RESUMEN
Von Willebrand factor (VWF) is a chaperone molecule for procoagulant factor VIII (FVIII). Its role in the reduction of the immunogenicity of therapeutic FVIII in patients with hemophilia A has been evoked but lacks clear cellular and molecular rationale. Here, we demonstrate that VWF protects FVIII from being endocytosed by human dendritic cells (DCs) and subsequently presented to FVIII-specific T cells. The immunoprotective effect of VWF requires a physical interaction with FVIII because the endocytosis of FVIII was significantly restored on hindering the formation of the VWF-FVIII complex. Interestingly, VWF had no direct inhibitory effect either on the ability of DCs to present antigenic peptides or on the activation potency of CD4+ T cells. We thus propose that VWF may reduce the immunogenicity of FVIII by preventing, upstream from the activation of immune effectors, the entry of FVIII in professional antigen-presenting cells.
Asunto(s)
Presentación de Antígeno/inmunología , Células Dendríticas/inmunología , Endocitosis/inmunología , Factor VIII/inmunología , Linfocitos T/inmunología , Factor de von Willebrand/fisiología , Células Cultivadas , Células Dendríticas/ultraestructura , HumanosRESUMEN
The present study was conducted to evaluate in vitro and in vivo the antioxidant and anti-inflammatory properties of a cantaloupe melon (Cucumis melo LC., Cucurbitaceae) extract (CME) selected for its high superoxide dismutase activity. Peritoneal macrophages were pre-activated in vitro with 300 IU of interferon-gamma (IFN-gamma) and were then challenged in culture with IgGl/anti-IgG1 immune complexes (IgG1IC) in presence of various CME extracts. The subsequent production of free radicals (superoxide anion, nitric oxide, and peroxynitrite) and of pro-(TNF-alpha) and anti-(IL-10) inflammatory cytokines was evaluated. The CME inhibited in a dose-dependent manner the production of superoxide anion with a maximal effect at 100 microg/ml. This inhibitory effect of CME appeared to be closely linked to the SOD activity because it was dramatically decreased after heat inactivation of the SOD activity (HI-CME). In addition, the CME inhibited the production of peroxynitrite strengthening the antioxidant properties of this CME rich in SOD activity. The production of the pro- and anti-inflammatory cytokines, namely TNF-alpha and IL-10, being conditioned by the redox status of macrophages we also evaluated the effect of CME and HI-CME on the IgG1IC-induced cytokine production. When the SOD activity was present in the CME it promoted the IgG1IC-induced production of IL-10 instead of TNF-alpha. These data demonstrated that, in addition to its antioxidant properties, the anti-inflammatory properties of the CME extract were principally related to its capacity to induce the production of IL-10 by peritoneal macrophages. The particular properties of wheat gliadin (Triticum vulgare, Poaceae) for the oral delivery of functional proteins led us to test it in a new nutraceutical formula based on its combination with the CME thus monitoring the SOD activity release during the gastro-intestinal digestive process. In these experiments C57BL/6 mice were supplemented orally everyday during 28 days with: (1) the placebo, (2) the CME extract alone, (3) the gliadin, (4) the CME/gliadin combination, or (5) the HI-CME/gliadin combination (SOD inactivated). At the end of the supplementation period all the animals were injected intra-peritoneal (i.p.) with the pro-inflammatory cytokine IFN-gamma (300 IU) and peritoneal macrophages were harvested 24 h after to test their capacities to produce free radicals, TNF-alpha and IL-10 after triggering with IgG1IC. We demonstrated that animals supplemented during 28 days with the CME/gliadin combination were protected against the pro-inflammatory properties of IFN-gamma while the other products were inefficient. These data did not only indicate that the SOD activity is important for the antioxidant and anti-inflammatory properties of the CME extract, but also demonstrated that when the SOD activity is preserved during the digestive process by its combination with wheat gliadin it is possible to elicit in vivo the pharmacological effects of this antioxidant enzyme.
