Evaluation of hydrophobic chitosan-based particulate formulations of porcine reproductive and respiratory syndrome virus vaccine candidate T cell antigens.

PRRS control is hampered by the inadequacies of existing vaccines to combat the extreme diversity of circulating viruses. Since immune clearance of PRRSV infection may not be dependent on the development of neutralising antibodies and the identification of broadly-neutralising antibody epitopes have proven elusive, we hypothesised that conserved T cell antigens represent potential candidates for development of a novel PRRS vaccine. Previously we had identified the M and NSP5 proteins as well-conserved targets of polyfunctional CD8 and CD4 T cells. To assess their vaccine potential, peptides representing M and NSP5 were encapsulated in hydrophobically-modified chitosan particles adjuvanted by incorporation of a synthetic multi-TLR2/TLR7 agonist and coated with a model B cell PRRSV antigen. For comparison, empty particles and adjuvanted particles encapsulating inactivated PRRSV-1 were prepared. Vaccination with the particulate formulations induced antigen-specific antibody responses, which were most pronounced following booster immunisation. M and NSP5-specific CD4, but not CD8, T cell IFN-γ reactivity was measurable following the booster immunisation in a proportion of animals vaccinated with peptide-loaded particles. Upon challenge, CD4 and CD8 T cell reactivity was detected in all groups, with the greatest responses being detected in the peptide vaccinated group but with limited evidence of an enhanced control of viraemia. Analysis of the lungs during the resolution of infection showed significant M/NSP5 specific IFN-γ responses from CD8 rather than CD4 T cells. Vaccine primed CD8 T cell responses may therefore be required for protection and future work should focus on enhancing the cross-presentation of M/NSP5 to CD8 T cells.

Expression of estrogen receptor subtypes and neuronal nitric oxide synthase in neutrophils from women and men: regulation by estrogen.

OBJECTIVES: (a) To identify the subtype of estrogen receptor (ER) expressed in neutrophils from premenopausal women and in neutrophils from men under different estrogen conditions and (b) to analyze the association between the modifications in the expression of ER subtypes and neuronal nitric oxide synthase (nNOS) expression induced by estrogen. METHODS: Neutrophils were isolated from pre-menopausal women during different stages of the menstrual cycle and from ten men for in vitro estrogen incubations. RESULTS: Neutrophils from premenopausal women expressed both ERalpha and ERbeta subtypes which were increased in the ovulatory phase of the menstrual cycle. Neutrophils derived from men also expressed ERalpha and ERbeta but only ERalpha expression was enhanced by in vitro incubation with 17beta-estradiol (10(-8) mol/l). In vitro incubation of neutrophils from women with 17beta-estradiol enhanced expression of both ER-alpha and ER-beta subtypes. nNOS protein was overexpressed in neutrophils from premenopausal women during the ovulatory phase. 17beta-Estradiol (10(-8) mol/l) also increased nNOS protein expression in neutrophils derived from men. Mithramycin A (10(-6) mol/l) and curcumin (10(-6) mol/l), prevented the upregulation of nNOS and ERalpha in neutrophils derived from men, suggesting the involvement of AP-1 and Sp-1 transcription factors. CONCLUSIONS: Although the in vivo levels of circulating estrogen concentrations seem to be associated with overexpression of both ERalpha and ERbeta in neutrophils from premenopausal women, which was further confirmed by the in vitro experiments with neutrophils from women, in vitro incubation of neutrophils from men with 17beta-estradiol only increased ERalpha protein expression which was associated with enhanced expression of nNOS protein.

Efecto del triflusal sobre la agregación y secreción de las plaquetas humanas: papel del óxido nítrico / Effect of Triflusal on Human Platelet Aggregation and Secretion: Role of Nitric Oxide

Introducción y objetivos. Recientes estudios in vitro realizados en nuestro laboratorio demostraron que el triflusal reduce la agregación plaquetaria mediante la estimulación de la producción de óxido nítrico (NO) por los neutrófilos. El objetivo de este trabajo fue evaluar si el tratamiento in vivo con triflusal también aumenta la capacidad de los neutrófilos de generar NO analizando el papel del NO liberado por los neutrófilos sobre la agregación y secreción plaquetaria. Métodos. El estudio se realizó en 12 voluntarios sanos de 32 ñ 6 años a los que se administró triflusal (600 mg/día) durante 5 días, extrayéndoles plaquetas y neutrófilos antes y después del tratamiento y midiéndoles su capacidad de producir NO, el porcentaje de agregación de sus plaquetas frente a ADP y la capacidad de liberar factor transformante del crecimiento (TGF- ). Resultados. Tras el tratamiento con triflusal se obtuvieron los siguientes resultados: a) aumento de la producción de NO en los neutrófilos; b) potenciación de la inhibición de la agregación plaquetaria en presencia de neutrófilos, efecto que se revertía al incubar los neutrófilos con un antagonista de L-arginina, L-NAME, y c) la presencia de neutrófilos redujo la liberación del TGF- por las plaquetas determinado como medida de secreción plaquetaria, por un mecanismo independiente del NO. Conclusiones. Nuestro estudio demuestra que el tratamiento con triflusal (600 mg/día/5 días) estimula la producción de NO por los neutrófilos. Tras el tratamiento con triflusal los neutrófilos inhiben la agregación y la secreción de las plaquetas. El efecto antiagregante plaquetario demostrado por los neutrófilos fue dependiente del NO, pero no así la inhibición de la desgranulación plaquetaria (AU)