The aim of this study was to investigate the use of a standardized animal model subjected to antibiotic treatment, and the effects of this treatment on the course of dextran sodium sulphate (DSS)-induced colitis in mice. By decontamination with selective antibiotics and observation of pathogenesis of ulcerative colitis (UC) induced chemically by exposure of mice to various concentrations of DSS, we obtained an optimum animal PGF model of acute UC manifested by mucin depletion, epithelial degeneration and necrosis, leading to the disappearance of epithelial cells, infiltration of lamina propria and submucosa with neutrophils, cryptitis, and accompanied by decreased viability of intestinal microbiota, loss of body weight, dehydration, moderate rectal bleeding, and a decrease in the selected markers of cellular proliferation and apoptosis. The obtained PGF model did not exhibit changes that could contribute to inflammation by means of alteration of the metabolic status and the induced dysbiosis did not serve as a bearer of pathogenic microorganisms participating in development of ulcerative colitis. The inflammatory process was induced particularly by exposure to DSS and its toxic action on compactness and integrity of mucosal barrier in the large intestine. This offers new possibilities of the use of this animal model in studies with or without participation of pathogenic microbiota in IBD pathogenesis.

There is a lack of in vitro models able to plausibly represent the inflammation microenvironment of knee osteoarthritis (OA). We analyzed the molecules released from OA tissues (synovial membrane, cartilage, infrapatellar fat pad) and investigated whether the stimulation of human synovial fibroblasts (SFs), with synthetic cytokines (IL-1β and TNF-α or IFN-γ) or conditioned media (CM) from OA tissues, influence the SFs’ response, in the sense of pro-inflammatory cytokines, chemokines, growth factors, and degradative enzymes modulation. Human SFs were obtained from OA synovial membranes. SFs and their CM were analyzed for biomarkers, proliferation rate, protein profile and gene expression, before and after stimulation. Real-time PCR and multiplex assays quantified OA-related gene expression and biomolecule production. Unlike other activators, CM from OA synovial membrane (CM-SM), significantly up-regulated all genes of interest (IL-6, IL-8, MMP-1, MMP-3, RANTES, MCP-1, TSG-6, YKL-40) in SFs. Multiplex immunoassay analysis showed that levels of OA-related cytokines (IL-6, IL-8, MCP 1, IL-1Ra), chemokine (RANTES) and growth factor (VEGF), produced by CM-SM stimulated SFs, increased significantly compared to non-stimulated SFs. Molecules released from the SM from OA patients induces OA-like changes in vitro, in specific OA synovial populations (SFs). These findings promote the use and establish a compelling in vitro model that simulates the versatility and complexity of the OA disease. This model, in the future, will allow us to study new cell therapies or test drugs by reducing or avoiding animal models.

Transplantation of faecal microbiota (FMT) is generally considered a safe therapeutic procedure with few adverse effects. The main factors that limit the spread of the use of FMT therapy for idiopathic inflammatory bowel disease (IBD) are the necessity of minimising the risk of infection and transfer of another disease. Obtaining the animal model of UC (ulcerative colitis) by exposure to DSS (dextran sodium sulphate) depends on many factors that significantly affect the result. Per os intake of DSS with water is individual for each animal and results in the development of a range of various forms of induced UC. For this reason, the aim of our study was to evaluate the modulation and regenerative effects of FMT on the clinical and histopathological responses and the changes in the bowel microenvironment in pseudo germ-free (PGF) mice of the BALB/c line subjected to chemical induction of mild, moderate and serious forms of UC. The goal was to obtain new data related to the safety and effectiveness of FMT that can contribute to its improved and optimised use. The animals with mild and moderate forms of UC subjected to FMT treatment exhibited lower severity of the disease and markedly lower damage to the colon, including reduced clinical and histological disease index and decreased inflammatory response of colon mucosa. However, FMT treatment failed to achieve the expected therapeutic effect in animals with the serious form of UC activity. The results of our study indicated a potential safety risk involving development of bacteraemia and also translocation of non-pathogenic representatives of bowel microbiota associated with FMT treatment of animals with a diagnosed serious form of UC.

