RESUMEN
Plasmacytoid dendritic cells (pDCs) play a major role in shaping both innate and adaptive immune responses, mainly via their production of large amounts of type I IFNs. pDCs are considered to primarily present endogenous Ags and are thought not to participate in the uptake and presentation of Ags from the extracellular environment, in contrast to their myeloid counterparts, which efficiently endocytose extracellular particulates. In this study, we show that human pDCs are able to phagocytose and process particulate forms of Ag entrapped in poly(lactic-coglycolic acid) microparticles. Furthermore, pDCs were also able to sense TLR ligands (TLR-Ls) incorporated in these particles, resulting in rapid pDC activation and high IFN-alpha secretion. Combining a tetanus toxoid peptide and TLR-Ls (CpG C and R848) in these microparticles resulted in efficient pDC activation and concomitant Ag-specific T cell stimulation. Moreover, particulate Ag was phagocytosed and presented more efficiently than soluble Ag, indicating that microparticles can be exploited to facilitate efficient delivery of antigenic cargo and immunostimulatory molecules to pDCs. Together, our results show that in addition to their potency to stimulate innate immunity, pDCs can polarize adaptive immune responses against exogenous particulate Ag. These results may have important consequences for the development of new immunotherapeutic strategies exploiting Ag and TLR-Ls encapsulated in microparticles to target APC subsets.
Asunto(s)
Presentación de Antígeno/inmunología , Micropartículas Derivadas de Células/inmunología , Células Dendríticas/inmunología , Glicolatos/inmunología , Fagocitosis/inmunología , Albúmina Sérica Bovina/inmunología , Animales , Cápsulas , Bovinos , Diferenciación Celular/inmunología , Micropartículas Derivadas de Células/metabolismo , Células Cultivadas , Células Dendríticas/citología , Células Dendríticas/metabolismo , Epítopos de Linfocito T/inmunología , Glicolatos/metabolismo , Humanos , Interferón-alfa/biosíntesis , Ácido Láctico , Ligandos , Activación de Linfocitos/inmunología , Ácido Poliglicólico , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Albúmina Sérica Bovina/metabolismo , Toxoide Tetánico/inmunología , Toxoide Tetánico/metabolismo , Receptores Toll-Like/metabolismoRESUMEN
Specific immunotherapy (SIT) is the only potentially curative treatment for those allergic processes mediated by IgE. We compared the effects of different SITs in mice sensitised with ovalbumin (OVA) Al (OH)(3) : 1) OVA entrapped in particles of poly (D,L-lactic-co-glycolic acid) (PLGA-OVA), 2) Soluble OVA (OVA-sol) and 3) Polymerised OVA (OVA-pol). Serum levels of specific IgE, IgG1, IgG2a and asymmetric IgG, the cutaneous anaphylaxis test (PCA), and the IL-10, IFNgamma and IL-4 levels in culture supernatants of splenocytes challenged with OVA were assessed. Mice treated with PLGA-OVA had higher levels of asymmetric antibodies than non-desensitised mice; a low IgG1 and high IgG2a level was observed together with inhibitory effect in the PCA reaction that reversed in the absence of asymmetric IgG. IL-10 and IFNgamma levels were higher in supernatants from mice treated with PLGA-OVA and OVA-sol than those obtained from non-desensitised controls. Our results suggest that among the different SITs evaluated, PLGA-OVA is the one that best showed an increase in the asymmetric IgG molecules and an effective deviation of the immune response. Furthermore, the increase in the proportion of asymmetric antibodies would be of importance when designing new vaccination strategies for allergy.
