No abstract

Outer membrane vesicles (OMVs) are nanoparticles released by Gram-negative bacteria, which contain different cargo molecules and mediate several biological processes. Recent studies have shown that OMVs are involved in antibiotic-resistance (AR) mechanisms by including β-lactamase enzymes in their lumen. Since no studies have as yet been conducted on Salmonella enterica subs. enterica serovar Infantis’ OMVs, the aim of the work was to collect OMVs from five S. Infantis β-lactam resistant strains isolated from a broiler meat production chain and to investigate whether β-lactamase enzymes are included in OMVs during their biogenesis. OMVs were isolated by means of ultrafiltration and a Nitrocefin assay quantified the presence of β-lactamase enzymes in the OMVs. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) were used to identify the OMVs. The results showed that all strains release spherical OMVs, ranging from 60 to 230 nm. The Nitrocefin assay highlighted the presence of β-lactamase enzymes within the OMVs. This suggests that β-lactamase enzymes also get packaged into OMVs from bacterial periplasm during OMV biogenesis. An investigation into the possible role played by OMVs in AR mechanisms would open the door for an opportunity to develop new, therapeutic strategies.

Adipose-derived mesenchymal stromal cells (MSCs) are extensively studied in both human and veterinary medicine. Their isolation is usually performed by collagenase digestion followed by filtration and removal of nonadherent tissue remnants 48 h after seeding. We observed that waste tissue fragments contain cells that adhere belatedly to the plastic. We aimed to investigate their basic properties to speculate on the possible existence of MSC subpopulations. Adipose tissue from three dogs was enzymatically digested. Three cell populations that adhered to the culture plastic 48, 96, and 144 h after seeding were obtained. After expansion, they were analyzed by flow cytometry for MSC-positive (CD90, CD44, and CD29) and -negative (CD14, MHCII, and CD45) markers as well as for endothelial, pericyte, and smooth muscle cell markers (CD31, CD146, and alpha-SMA). Furthermore, cells were assessed for viability, doubling time, and trilineage differentiation ability. No significant differences were found between the three subpopulations. As a result, this procedure has proven to be a valuable method for dramatically improving MSCs yield. As a consequence of cell recovery optimization, the amount of tissue harvested could be reduced, and the time required to obtain sufficient cells for clinical applications could be shortened. Further studies are needed to uncover possible different functional properties.

BACKGROUD: Mesenchymal Stromal Cells (MSC) from adipose tissue are among the most extensively investigated cells for clinical applications both in human and veterinary medicine. Their isolation is mostly carried out by collagenase digestion followed by filtration and seeding. Non-adherent cells are then removed from culture 48 hours upon seeding. Our hypothesis is that fragments eliminated by filtration or discarded after 48h might contain residual MSC that adhere later to the plastic, also after 6 days (144 hours) upon isolation. The aim of this study is to evaluate the basic features of cells that adhere to the plastic at 3 different time points after isolation in order to speculate on the possible existence of MSC subpopulations that are located in different tissue niches.METHODS: Subcutaneous adipose tissue collected from 3 dogs was minced and digested with collagenase I. Three cell populations were obtained at different time points from isolation (48h, 96h, and 144h). They were expanded until passage 3 and characterized by flow cytometry for positive (CD90, CD44, CD29) and negative (CD14, MHC2, CD45) MSC markers as well as for CD31 (endothelial cell marker), CD146 (pericyte marker), alpha-SMA (smooth muscle cell marker). At passage 3, cells were evaluated for viability (MTT assay), doubling time, and trilineage differentiation ability.RESULTS: No significant differences between the 3 subpopulations were observed. They were all characterized by the expression of MSC positive markers and by the absence of negative markers. CD31, CD146, and alpha-SMA were expressed by less than 5% of the cells in all the 3 sub-populations. No differences in differentiation ability and viability were detected. Doubling time ranged between 25 and 30 hours in all the 3 experimental groups.CONCLUSIONS: The 3 cell subpopulations were similar in terms of immunophenotype, proliferation, and differentiation potential. In this view, the procedure of sequential adhesions seems to be a useful method to efficiently improve MSC yield. Indeed, it allows for optimized cell recovery, reducing the amount of sampled tissue and shortening the time necessary to obtain an adequate number of cells for clinical applications. However, functional differences cannot be excluded, and potency assays are required in order to explore possible distinct biological attitudes.