RESUMEN
Cytolytic ETosis is a type of programmed cell death distinct from apoptosis and necrosis and plays a major role in the innate immune system and disease progression. Through the process of ETosis, cells release their chromatin with diverse antimicrobial proteins into the extracellular milieu, forming extracellular traps (ETs). Although ETosis has been reported in several leukocyte types, few studies have compared ETosis and the component proteins of ETs in leukocytes. The aim of this study was to better understand the characteristics of eosinophil ETosis (EETosis) compared with other leukocytes. We isolated human blood eosinophils, neutrophils, basophils, monocytes, and lymphocytes and stimulated them with known ETosis inducers, a protein kinase C activator PMA, or a calcium ionophore A23187. Both stimuli induced eosinophil cell death and ET release after 180 minutes of stimulation in a NADPH-oxidase-dependent manner. PMA also induced NADPH-oxidase-dependent ETosis in neutrophils, whereas little or no significant ETosis was observed in basophils, monocytes, or lymphocytes at 180 minutes. Mass spectrometry-based proteomic analysis of eosinophil- and neutrophil-derived ETs identified 997 and 1415 proteins, respectively. Among the physiological stimuli tested, immobilized IgA and IgG induced EETosis. C-C motif chemokine ligand 11 (CCL11) and interleukin 5 (IL-5) were weak inducers of EETosis, but co-stimulation significantly induced rapid EETosis. Under high serum or albumin conditions, co-stimulation with CCL11 and IL-5 paradoxically prolonged cell survival by preventing spontaneous apoptosis. This study provides an in-depth characterization of EETosis and highlights the precise regulation of eosinophil survival and cell death pathways.
RESUMEN
SUMMARY: Although supermicrosurgery techniques are essential skills for lymphatic surgery or perforator-flap surgery, an ideal training model is yet to be introduced. Living animal models, such as rodents, are considered to be ideal microsurgical training models. However, the use of living animal models is costly and involves bioethical considerations. Hence, the authors developed a novel, cost-effective, highly reproducible, and easy-to-handle supermicrosurgical anastomosis training system using the chicken embryo within the egg-in-cube system. Chick embryos were fertilized in the artificial cubic eggshell, which was fabricated by integrating a polycarbonate frame structure and five polydimethylsiloxane membranes. Seven days later, the trainees underwent supermicrosurgical training using the vitelline artery of the chick embryo. The trainees were able to perform supermicrosurgical training using all 11 surviving chicken embryos. The average diameter of the vitelline artery was 0.43 mm. Patency and pulsation were observed after the anastomosis in four of the 11 cases. The supermicrosurgical training system using the chicken embryo within the egg-in-cube system has several advantages. This system is ethically acceptable, less costly and easier to manage than other animal models, and suitable as a supermicrosurgical training model, such as for lymphovenular anastomosis, because the diameter of the vitelline artery was similar to that of the lymphatic vessels in patients with lymphedema. Moreover, the trainee can confirm patency and leakage after the anastomosis because this model has a circulation system. The trainee can practice the supermicrosurgical technique efficiently with simultaneous feedback on anastomosis results. CLINICAL RELEVANCE STATEMENT: This study introduces a novel, cost-effective supermicrosurgical training system using chicken embryos within an egg-in-cube, offering a practical and ethical alternative. Its close simulation to human lymphatic vessels supports skill enhancement for practicing surgeons.