RESUMEN
Cell penetrating peptides (CPP) represent a novel approach to facilitate cytoplasmic delivery of macromolecules. The DNA binding domain of Drosophila Antennapedia contains 60 amino acids and consists of 3 α-helices, with internalizing activity mapped to a 16-amino acid peptide penetratin (Antp) within the third α-helix. Here, we report on the use of penetratin to deliver a multiple antigen peptide (MAP) incorporating the immunodominant CD8 epitope of ovalbumin, SIINFEKL (MAPOVACD8). We demonstrate that penetratin linked to the MAPOVACD8 construct either by a disulfide (SS) or thioether (SC) linkage promotes the uptake, cross presentation and subsequent in vivo proliferation and generation of OVACD8 (SIINFEKL)-specific T cells. The MAPOVACD8 construct without penetratin is not presented by MHC class I molecules nor does it generate an in vivo IFN-γ response in C57BL/6 mice. Moreover, we clearly define the uptake and intracellular processing pathways of AntpMAPOVACD8 SS and SC revealing the majority of AntpMAPOVACD8 is taken up by DC via an endocytic, proteasome and tapasin independent mechanism. We also show that the uptake mechanism of AntpMAPOVACD8 is dose dependent and uptake or intracellular processing is not altered by the type of chemical linkage.
Asunto(s)
Proteínas Portadoras , Péptidos de Penetración Celular , Epítopos , Ovalbúmina , Linfocitos T/inmunología , Animales , Presentación de Antígeno/efectos de los fármacos , Presentación de Antígeno/inmunología , Proteínas Portadoras/química , Proteínas Portadoras/inmunología , Proteínas Portadoras/farmacología , Proliferación Celular/efectos de los fármacos , Péptidos de Penetración Celular/química , Péptidos de Penetración Celular/inmunología , Péptidos de Penetración Celular/farmacología , Células Dendríticas/inmunología , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta Inmunológica , Drosophila , Endocitosis/efectos de los fármacos , Endocitosis/inmunología , Epítopos/química , Epítopos/inmunología , Epítopos/farmacología , Antígenos de Histocompatibilidad Clase I/inmunología , Interferón gamma/inmunología , Proteínas de Transporte de Membrana/inmunología , Ratones , Ovalbúmina/química , Ovalbúmina/inmunología , Ovalbúmina/farmacología , Complejo de la Endopetidasa Proteasomal/inmunología , Estructura Secundaria de ProteínaRESUMEN
Recent years have seen a surge in interest in cell-penetrating peptides (CPP) as an efficient means for delivering therapeutic targets into cellular compartments. The cell membrane is impermeable to hydrophilic substances yet linking to CPP can facilitate delivery into cells. Thus the unique translocatory property of CPP ensures they remain an attractive carrier, with the capacity to deliver cargoes in an efficient manner having applications in drug delivery, gene transfer and DNA vaccination. Fundamental for an effective vaccine is the delivery of antigen epitopes to antigen-presenting cells, ensuing processing and presentation and induction of an immune response. Vaccination with proteins or synthetic peptides incorporating CTL epitopes have proven limited due to the failure for exogenous antigens to be presented efficiently to T cells. Linking of antigens to CPP overcomes such obstacles by facilitating cellular uptake, processing and presentation of exogenous antigen for the induction of potent immune responses. This review will encompass the various strategies for the delivery of whole proteins, T cell epitopes and preclinical studies utilizing CPP for cancer vaccines.
Asunto(s)
Vacunas contra el Cáncer/administración & dosificación , Proteínas Portadoras/administración & dosificación , Sistemas de Liberación de Medicamentos , Neoplasias/tratamiento farmacológico , Fragmentos de Péptidos/administración & dosificación , Péptidos de Penetración Celular , Humanos , Neoplasias/inmunologíaRESUMEN
OBJECTIVE: To examine the estrogenic and androgenic activity of Lepidium meyenii (Maca) and its effect on the hormonal profile and symptoms in postmenopausal women. DESIGN: Fourteen postmenopausal women completed a randomized, double-blind, placebo-controlled, crossover trial. They received 3.5 g/day of powered Maca for 6 weeks and matching placebo for 6 weeks, in either order, over a total of 12 weeks. At baseline and weeks 6 and 12 blood samples were collected for the measurement of estradiol, follicle-stimulating hormone, luteinizing hormone, and sex hormone-binding globulin, and the women completed the Greene Climacteric Scale to assess the severity of menopausal symptoms. In addition, aqueous and methanolic Maca extracts were tested for androgenic and estrogenic activity using a yeast-based hormone-dependent reporter assay. RESULTS: No differences were seen in serum concentrations of estradiol, follicle-stimulating hormone, luteinizing hormone, and sex hormone-binding globulin between baseline, Maca treatment, and placebo (P > 0.05). The Greene Climacteric Scale revealed a significant reduction in scores in the areas of psychological symptoms, including the subscales for anxiety and depression and sexual dysfunction after Maca consumption compared with both baseline and placebo (P < 0.05). These findings did not correlate with androgenic or alpha-estrogenic activity present in the Maca as no physiologically significant activity was observed in yeast-based assays employing up to 4 mg/mL Maca extract (equivalent to 200 mg/mL Maca). CONCLUSIONS: Preliminary findings show that Lepidium meyenii (Maca) (3.5 g/d) reduces psychological symptoms, including anxiety and depression, and lowers measures of sexual dysfunction in postmenopausal women independent of estrogenic and androgenic activity.