RESUMEN
Type I interferons are secreted by infected cells and promote an antiviral state in neighbouring cells by the induction of numerous genes, some of which present antiviral activity, as the Mx proteins. In this study, three different Mx cDNAs (Mx1, Mx2 and Mx3) from gilthead seabream (Sparus aurata), the most important fish species in Southern European aquaculture, have been cloned and characterized. A Southern blot assay revealed the existence of three Mx loci, thus the three Mx isoforms correspond to three different genes that seem to have a common origin. The genomic sequences of Mx1, Mx2 and Mx3 have been completely obtained, and consist on 11 introns and 12 exons in a full length of 5971 bp, 7391 bp and 6938 bp, respectively. As a first approach to the functional meaning of these three genes, their response to the viral nervous necrosis virus (VNNV) infection was tested. Important differences in terms of tissue, time course and level of induction were found between them, thus suggesting a differential functional role for each isoform, which can represent a key point in the natural resistance of this fish species, that has been repeatedly reported as an asymptomatic carrier of VNNV.
Asunto(s)
Enfermedades de los Peces/virología , Proteínas de Peces/genética , Proteínas de Unión al GTP/genética , Nodaviridae/inmunología , Infecciones por Virus ARN/virología , Dorada/inmunología , Dorada/virología , Secuencia de Aminoácidos , Animales , Southern Blotting , Clonación Molecular , ADN Complementario/genética , Exones/genética , Enfermedades de los Peces/genética , Enfermedades de los Peces/inmunología , Proteínas de Peces/metabolismo , Proteínas de Unión al GTP/química , Proteínas de Unión al GTP/metabolismo , Genoma/genética , Intrones/genética , Datos de Secuencia Molecular , Proteínas de Resistencia a Mixovirus , Infecciones por Virus ARN/genética , Infecciones por Virus ARN/inmunología , Dorada/genética , Alineación de Secuencia , Homología de Secuencia de Aminoácido , Factores de Tiempo , Transcripción GenéticaRESUMEN
According to the Edmonton protocol, human islet transplantation can result in insulin independency for periods longer than 3 years. However, this therapy for type 1 diabetes is limited by the scarcity of cadaveric donors. Owing to the ability of embryonic stem cells to expand in vitro and differentiate into a variety of cell types, research has focused on ways to manipulate these cells to overcome this problem. It has been demonstrated that mouse embryonic stem cells can differentiate into insulin-containing cells, restoring normoglycaemia in diabetic mice. To this end, mouse embryonic stem cells were transfected with a DNA construct that provides resistance to neomycin under the control of the regulatory regions of the human insulin gene. However, this protocol has a very low efficiency, needing improvements for this technology to be transferred to human stem cells. Optimum protocols will be instrumental in the production of an unlimited source of cells that synthesise, store and release insulin in a physiological manner. The review focuses on the alternative source of tissue offered by embryonic stem cells for regenerative medicine in diabetes and some key points that should be considered in order for a definitive protocol for in vitro differentiation to be established.
Asunto(s)
Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Células Madre , Ingeniería de Tejidos/métodos , Técnicas de Cultivo de Célula , Diferenciación Celular , Humanos , Secreción de Insulina , Trasplante de Islotes Pancreáticos , Trasplante HeterólogoRESUMEN
The various cell types involved in fish phagocytic defence have not been properly established because of the morphological heterogeneity of leucocytes and the lack of appropriate cell-surface markers. In this study, we report the production and characterisation of a monoclonal antibody, G7, which specifically recognises gilthead seabream acidophilic granulocytes, as assayed by immunofluorescence and immunoelectron microscopy. The antibody reacted with 40%-50% of head-kidney and peritoneal exudate leucocytes and 10%-20% of spleen and peripheral blood leucocytes. More importantly, G7(+) acidophils constituted 85% of the head-kidney leucocytes showing phagocytic activity towards the fish pathogenic bacterium Vibrio anguillarum. The results are discussed in relation to the role played by this cell type in fish immune responses.
Asunto(s)
Anticuerpos Monoclonales/farmacología , Granulocitos/inmunología , Fagocitosis , Dorada/inmunología , Animales , Granulocitos/clasificación , Granulocitos/ultraestructura , Dorada/anatomía & histologíaRESUMEN
Cell adhesion molecules play a key role in the inflammatory response. Selectins, integrins and immunoglobulin gene superfamily adhesion receptors mediate the different steps of leukocyte migration from the blood-stream towards inflammatory foci. In addition to their adhesive function, these receptors modulate major cellular processes such as cell activation, growth, differentiation and death. To characterise the fish molecules involved in cell adhesion, a panel of mAbs was raised by immunising mice with macrophages from the marine fish gilthead seabream (Sparus aurata L.). One of these mAbs, which we named anti-Aggregatin, was found to induce a rapid heterotypic aggregation of seabream leukocytes. Anti-Aggregatin defined a 140-kDa cell surface receptor which was highly expressed by macrophages and was up-regulated after co-stimulation with LPS and MAF. Functionally, the cell adhesion which occurred upon exposure to anti-Aggregatin required Ca(2+), an intact cytoskeleton and an active cell metabolism. More importantly, Aggregatin engagement resulted in strong inhibition of the phagocyte respiratory burst, although the cells showed neither loss of viability nor DNA fragmentation. The results are discussed in relation to the potential role of cell adhesion molecules in fish immune responses.