Asunto(s)
Antiinflamatorios/farmacología , Antioxidantes/farmacología , Cucumis melo , Superóxido Dismutasa/farmacología , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Radicales Libres/metabolismo , Gliadina/farmacología , Interleucina-10/biosíntesis , Macrófagos Peritoneales/efectos de los fármacos , Macrófagos Peritoneales/metabolismo , Ratones , Ratones Endogámicos C57BL , Extractos Vegetales/farmacología , Precursores de Proteínas/antagonistas & inhibidores , Factor de Necrosis Tumoral alfa/antagonistas & inhibidoresRESUMEN
The potential benefits to health of antioxidant enzymes supplied either through dietary intake or supplementation is still a matter of controversy. The development of dietary delivery systems using wheat gliadin biopolymers as a natural carrier represents a new alternative. Combination of antioxidant enzymes with this natural carrier not only delayed their degradation (i.e. the superoxide dismutase, SOD) during the gastrointestinal digestive process, but also promoted, in vivo, the cellular defences by strengthening the antioxidant status. The effects of supplementation for 28 days with a standardized melon SOD extract either combined (Glisodin) or not with gliadin, were evaluated on various oxidative-stress biomarkers. As already described there was no change either in superoxide dismutase, catalase or glutathione peroxidase activities in blood circulation or in the liver following non-protected SOD supplementation. However, animals supplemented with Glisodin showed a significant elevation in circulated antioxidant enzymes activities, correlated with an increased resistance of red blood cells to oxidative stress-induced hemolysis. In the presence of Sin-1, a chemical donor of peroxynitrites, mitochondria from hepatocytes regularly underwent membrane depolarization as the primary biological event of the apoptosis cascade. Hepatocytes isolated from animals supplemented with Glisodin presented a delayed depolarization response and an enhanced resistance to oxidative stress-induced apoptosis. It is concluded that supplementation with gliadin-combined standardized melon SOD extract (Glisodin) promoted the cellular antioxidant status and protected against oxidative stress-induced cell death.
Asunto(s)
Antioxidantes/farmacología , Cucumis melo , Gliadina/farmacología , Fitoterapia , Extractos Vegetales/farmacología , Triticum , Animales , Antioxidantes/administración & dosificación , Antioxidantes/uso terapéutico , Apoptosis/efectos de los fármacos , Suplementos Dietéticos , Gliadina/administración & dosificación , Gliadina/uso terapéutico , Hepatocitos/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Mitocondrias Hepáticas/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Ácido Peroxinitroso , Extractos Vegetales/administración & dosificación , Extractos Vegetales/uso terapéutico , Superóxido Dismutasa/administración & dosificación , Superóxido Dismutasa/farmacología , Superóxido Dismutasa/uso terapéuticoRESUMEN
Subcutaneous in vivo injections of cells of the mastocytoma line P815 in syngenic DBA/2 mice induce locally fast growing solid tumors. These have been used extensively as a cancer model to analyze and manipulate the relationship between tumor cells and host's immune defenses. We report that progression of P815 tumors in vivo was accompanied by a burst (Days 5-7) of local inflammatory cells recruitment and angiogenesis observed histologically, corroborated in vivo by MRI with gadolinium, overtranscription of macrophage activation marker genes, secretion of TNF-alpha by regional lymph node cells and concomitant systemic inflammation. No substantial overtranscriptions of either VEGF or IL-10 or TGF-beta genes were observed. Induction of COX-2 gene was a late event. To establish a possible relationship between the tumor-induced local, regional and systemic increase of pro-inflammatory mediators and progression of tumors in vivo, we carried out experiments deliberately modulating the inflammatory status of the recipient animals. Pretreatment of recipient animals by i.p. injection of thioglycolate accelerated P815 tumor growth. At the opposite, treatment of mice with either a COX-1 + COX-2 inhibitor (aspirin, 1 mg/day/mouse) or a specific COX-2 inhibitor (celecoxib, 0.13 mg/day/mouse) for 2 weeks after injection of tumor cells, significantly reduced the size and growth rate of tumors compared to control mice. Experiments carried out in vitro indicated that peritoneal macrophages from untreated animals were strongly activated by live P815 cells and by P815 membrane preparations. The tumor-induced inflammatory reaction could establish a local micro environment favoring tumor progression. The P815 tumor model might be helpful to recognize important factors controlling host/tumor relationship.