, strongly suggested that 29-35 and 122-139 interferon-alpha2b fragments constitute the conformational epitope or mimotope that interacts with the cytokine-specific receptor.
Asunto(s)
Interferón-alfa/química , Péptidos/química , Sitios de Unión , División Celular , Línea Celular , Membrana Celular/metabolismo , Cromatografía en Gel/métodos , Cisteína/química , Ditiotreitol/farmacología , Relación Dosis-Respuesta a Droga , Electroforesis en Gel de Poliacrilamida , Epítopos , Humanos , Interferón alfa-2 , Interferón-alfa/aislamiento & purificación , Mercaptoetanol/farmacología , Péptidos/síntesis química , Péptidos/aislamiento & purificación , Unión Proteica , Proteínas Recombinantes , Urea/farmacologíaRESUMEN
Monoclonal antibodies (MAb) anti-human growth hormone (hGH) termed MAb AE5, AC8 and F11 recognize a cluster of epitopes left exposed after hormone binding to receptors. Since these MAb were able to produce either positive (MAb AE5) or negative (MAb AC8 and F11) allosteric effects on hGH binding, the purpose of this work was to further characterize MAb behavior. Results indicated a straight correlation between MAb allosteric effects and affinity constant values for binding of different hGH:MAb complexes to lactogenic receptors from rat liver. Affinity of hGH:MAb AE5 as well as hGH:Fab AE5 complexes enhanced proportionally to the fraction of occupied receptors and Hill coefficients higher than 1 were obtained, suggesting the induction of positive cooperative effects between membrane-bound receptors. On the other hand, hGH:MAb AC8 and hGH:MAb F11 complexes binding affinity to lactogenic sites could not be related to receptor occupancy degree. It is proposed that binding of hGH:MAb AE5 complexes to receptors would elicit a conformational change on adjacent receptor molecules leading to an increase of their affinity to bind subsequent hGH:MAb AE5 complexes.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Hormona del Crecimiento/inmunología , Regulación Alostérica , Animales , Femenino , Hormona del Crecimiento/metabolismo , Humanos , Microsomas Hepáticos/metabolismo , Ensayo de Unión Radioligante , Ratas , Receptores de Somatotropina/metabolismoRESUMEN
Four monoclonal antibodies (mAbs) directed against the recombinant human interferon-alpha2b (IFN-alpha2b) were used as probes to study the interaction of the IFN molecule to its receptors. The [125I]IFN-alpha2b binding to immobilized mAbs was completely inhibited by IFN-alpha2b and IFN-alpha2a but neither IFNbeta nor IFNgamma showed any effect. Gel-filtration HPLC of the immune complexes formed by incubating [125I]IFN-alpha2b with paired mAbs revealed the lack of simultaneous binding of two different antibodies to the tracer, suggesting that all mAbs recognize the same IFN antigenic domain. Furthermore, the mAbs were also able to neutralize the IFN-alpha2b anti-viral and anti-proliferative activities as well as [125I]IFN-alpha2b binding to WISH cell-membranes. As [125I]mAbs did not recognize IFN exposed epitopes in the IFN:receptor complexes, mAb induction of a conformational change in the IFN binding domain impairing its binding to receptors was considered unlikely. In order to identify the IFN region recognized by mAbs, IFN-alpha2b was digested with different proteolytic enzymes. Immunoreactivity of the resulting peptides was examined by Western blot and their sequences were established by Edman degradation after blotting to poly(vinylidene difluoride) membranes. Data obtained indicated that the smallest immunoreactive region recognized by mAbs consisted of residues 107-132 or 107-146. As this zone includes the sequence 123-140, which has been involved in the binding to receptors, and our mAbs did not show an allosteric behaviour, it is concluded that they are directed to overlapping epitopes located close to or even included in the IFN binding domain.
Asunto(s)
Interferón-alfa/inmunología , Interferón-alfa/metabolismo , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Especificidad de Anticuerpos , Sitios de Unión , Unión Competitiva , Mapeo Epitopo , Epítopos , Interferón alfa-2 , Interferón beta/metabolismo , Interferón gamma/metabolismo , Datos de Secuencia Molecular , Pruebas de Neutralización , Unión Proteica , Receptor de Interferón alfa y beta , Receptores de Interferón/metabolismo , Proteínas RecombinantesRESUMEN
The ability of monoclonal antibodies (MAb) to bind or not simultaneously to the antigen (Ag) is used to establish antigenic maps considering that two different MAb do not bind to the Ag when the corresponding epitopes are overlapped (steric effect). Nevertheless, MAb inducing negative allosteric effect on the Ag could prevent the binding of the second MAb even if it is directed to a separate epitope. We report here that a knowledge-based expert module included in our previously described antigenic model-builder program (MAPAG) was able to differentiate between steric and negative allosteric effects between some MAb.
Asunto(s)
Sitio Alostérico/inmunología , Anticuerpos Monoclonales/inmunología , Sitios de Unión de Anticuerpos/inmunología , Simulación por Computador , Epítopos/inmunología , Programas Informáticos , Algoritmos , Complejo Antígeno-Anticuerpo/inmunología , Inteligencia Artificial , Sistemas Especialistas , Humanos , Procesamiento de Imagen Asistido por Computador , Modelos InmunológicosRESUMEN
This paper describes a study of whether accident risks were equally distributed across age categories among a population of mining workers whose work activities were suspected to be age-impaired. The impairment factors in focus are the transformation of production technology during the 80s and consequent changes in job content. It was hypothesized that the combined effect of these factors might lead accident risks, both non-specific (aggregated) and specific (by kind), to increase with age. Accident risk ratios (ARRs), however, proved to be higher for younger workers than older ones, in both the non-specific and the specific cases. However, two accident patterns (specific risks) also show relatively high ARRs among workers in their 40s (and even 30s), results that might be explained by particular exposures and/or age-related performance problems. The findings suggest that technological changes designed to increase productivity and reduce staffing levels more rapidly affect efficiency and productivity than they do accident occurrence, and that they penalize young workers in the first instance.
Asunto(s)
Accidentes de Trabajo/estadística & datos numéricos , Minería/métodos , Adulto , Factores de Edad , Intervalos de Confianza , Eficiencia , Humanos , Masculino , Persona de Mediana Edad , Minería/tendencias , Distribución de Poisson , Riesgo , Suecia/epidemiología , Tecnología , Heridas y Lesiones/clasificaciónRESUMEN
Foram estudados, durante 1980, o risco de morte e os acidentes de trabalho na industria eletrica do Rio de Janeiro, observando-se elevado coeficiente de mortalidade padronizado por idade nos eletricitarios assim como elevada mortalidade proporcional por neoplasias. A analise dos acidentes de trabalho do ramo mostrou elevada taxa de frequencia e um sub-registro importante dos acidentes sem afastamento do trabalho, uma possivel relacao dos acidentes com a continuidade da jornada de trabalho e uma maior frequencia dos acidentes sob responsabilidade virtual da empresa