COLOSTRUM AND MILK EVS FROM WATER BUFFALO SAMPLES: PRELIMINARY CHARACTERIZATION AND EVALUATION OF THEIR EFFECTS ON BOVINE MACROPHAGES AND HUMAN INTESTINAL CELLS

Colostrum and milk are nutrient-rich liquids produced by mammals, beyond traditional nutritional aspects, recent research has focused on extracellular vesicles (EVs) and their potential health benefits, especially for gastrointestinal health. This study aims to characterize and evaluate the effects of EVs from colostrum and milk of water buffaloes, on bovine macrophages and human intestinal cells. Briefly, 15 and 60 days milk (15m and 60m) and colostrum (colo) from water buffaloes (Bubalus bubalis) were obtained by Istituto Zooprofilattico Sperimentale del Mezzogiorno and after centrifugation steps, serum samples were shipped to laboratory of University of Urbino for the characterization of EVs. The samples were filtered (450 nm) and labelled by LCD, anti-CD63, anti-CD81 and anti-CD9. Two different clones of anti-human -CD9 antibody (M-L13 and MM2-57) were screened for cross reactivity with buffalo species. The MM2-57, known as cross reactive with bovine, was the only one that showed cross reactivity with buffalo leukocytes. Furthermore, EVs from utracentrifugation of selected colo, 15m and 60m samples, were analysed by means of NTA, FC and Transmission Electron Microscopy (TEM) and employed for conditioning (4 and 24h) both BOMAC bovine macrophages and Caco-2 human intestinal cells. Uptake experiments through PKH67 labelled EVs, highlights a greater internalization for colostrum EVs, at both investigated time-points (4h and 24h), indeed only colo EVs demonstrated to moderately increase the scarce phagocytic activity of BoMAC cells (Barletta RG, et al. 2023; Woo, S. R., et al.2006). On water buffalo macrophages, colo EVs lowered FITC-labelled E coli cell adhesion. On the contrary, all the studied EVs revealed to modulate basal mitochondrial ROS production, particularly after 24h. Intriguingly, at the same time point, the lysosome compartment is reduced by colo EV addition (with the highest uptake) and also by 60m EV addition, characterised by mild uptake, suggesting a different EV cargo. In human intestinal Caco-2 cells, our preliminary data highlight a more pronounced uptake, and EV treatment leads to a decrease in basal hydrogen peroxide, particularly after 4h. These preliminary findings underscore the promising role of colostrum and milk-derived EVs in supporting human gastrointestinal health and different functions in bovine and bubaline cells. Further experiments are ongoing to confirm and extend the above mentioned results. This research was funded by the Italian Ministry for Health (grant number RC IZSME 03/23 RC).