Asunto(s)
Desensibilización Inmunológica/métodos , Modelos Animales de Enfermedad , Hipersensibilidad/inmunología , Inmunoglobulina G/sangre , Animales , Células Cultivadas , Cromatografía de Afinidad , Glicolatos/inmunología , Hipersensibilidad/sangre , Inmunoglobulina E/sangre , Interferón gamma/metabolismo , Interleucina-10/metabolismo , Interleucina-4/metabolismo , Ácido Láctico , Ratones , Ratones Endogámicos BALB C , Ovalbúmina/inmunología , Anafilaxis Cutánea Pasiva , Ácido Poliglicólico , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Bazo/citología , Bazo/inmunología , Bazo/metabolismo , Factores de TiempoRESUMEN
Poly(lactide-co-glycolic acid) (PLGA) has been widely applied to tissue engineering as a good biocompatible material because of its biodegradability and nontoxic metabolites, but how the inflammatory reaction of PLGA on the surrounding tissue in vivo is reduced has not been discussed sufficiently. We hypothesized that the cells neighboring the PLGA implant might have an inflammatory response that could be reduced by impregnating demineralized bone particles (DBPs) into the PLGA. We manufactured five different ratios of DBP/PLGA hybrid materials, with each material containing 0, 10, 20, 40, and 80 wt% of DBPs of PLGA. For biocompatibility test, NIH/3T3 mouse fibroblasts were cultured on the DBP/PLGA scaffold for 3 days. The inflammatory potential of PLGA was evaluated using messenger ribonucleic acid expression of tumor necrosis factor alpha (TNF-alpha) and interleukin 1-beta (IL-1beta) on a human acute promyelocytic leukemic cell (HL-60). The in vivo response of DBP/PLGA film was compared with that of PLGA film implanted subcutaneously; the local inflammatory response was observed according to histology. The DBP/PLGA scaffold had no adverse effect on NIH/3T3 initial cell attachment and did not affect cell viability. DBP/PLGA films, especially PLGA films containing 80% DBP, elicited a significantly lower expression of IL-1beta and TNF-alpha from HL-60 cells than PLGA film alone. In vivo, DBP/PLGA film demonstrated a more favorable tissue response profile than PLGA film, with significantly less inflammation and fibrous capsule formation as below only 20% of DBP in PLGA film during implantation. This study shows that application of DBPs reduces the fibrous tissue encapsulation and foreign body giant cell response that commonly occurs at the interface of PLGA.
Asunto(s)
Reacción de Fase Aguda/inmunología , Técnica de Desmineralización de Huesos , Glicolatos/inmunología , Animales , Células Cultivadas , Fibrosis/patología , Expresión Génica/genética , Células Gigantes de Cuerpo Extraño/citología , Células HL-60 , Humanos , Implantes Experimentales , Interleucina-1beta/genética , Ácido Láctico , Macrófagos/citología , Ensayo de Materiales , Ratones , Células 3T3 NIH , Infiltración Neutrófila , Ácido Poliglicólico , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Ratas , Ratas Wistar , Tejido Subcutáneo , Andamios del Tejido , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
In this study we demonstrated the use of an oculonasally delivered poly(D,L-lactic-co-glycolic acid) microparticle (PLGA-MP)-based and genetically engineered vaccination strategy in the avian system. An avian Metapneumovirus (aMPV) fusion (F) protein-encoding plasmid vaccine and the corresponding recombinant protein vaccine were produced and bound to or encapsulated by PLGA-MP, respectively. The PLGA-MP as the controlled release system was shown in vitro to not induce any cytopathic effects and to efficiently deliver the F protein-based aMPV-vaccines to avian cells for further processing. Vaccination of turkeys was carried out by priming with an MP-bound F protein-encoding plasmid vaccine and a booster-vaccination with an MP-encapsulated recombinant F protein. Besides the prime-boost F-specific vaccinated birds, negative control birds inoculated with a mock-MP prime-boost regimen as well as non-vaccinated birds and live vaccinated positive control birds were included in the study. The MP-based immunization of turkeys via the oculonasal route induced systemic humoral immune reactions as well as local and systemic cellular immune reactions, and had no adverse effects on the upper respiratory tract. The F protein-specific prime-boost strategy induced partial protection. After challenge the F protein-specific MP-vaccinated birds showed less clinical signs and histopathological lesions than control birds of mock MP-vaccinated and non-vaccinated groups did. The vaccination improved viral clearance and induced accumulation of local and systemic CD4+ T cells when compared to the mock MP-vaccination. It also induced systemic aMPV-neutralizing antibodies. The comparison of mock- and F protein-specific MP-vaccinated birds to non-vaccinated control birds suggests that aMPV-specific effects as well as adjuvant effects mediated by MP may have contributed to the overall protective effect.
