RESUMO
OBJECTIVE: Abnormalities in the coagulation pathway are often included in the diagnostic work-up of stroke patients, especially in young adults with cryptogenic stroke. METHODS: Three common genetic variants within the coagulation cascade were investigated in 500 control subjects and in 167 patients with ischemic stroke defined by TOAST subclassification. Analysed variants were factor V Leiden, prothrombin 20210G-->A and factor XIII Val34Leu. RESULTS: The factor V Leiden mutation was over-represented in patients with cardioembolic stroke for trend, whereas the prothrombin 20210G-->A variant and the factor XIII polymorphism Val34Leu were not associated with stroke of any subtype. The three polymorphisms showed no association with stroke in subgroups of patients defined by age (<40, 40-49, 50-59, > or =60 years). DISCUSSION: This study suggests that the analysis of prothrombin 20210G-->A and factor XIII Val34Leu is not a useful diagnostic procedure in the work-up of ischemic stroke.
Assuntos
Isquemia Encefálica/genética , Fator V/genética , Fator XIII/genética , Polimorfismo Genético , Protrombina/genética , Acidente Vascular Cerebral/genética , Adulto , Fatores Etários , Coagulação Sanguínea/genética , Coagulação Sanguínea/fisiologia , Estudos de Casos e Controles , Fator V/metabolismo , Fator XIII/metabolismo , Feminino , Estudos de Associação Genética , Variação Genética , Alemanha , Humanos , Embolia Intracraniana/genética , Masculino , Pessoa de Meia-Idade , Modelos Cardiovasculares , Protrombina/metabolismo , População Branca/genéticaRESUMO
The skin is an attractive target for antigen-specific vaccination. Particle bombardment of the epidermis with plasmid DNA using the gene gun results in antigen expression in keratinocytes of the epidermis leading to antigen presentation in the draining lymph nodes by migratory dendritic cells (DC). In order to better understand the role of the skin in stimulating antigen-specific CD8+cytotoxic T cells (CTL), we compared gene gun immunization with intracutaneous injections of antigen-transduced DC. A single intracutaneous injection of antigen-transduced DC was able to induce in vivo expansion of CD8+CTL specific for the model antigen chicken ovalbumin while four simultaneous shots with the gene gun were not effective. Antigen-transduced DC were much more efficient than particle bombardment of the epidermis in stimulating adoptively transferred TCR-transgenic CD8+CTL in the draining lymph nodes. Employing the novel technique of in vivo bioluminescence imaging, we demonstrated efficient gene transfer to the skin following gene gun bombardment and confirmed that a similar amount of antigen reached the lymph node when compared with injection of antigen-transduced DC. Our results suggest that direct transfection of the skin does not optimally reach and activate appropriate antigen-presenting DC. We believe that this reflects the immunological function of the epidermis which must balance immunity and tolerance to foreign antigens. Further investigations will have to address the role of Langerhans cells for the activation of cellular immunity in the skin.
Assuntos
Antígenos/administração & dosagem , Biolística/métodos , Células Dendríticas/imunologia , Imunização/métodos , Pele/imunologia , Linfócitos T Citotóxicos/imunologia , Transdução Genética , Transferência Adotiva , Animais , Proliferação de Células , Células Dendríticas/citologia , Injeções Intradérmicas , Luciferases/metabolismo , Proteínas Luminescentes/metabolismo , Linfonodos/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Ovalbumina , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Citotóxicos/citologia , Vacinas de DNA/imunologia , Imagem Corporal TotalRESUMO
Recombinant DNA vaccines are able to induce strong CD8+ T cell mediated immunity and have become increasingly attractive for the prevention and treatment of infectious diseases and cancer. Dendritic cells (DC), which critically control cellular immune responses, have been transduced with antigen ex vivo and used as 'nature's adjuvant' to enhance vaccine efficacy. The impact of the application route on the in vivo distribution of antigen and the stimulation of CD8+ T cells have been subjects of considerable debate. Here we report the construction of vectors expressing a fusion protein between EGFP, the H2-K(b)-binding peptide OVA(aa257-264) and green click beetle luciferase as a model antigen which allows for simultaneous quantitative assessment of antigen expression using fluorescence and bioluminescence imaging in correlation with CD8+ T cell stimulation in vivo. We applied this construct to evaluate DNA vaccination with recombinant adenoviral vectors, assess the impact of using cultured DC for vaccine delivery and investigate different application routes. Antigen expression was non-invasively followed in vivo by visualizing bioluminescence with an ultrasensitive CCD camera. CD8+ T cell stimulation was detected with H2-K(b)-OVA(aa257-264) tetramers. We found that intravenous injection of adenovirus-transduced DC stimulated the strongest OVA(aa257-264)-specific cytotoxic T-lymphocyte (CTL) responses although it delivered two orders of magnitude less antigen in vivo when compared to direct injection of recombinant adenovirus. We believe that our experimental approach has the potential to facilitate translational development of improved genetic immunization strategies targeting DC directly in vivo.