RESUMEN
Growth factor therapy is an emerging treatment modality that enhances tissue vascularization, promotes healing and regeneration and can treat a variety of inflammatory diseases. Both recombinant human growth factor proteins and their gene therapy are in human clinical trials to heal chronic wounds. As platelet-derived growth factor-bb (PDGF-BB) and fibroblast growth factor-2 (FGF-2) are known to induce chemotaxis, proliferation, differentiation, and matrix synthesis, we investigated a non-viral means for gene delivery of these factors using the cationic polysaccharide chitosan. Chitosan is a polymer of glucosamine and N-acetyl-glucosamine, in which the percentage of the residues that are glucosamine is called the degree of deacetylation (DDA). The purpose of this study was to express PDGF-BB and FGF-2 genes in mice using chitosan-plasmid DNA nanoparticles for the controlled delivery of genetic material in a specific, efficient, and safe manner. PDGF-BB and FGF-2 genes were amplified from human tissues by RT-PCR. To increase the secretion of FGF-2, a recombinant 4sFGF-2 was constructed bearing eight amino-acid residues of the signal peptide of FGF-4. PCR products were inserted into the expression vector pVax1 to produce recombinant plasmids pVax1-4sFGF2 and pVax1-PDGF-BB, which were then injected into BALB/C mice in the format of polyelectrolyte nanocomplexes with specific chitosans of controlled DDA and molecular weight, including 92-10, 80-10, and 80-80 (DDA-number average molecular weight or M(n) in kDa). ELISA assays on mice sera showed that recombinant FGF-2 and PDGF-BB proteins were efficiently expressed and specific antibodies to these proteins could be identified in sera of injected mice, but with levels that were clearly dependent on the specific chitosan used. We found high DDA low molecular weight chitosans to be efficient protein expressors with minimal or no generation of neutralizing antibodies, while lowering DDA resulted in greater antibody levels and correspondingly lower levels of detected recombinant protein. Histological analyses corroborated these results by revealing greater inflammatory infiltrates in lower DDA chitosans, which produced higher antibody titers. We found, in general, a more efficient delivery of the plasmids by subcutaneous than by intramuscular injection. Specific chitosan carriers were identified to be either efficient non-toxic therapeutic protein delivery systems or vectors for DNA vaccines.
Asunto(s)
Quitosano , Factor 2 de Crecimiento de Fibroblastos/genética , Técnicas de Transferencia de Gen , Nanopartículas/administración & dosificación , Factor de Crecimiento Derivado de Plaquetas/genética , Animales , Formación de Anticuerpos , Secuencia de Bases , Becaplermina , Femenino , Factor 2 de Crecimiento de Fibroblastos/inmunología , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Humanos , Inyecciones Intramusculares , Inyecciones Subcutáneas , Ratones , Ratones Endogámicos BALB C , Microscopía Electrónica de Rastreo , Datos de Secuencia Molecular , Músculo Esquelético/patología , Nanopartículas/ultraestructura , Plásmidos/administración & dosificación , Factor de Crecimiento Derivado de Plaquetas/inmunología , Factor de Crecimiento Derivado de Plaquetas/metabolismo , Proteínas Proto-Oncogénicas c-sis , Proteínas Recombinantes/genética , Piel/patologíaRESUMEN
Human immunodeficiency virus type 1 (HIV-1) infection triggers a cytotoxic T-lymphocyte (CTL) response mediated by CD8(+) and perhaps CD4(+) CTLs. The mechanisms by which HIV-1 escapes from this CTL response are only beginning to be understood. However, it is already clear that the extreme genetic variability of the virus is a major contributing factor. Because of the well-known ability of altered peptide ligands (APL) to induce a T-cell receptor (TCR)-mediated anergic state in CD4(+) helper T cells, we investigated the effects of HIV-1 sequence variations on the proliferation and cytotoxic activation of a human CD4(+) CTL clone (Een217) specific for an epitope composed of amino acids 410 to 429 of HIV-1 gp120. We report that a natural variant of this epitope induced a functional anergic state rendering the T cells unable to respond to their antigenic ligand and preventing the proliferation and cytotoxic activation normally induced by the original antigenic peptide. Furthermore, the stimulation of Een217 cells with this APL generated altered TCR-proximal signaling events that have been associated with the induction of T-cell anergy in CD4(+) T cells. Importantly, the APL-induced anergic state of the Een217 T cells could be prevented by the addition of interleukin 2, which restored their ability to respond to their nominal antigen. Our data therefore suggest that HIV-1 variants can induce a state of anergy in HIV-specific CD4(+) CTLs. Such a mechanism may allow a viral variant to not only escape the CTL response but also facilitate the persistence of other viral strains that may otherwise be recognized and eliminated by HIV-specific CTLs.