Asunto(s)
Glicolatos/inmunología , Metapneumovirus/inmunología , Infecciones por Paramyxoviridae/prevención & control , Infecciones por Paramyxoviridae/veterinaria , Enfermedades de las Aves de Corral/prevención & control , Proteínas Virales de Fusión/inmunología , Animales , Anticuerpos Antivirales/inmunología , Formación de Anticuerpos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Efecto Citopatogénico Viral/efectos de los fármacos , Efecto Citopatogénico Viral/inmunología , Preparaciones de Acción Retardada/farmacología , Ingeniería Genética , Glicolatos/química , Glicolatos/farmacología , Inmunización Secundaria , Ácido Láctico , Metapneumovirus/genética , Infecciones por Paramyxoviridae/genética , Infecciones por Paramyxoviridae/inmunología , Plásmidos/inmunología , Ácido Poliglicólico , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Enfermedades de las Aves de Corral/genética , Enfermedades de las Aves de Corral/inmunología , Pavos , Vacunación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Vacunas Sintéticas/farmacología , Proteínas Virales de Fusión/farmacologíaRESUMEN
Understanding of biomaterial adjuvant effect and its mechanisms is essential for the effective design and selection of appropriate materials for specific applications. We have previously shown that poly(lactic-co-glycolic acid) (PLGA), one of the most commonly studied polymers in tissue engineering, supports an adjuvant effect as measured by enhanced immune response against a co-delivered model antigen, which was dependent on the form of the biomaterial. Furthermore, we have shown that PLGA induces the maturation of human peripheral blood mononuclear cell-derived dendritic cells (DCs) in vitro. In this study, the effect of PLGA contact on the maturation of murine bone marrow-derived DCs was investigated in part to explain the biomaterial adjuvant effect observed. Treatment of bone marrow-derived DCs from C57BL6 mice with PLGA microparticles or films lead to maturation of these cells as exemplified by increased expression of co-stimulatory molecules CD80 and CD86 and production of proinflammatory cytokines TNF-alpha and IL-6. These results suggest that PLGA contact induces maturation of murine DCs, supporting our observations with human DCs. With the techniques developed in this study and given the results, our future goal is to utilize transgenic murine models to delineate the mechanisms of biomaterial-induced DC maturation.
Asunto(s)
Adyuvantes Inmunológicos , Materiales Biocompatibles , Células de la Médula Ósea/inmunología , Células Dendríticas/inmunología , Glicolatos/inmunología , Ensayo de Materiales , Animales , Antígeno B7-2/biosíntesis , Antígeno B7-2/inmunología , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Células Cultivadas , Células Dendríticas/citología , Células Dendríticas/metabolismo , Humanos , Interleucina-6/biosíntesis , Interleucina-6/inmunología , Ácido Láctico , Ratones , Ácido Poliglicólico , Copolímero de Ácido Poliláctico-Ácido Poliglicólico , Ingeniería de Tejidos , Factor de Necrosis Tumoral alfa/biosíntesis , Factor de Necrosis Tumoral alfa/inmunologíaRESUMEN
Immature dendritic cells (iDCs) were derived from human peripheral blood monocytes, and treated with 75:25 poly(lactic-co-glycolic acid) (PLGA) microparticles (MPs) or film to assess the resultant dendritic cell (DC) maturation as compared to positive control of lipopolysaccharide (LPS) treatment for DC maturation or negative control of untreated iDCs. The effect of PLGA contact on DC maturation was examined as one possible explanation for the PLGA adjuvant effect we have observed in the enhancement of an immune response to codelivered model antigen, as adjuvants act through the maturation of DCs. Culturing iDCs with PLGA MPs or PLGA film resulted in morphology similar to that of LPS-matured DCs and the association, or possible internalization, of PLGA MPs. Furthermore, biomaterial-treated iDCs demonstrated an increase in MHC class II and costimulatory molecule expression compared to iDCs but to a lower level than that of LPS-matured DCs. Direct iDC contact with PLGA MPs was necessary for maturation. Immature DCs exposed to PLGA MPs were stimulatory of allogeneic T-cell proliferation, whereas cells exposed to PLGA film were not. Further, PLGA MPs supported a moderate delayed type hypersensitivity reaction in mice indicative of in vivo DC maturation. Taken together, these results suggest that PLGA is a DC maturation stimulus and that the form of the biomaterial may influence the extent of DC maturation.
