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University of Macau / Institute of Chinese Medical Sciences

Memoria para optar el título de Bioquímico / Los organismos regulan su número de células a través de un balance entre la división y muerte celular. Con respecto a este último, existen diversos mecanismos de muerte celular, como la apoptosis, autofagia y necrosis, presentando cada uno características morfológicas y moleculares diferentes. Se ha considerado a la necrosis como un mecanismo de muerte celular “accidental” y, por tanto, menos regulado. Sin embargo existen evidencias de que la necrosis también puede ser considerada un tipo de muerte celular programada. Es así que las ceramidas, precursores de los esfingolípidos eucariontes, han sido reconocidas como importantes segundos mensajeros implicados en el gatillamiento de procesos apoptótico/necróticos en diversos tipos celulares. Recientemente se ha descrito que células de linfoma B humano (línea celular A20) al ser estimuladas con ligando de Fas en presencia de ceramidas van a un destino de muerte necrótico. Además de esto, se ha descrito una relación entre estrés oxidativo y la generación de ceramidas, relación que se encontraría íntimamente ligada a procesos de señalización de muerte celular. Con estos datos se postuló que las ceramidas intervienen en la decisión apoptóticonecrótica en células epiteliales. Utilizando métodos de citometría de flujo y ensayos enzimáticos se encontró que las ceramidas aumentaron la muerte por necrosis en el tiempo tanto para células HeLa como para HTC, este aumento es dosis dependiente. Ácido flufenámico, un inhibidor inespecífico de canales catiónicos no selectivos involucrados en regulación del volumen celular por estrés oxidativo, en conjunto con ceramidas no cambió el porcentaje de muerte por necrosis y aumentó la población apoptótica. Al reemplazar el Na+ extracelular por el catión monovalente NMDG+ no se observó diferencia entre los porcentajes de las distintas poblaciones celulares. La aplicación en conjunto de H2O2 con ceramidas aumentó la muerte celular, pero sólo a altas concentraciones (10 mM). El efecto de ceramidas sobre la apoptosis se ensayó utilizando el inhibidor genérico de caspasas zVAD-fmk. No hubo cambios significativos en los porcentajes de muerte celular. Efectos similares se observaron al depletar los depósitos intracelulares de calcio. Estos resultados permiten concluir que las ceramidas inducen muerte celular necrótica de manera tiempo y dosis dependiente / Organisms regulate their number of cells through fine a balance between cell division and cell death. In respect to cell death, diverse mechanisms exist, such as apoptosis, autophagia and necrosis, presenting each one different morphologic and molecular characteristics. Necrosis has been considered as a mechanism of "accidental" cell death and therefore, less regulated. Nevertheless, evidences exist of which necrosis can be also considered as a type of programmed cell death. For example, ceramides, precursors of sphingolipids in euchariotic cells, have been recognized as important second messengers implicated in the development of apoptotic/necrotic processes in diverse cell types. It has been recentely described that human lymphoma B cells (A20 cell line),upon stimulation with Fas ligand in the presence of ceramides, induce necrotic cell death. Also, it has been described a relationship between oxidative stress and the generation of ceramides which are intimately linked to cell death signaling processes. Based on this information, it was postulated that ceramides take part in the apoptotic-necrotic decision in epithelial cells. Using flow citometry and enzymatic assays we found that ceramides increased cell death by necrosis in HeLa and HTC cells in a dose-dependent manner. Flufenamic acid, a non specific inhibitor of nonselective cation channels involved in regulation of the oxidative stress-dependent cell volume, used in conjunction with ceramides did not produce changes in the percentage of necrotic cell death and increased the population of apoptotic cells. Upon replacement of external Na+ by the monovalent cation NMDG+, the percentage of different cell populations was not altered. The application of ceramides with H2O2 produced an increase in the cell death, but only at high concentrations of H2O2 (10 mM). The effect of ceramides upon apoptosis was assayed using the generic caspase inhibitor zVAD- fmk. No significant changes were observed in the percentage of cell death. Similar effects were observed when depleting the intracellular calcium stores. These results allow us to conclude that ceramides induces necrotic cell death in a time and dose-dependent manner

The activation and effector function of CD4+ T cells are critical points of regulation during an antigen specific T cell response. Dysregulation of these processes can lead to the development of human diseases, encompassing both immunodeficiency and autoimmunity. Members of the TNF superfamily have recently emerged as important regulators of T cell responses, with their overexpression causing autoimmune inflammation in animal models. As overproduction of the novel TNF superfamily ligand BAFF is associated with several autoimmune conditions, we sought to examine the potential role of BAFF as a regulator of T cell activation and effector function. We initially demonstrated BAFF costimulation of T cell activation in vitro. Generation of specific monoclonal antibodies identified BAFF-R as the only BAFF receptor present on T cells, and showed that it was expressed in an activation-dependent and subset-specific manner. Impaired BAFF costimulation in BAFF-R deficient mice indicated that BAFF-R was crucial for mediating BAFF effects in T cells. Analysis of T cell responses in vivo revealed that BAFF transgenic mice have increased T cell priming and recall responses to protein antigens, and showed a corresponding increase in the DTH model of Th1 cell-dependent inflammation. In addition, Th2-dependent allergic airway responses are suppressed in BAFF transgenic mice. Crossing to a B cell deficient background revealed that the proinflammatory effects of BAFF on T cell priming and DTH rely on the presence of B cells, while the suppressive effects during allergic airway inflammation are B cell independent. These data demonstrated that BAFF regulated the outcome of T cell responses in vivo and identified BAFF dependent crosstalk between T and B cells. Stimulation of B cells with BAFF induced the upregulation of MHC class II and ICOS-L both in vitro and in vivo. Induction of these cell surface molecules was associated with an increased capacity to induce T cell proliferation, however this effect was independent of ICOS-L expression. Thus it was demonstrated that BAFF regulated T cell activation and effector function both directly, via stimulation of BAFF-R, and indirectly, by altering the function of B cells. These data suggest that BAFF dependent alterations in T cell function may be an additional causative factor in the association between elevated BAFF levels and the generation of autoimmunity.

Nervous necrosis virus of Epinephelus malabaricus (MGNNV) belongs to the genus of Betanodavirus that causes vacuolating encephalopathy and retinopathy and viral nervous necrosis. A method to purify MGNNV was proposed. The cellular components of SSN-1 reprent few viral receptors for Betanodavirus. Several cellular virus binding proteins (VBPs) were detected by employing the technique of immobilizing virus on nitrocellulose. The least VBPs were found in SSN-1 cell lysate that was treated with proteinase K. The approaches used receptor antagonists to identify the cell receptor. The antagonists are able to block the viral binding and thus potentially directly against the receptor. The results implied that the receptor of serotonin 5-HT1A or £\2 adrenergic may act as the receptor of MGNNV.