RESUMO
Survival benefit and long-term duration of clinical response have been seen using the epidermal growth factor receptor (EGFR)-targeted monoclonal antibody (mAb) nimotuzumab. Blocking EGFR signaling may not be the only mechanism of action underlying its efficacy. As an IgG1 isotype mAb, nimotuzumab's capacity of killing tumor cells by antibody dependent cellular cytotoxicity (ADCC) and to induce an immune response in cancer patients have not been studied. ADCC-induced by nimotuzumab was determined using a 51Cr release assay. The in vitro effect of nimotuzumab on natural killer (NK) cell activation and dendritic cell (DC) maturation and the in vivo frequency of circulating regulatory T cells (Tregs) and NK cells were assessed by flow cytometry. Cytokine levels in supernatants were determined by ELISA. ELISpot was carried out to quantify EGFR-specific T cells in nimotuzumab-treated head and neck cancer (HNSCC) patients. Nimotuzumab was able to kill EGFR+ tumor cells by NK cell-mediated ADCC. Nimotuzumab-activated NK cells promoted DC maturation and EGFR-specific CD8+ T cell priming. Interestingly, nimotuzumab led to upregulation of some immune checkpoint molecules on NK cells (TIM-3) and DC (PD-L1), to a lower extent than another EGFR mAb, cetuximab. Furthermore, circulating EGFR-specific T cells were identified in nimotuzumab-treated HNSCC patients. Notably, nimotuzumab combined with cisplatin-based chemotherapy and radiation increased the frequency of peripheral CD4+CD39+FOXP3+Tregs which otherwise were decreased to baseline values when nimotuzumab was used as monotherapy. The frequency of circulating NK cells remained constant during treatment. Nimotuzumab-induced, NK cell-mediated DC priming led to induction of anti-EGFR specific T cells in HNSCC patients. The association between EGFR-specific T cells and patient clinical benefit with nimotuzumab treatment should be investigated.
RESUMO
Es necesario investigar drogas naturales nuevas que aporten principios farmacológicos activos para ser utilizadas como una alternativa terapéutica. Por este motivo nos propusimos estudiar a Ficus insipida, cuyo látex ha sido usado como antihelmíntico durante muchos años en la amazonía, pero se investigó solo superficialmente su efecto anticoagulante in vitro. Objetivo: Comprobar el efecto anticoagulante in vitro y determinar la vía de la coagulación sobre la que actúa el látex de Ficus insípida. Material y métodos: Se obtuvo el látex de Ficus insipida y se prepararon diferentes concentraciones del mismo. Se obtuvieron muestras de sangre venosa periférica de 5 donantes voluntarios, anticoagulándolas con citrato de sodio. Luego, éstas se mezclaron con las diluciones del látex, se centrifugaron y se extrajo el plasma. Posteriormente, se obtuvo un pool de plasma para cada concentración del látex y se procedió a determinar el Tiempo de Protrombina (TP) y el Tiempo de Tromboplastina Parcial activada (TTPa), respectivamente. Resultados: Se encontró que el látex de Ficus insipida prolongó el TP a una concentración mayor o igual a 0,03125% (V/V), y ambos, el TP y TTPa a una concentración mayor o igual a 0,15% (V/V). Conclusiones: Los resultados obtenidos nos permiten afirmar que el látex de Ficus insipida posee un efecto anticoagulante in vitro dosis dependiente sobre la vía extrínseca de la coagulación sanguínea a una concentración igual o mayor a 0,03125% (V/V) y que a una concentración igual o mayor a 0,15% (V/V) posee un potente efecto anticoagulante sobre ambas vías de la coagulación.
We need to search for new natural drugs yielding pharmacological active principles to be used as an alternative to conventional therapies. For this reason, we proposed to study Ficus insipida, an amazonian plant that its latex has been used for years as an Antihelmintic agent but its anticoagulant effect has been only superficially studied. Objective: To corroborate the in vitro anticoagulant effect and to determine upon which of the coagulation pathways acts the latex of Ficus insipida. Material and methods: The latex of Ficus insipid was obtained and prepared at different concentrations. Then, samples of peripheral blood from 5 donors were taken using sodium citrate to avoid blood clotting; the samples were mixed with the latex dilutions and centrifuged, after that, the plasma was extracted to form a plasma pool from each latex concentration. Finally, the Prothrombin Time (PT) and the Partial Thromboplastin Time (PTT) were determined, respectively. Results: The results obtained demonstrated that the latex of Ficus insipida delays the PT at concentrations equal or higher than 0.03125% (V/V), and both the PT and PTT at concentrations equal or higher than 0.15% (V/V). Conclusions: This data allow us to affirm that the latex of Ficus insipida has a dose-dependent in vitro anticoagulant effect upon the extrinsic coagulation pathway at concentrations equal or higher than 0.03125% (V/V). Additionally, at concentrations equal or higher than 0.15% (V/V) it has a potent anticoagulant effect over both coagulation pathways.