Asunto(s)
Materiales Biocompatibles/farmacología , Células Dendríticas/efectos de los fármacos , Glicolatos/farmacología , Monocitos/efectos de los fármacos , Animales , Antígenos CD/inmunología , Biomarcadores , Técnicas de Cultivo de Célula/métodos , Forma de la Célula , Células Cultivadas , Células Dendríticas/citología , Células Dendríticas/inmunología , Células Dendríticas/fisiología , Glicolatos/inmunología , Humanos , Hipersensibilidad Tardía , Ácido Láctico , Prueba de Cultivo Mixto de Linfocitos , Ensayo de Materiales , Ratones , Monocitos/citología , Monocitos/inmunología , Monocitos/fisiología , Fenotipo , Ácido Poliglicólico , Copolímero de Ácido Poliláctico-Ácido PoliglicólicoRESUMEN
The present work describes the design and synthesis of the structurally unique hapten, "bifunctional hapten", to produce a group-specific polyclonal antiserum to triclopyr and 3,5,6-trichloro-2-pyridinol. A bifunctional hapten was designed and synthesized by conjugating commercially available Nepsilon-2,4-dinitrophenyl (DNP)-L-lysine to triclopyr, and then coupling this to carrier proteins such as bovine serum albumin (BSA). The synthesized bifunctional hapten greatly raised the antiserum titer in comparison with that of the conventional hapten, triclopyr. Antiserum with a sufficiently high titer to provide the determinations of targeted compounds was obtained only 63 days after the primary immunization. The obtained antiserum showed the highest affinity to triclopyr (IC(50) = 3.5 nM) and 3,5,6-trichloro-2-pyridinol (IC(50) = 5.1 nM) in homologous ELISA. The cross-reactivities to various agrochemicals and some chlorinated phenolic compounds were determined. Significant cross-reactivity was found to the herbicide 2,4,5-T. The antiserum reacted to both triclopyr and its metabolite. Assay sensitivity was evaluated for effects of various assay conditions, including pH value and concentrations of organic solvents and detergents. Under optimized assay conditions, the quantitative working range of triclopyr ELISA was from 0.1 to 5.2 ng/mL with a limit of detection (LOD) of 0.037 ng/mL, and an IC(50) of 0.72 ng/mL. On the other hand, the quantitative working range of 3,5,6-trichloro-2-pyridinol ELISA was from 0.13 to 6.0 ng/mL with a LOD of 0.052 ng/mL, and an IC(50) of 0.95 ng/mL. Water samples fortified with triclopyr or its metabolite at 1, 5, and 10 ng/mL were directly analyzed without extraction and cleanup by the proposed ELISA. The mean recovery was 101.6%, and the mean coefficient of variation (CV) was 7.1% in the case of the triclopyr ELISA. In the case of the 3,5,6-trichloro-2-pyridinol ELISA, the mean recovery was 99.8%, and the mean CV was 9.5%. The proposed ELISA turned out to be a powerful tool for monitoring of residual triclopyr or 3,5,6-trichloro-2-pyridinol in water samples at trace level.
Asunto(s)
Ensayo de Inmunoadsorción Enzimática/métodos , Glicolatos/análisis , Haptenos , Sueros Inmunes , Piridonas/análisis , 2,4-Dinitrofenol/inmunología , Agroquímicos/análisis , Especificidad de Anticuerpos , Detergentes , Glicolatos/inmunología , Haptenos/química , Haptenos/inmunología , Concentración de Iones de Hidrógeno , Piridonas/inmunología , Control de Calidad , Sensibilidad y Especificidad , Albúmina Sérica Bovina/inmunología , Solventes , Agua/análisisRESUMEN
In order to investigate the mechanisms underlying the hepatotoxicity associated with tienilic acid (Ticrynafen) ingestion we have looked for evidence of sensitisation to drug altered liver cell determinants using an indirect antibody dependent, cell mediated cytotoxicity assay (ADCC). As targets, hepatocytes were isolated from rabbits pretreated with either tienilic acid or its isomer with or without previous enzyme induction with either phenobarbitone or B-naphthoflavone (BNF). Sera from 16 of 36 patients with presumed tienilic acid hepatotoxicity induced significant cytotoxicity to hepatocytes isolated from rabbits pretreated with BNF and subsequently tienilic acid. Three of 10 sera from patients receiving tienilic acid but without overt liver damage also induced significant cytotoxicity to these hepatocytes, however, although none of 20 normal controls or of 16 patients with other liver diseases did so. Non-organ specific autoantibodies, classified as anti-LKM2, were also detectable. These were present in association with tienilic acid associated antibodies: of the 36 patients with presumed tienilic acid hepatotoxicity, 38% had both antibodies, 18% had only anti-LKM2 antibodies and 9% only tienilic acid associated antibodies. These results suggest that this drug reaction is associated with sensitisation to drug altered liver cell antigens and autoantigens. If ticrynafen associated hepatotoxicity is immune mediated, then one possible mechanism is that the drug induced antigens break tolerance, leading to an immune attack on normal liver cell components.