Surface marker expression in small and medium/large mesenchymal stromal cell-derived extracellular vesicles in naive or apoptotic condition using orthogonal techniques

Extracellular vesicles released by mesenchymal stromal cells (MSC-EVs) are a promising resource for regenerative medicine. Small MSC-EVs represent the active EV fraction. A bulk analysis was applied to characterise MSC-EVs’ identity and purity, with the assessment of single EV morphology, size and integrity using electron microscopy. We applied different methods to quantitatively analyse the size and surface marker expression in medium/large and small fractions, namely 10k and 100k fractions, of MSC-EVs obtained using sequential ultracentrifugation. Bone marrow, adipose tissue and umbilical cord MSC-EVs were compared in naive and apoptotic conditions. As detected by electron microscopy, the 100k EV size < 100 nm was confirmed by super-resolution microscopy and ExoView. Single-vesicle imaging using super-resolution microscopy revealed heterogeneous patterns of tetraspanins. ExoView allowed a comparative screening of single MSC-EV tetraspanin and mesenchymal markers. A semiquantitative bead-based cytofluorimetric analysis showed the segregation of immunological and pro-coagulative markers on the 10k MSC-EVs. Apoptotic MSC-EVs were released in higher numbers, without significant differences in the naive fractions in surface marker expression. These results show a consistent profile of MSC-EV fractions among the different sources and a safer profile of the 100k MSC-EV population for clinical application. Our study identified suitable applications for EV analytical techniques

VALUTAZIONE DI PARAMETRI ANIMAL-BASED IN AZIENDE BOVINE DA LATTE SOTTOPOSTE A CATEGORIZZAZIONE DEL RISCHIO ATTRAVERSO IL SISTEMA CLASSYFARM

Nel presente studio sono stati misurati alcuni indicatori diretti di benessere animale in tre aziende di bovine da latte (azienda A, B e C) sottoposte a categorizzazione del rischio attraverso il sistema ClassyFarm, al fine di confermare la validità di tali indicatori a supporto della stima del rank di rischio aziendale. Pertanto, si è proceduto alla quantificazione dei seguenti parametri animal- based: (i) cortisolo plasmatico, (ii) livelli sierici di IL-6 e (iii) conta delle cellule somatiche (effettuata per campione individuale di latte). Il cortisolo plasmatico e l’IL-6 sono stati determinati con test immunologico ELISA per cortisolo e IL-6 (Bovine Cortisol ELISA; My- Bio-Source, San Diego, CA, USA, e Bovine IL-6 ELISA; My-Bio-Source, San Diego, CA, Stati Uniti, rispettivamente), seguendo le indicazioni del produttore ed utilizzando un sistema di elaborazione ELISA a quattro piastre completamente automatizzato DYNEX DSX®. I livelli medi di cortisolo sono risultati più elevati nell’azienda A rispetto alla B e alla C e, rispettivamente, pari a 9,34 ng/mL; 6 ng/mL e 6,77 ng/mL. Medesimo andamento si è osservato per la misurazione dell’IL 6 che presenta valori medi pari a 25,68 ng/mL; 24,04 ng/mL e 22,14 ng/mL. Infine, i valori medi di cellule somatiche, rilevati per campione individuale, risultano pari a 675000 CS/mL; 540000 CS/mL e 494000 CS/mL, rispettivamente per le aziende A, B e C. I dati ottenuti sono stati, quindi, confrontati con quelli forniti dal sistema ClassyFarm nelle tre aziende zootecniche considerate dimostrando la concordanza tra i parametri animal-based oggetto di indagine e la categorizzazione del rischio operata dal sistema integrato attualmente applicato in Italia

Religious slaughtering: Implications on pH and temperature of bovine carcasses

The main objective of this study was to investigate the influence of different types of slaughtering on the variations of pH and temperature. Ninety entire male beef cattle, belonging to the Charolais breed, were distributed in three groups based on method of slaughtering. A total of 30 subjects (group K) were slaughtered without stunning, according to the Koscher rite; thirty bovines (group T) were stunned with a captive bolt gun, following the common slaughtering reported by Council Regulation (EC) 1099/2009; other thirty animals (group H) were slaughtered without stunning, based on Halal procedures. The temperature and pH values of carcasses were determined at 3, 6 and 24 h post-mortem, respectively. At 24 h postmortem of the 30 carcasses evaluated for each group, muscle temperature decreased in all three types of slaughtering. In particular, the average temperature was 4.0, 4.9 and 3.0 degrees C for traditional, Halal and Koscher slaughtering, respectively. Moreover, at the same time step (24 h post-mortem), muscle pH decreased showing values equal to 5.72, 5.83, 5.81 for traditional, Halal and Koscher slaughtering, respectively. The findings of the present study may be useful to highlight the influence of slaughtering method on pH and, more generally, on meat quality. Additionally, our results show that, in slaughtering carried out without prior stunning, the carcass could be more susceptible to alterations because of the high pH values

Serum albumin fragmentation in end-stage renal disease patients - a pilot study

BACKGROUND: The goal of this study was to detect modification in the expression of plasma proteins and/or post-translational modifications of their structure in patients with end stage renal disease. METHODS: Serum samples from 19 adult patients treated by maintenance hemodialysis (MHD) were analyzed in comparison to sera from six healthy controls using sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis (2DE). Spots of interest were identified by mass spectrometry analysis. In addition, the 2DE maps were incubated with a human anti-albumin polyclonal antibody. RESULTS: SDS-PAGE gels, 2DE maps and matrix-assisted laser desorption/ionization time of flight analysis indicated over-expression of low-molecular weight proteins (LMWP) in sera from patients. Unexpectedly, another 15 spots with estimated M(r) of 12.5-29 kDa from the 2DE maps of six patients were identified as fragments of albumin. 2D immunoblotting of sera from 12 other patients detected numerous albumin fragments. CONCLUSIONS: These results indicate that in addition to increased expression of LMWP, a relevant amount of albumin fragments are detectable in the serum of patients undergoing MHD. Uremia appears to facilitate the fragmentation of albumin and/or the retention of albumin fragments in blood