Chorionic mesenchymal stromal cells (CHO-MSCs) and their extracellular vesicles (EVs) are becoming increasingly popular, since chorion is ethically harmless and an easily accessible source of MSCs. However, until now there is only a limited number of studies with a thorough characterization of CHO-MSCs derived EVs and their miRNA profile. In this study, we monitored changes in the EV-miRNA profile between early and late passage of human CHO-MSCs. First, senescence of CHO-MSCs was induced by serial passaging and confirmed by morphological changes, shortened telomeres and changes in the expression of selected genes. The expression of MSCs-specific surface markers CD73, CD90, CD105 did not change with increasing passages. Next, EVs and their miRNA profiles were compared between early vs late passage cells. Number of EVs and their size were not significantly changed. Seven of the top 10 most expressed EV-miRNAs were common to both early and late passages. A differential expression study between early and late passages identified 37 significantly differentially expressed EV-miRNAs, out of which 23 were found to be associated with pathways of cellular senescence based on KEGG pathway analysis. A set of 9 miRNAs were identified as the most frequently associated with senescence and/or with the most altered expression between early and late passages, out of which miR-145-5p, miR-335-5p and miR-199b-3p were the most significant downregulated miRNAs in late passages. The most upregulated EV-miRNAs were miR-1307-3p, miR-3615 and miR320b. Targeting these miRNAs in future experiments may prolong the therapeutic potential of CHO-MSCs and their EVs. Graphical Abstract

Currently, there are over 200 officially registered sulfur springs in Slovakia, most of which are cold hydrogen sulfide springs with a characteristic strong odor of rotten eggs. Their typical inhabitants are filamentous sulfur-oxidizing bacteria. The aim of this study was to identify the major bacterial populations inhabiting sulfur springs in Slovakia using a combination of non-cultivation and cultivation approaches. Non-cultivation analyses revealed high variability of sulfur-oxidizing bacteria in Slovak springs. Sulfur mats were usually dominated by chemolithotrophic sulfur-oxidizers from the phylum Proteobacteria (more than 87%), followed by photoautotrophic sulfur-oxidizers of the phylum Chlorobi. The group of nonsulfur-oxidizers was composed of typical freshwater species and bacteria with genetic potential for sulfur oxidation. The low similarity in the 16S rRNA gene sequence of the sequenced DGGE bands indicates that about half of the sulfur-oxidizing bacteria identified represent new species/genera of sulfur-oxidizing bacteria. In attempts to cultivate potential new species of sulfur-oxidizing bacteria, a very low overlap was observed between non-cultivation and cultivation analyses, and a new species of the Thiofaba genus was cultivated. Our results expand knowledge on the diversity of sulfur bacteria in cold sulfur springs in Slovakia and indicate that the diversity of sulfur-oxidizing bacteria is significantly underestimated and that sulfur springs could be inhabited by unique bacterial populations.

Background: Extracellular vesicles (EVs) are the foundation of modern regenerative medicine using a cell-free approach. While current research mainly explores EVs from biological fluids and cell culture supernatants, tissue-derived EVs hold great promise, but remain largely underexplored. Since healthy placental tissues such as the chorion are widely available after full-term delivery, ethically unobjectionable, and possess exceptional regenerative potential, we sought to compare the biological effects of EVs derived directly from chorion tissue with those from chorion-derived mesenchymal stromal cell EVs and plasma EVs. Method: We compared the biological impact of EVs from various sources (chorion tissue CHO-Ti, MSCs from chorion CHO-MSC and platelet-poor plasma PPP) and isolated by various techniques on the gene expression of osteoarthritic chondrocytes. Additionally, we assessed the effect of enriched soluble proteins of CHO-MSC and CHO-Ti secretome vs. their EVs. EVs were characterized by particle number and size (NTA), protein content (BCA assay) and immunophenotype (flow cytometry). Changes in gene expression of chondrocytes were quantified by RT-qPCR. Results: CHO-Ti-EVs and PPP-EVs showed particularly beneficial effect on the inflammatory process, with their biological impact surpassing that of CHO-MSC-EVs. Chondroprotective markers COL2A and ACAN were robustly upregulated by CHO-Ti-EVs and PPP-EVs but showed only modest or variable increases with CHO-MSC-EVs. COMP expression, however, was specifically enhanced by CHO-MSC-derived components. Furthermore, our results also indicate that the therapeutic properties of the CHO-Ti secretome are exclusively linked to EVs. Among CHO-MSC-EVs, purification combined with UC resulted in the highest purity, however EVs purified by SEC presented a more favourable surface marker profile and better biological effects. The observed variability suggests that different EV preparations harbour distinct subpopulations that influence regulatory pathways differently and highlight the importance of EV source and isolation methodology in determining biological activity. Conclusion: CHO-Ti-EVs showed promising effects on cartilage regeneration and inflammation modulation, suggesting they may represent a viable alternative to plasma- and CHO-MSC-EVs. Moreover, the chorion represents a readily accessible and abundant source of perinatal tissue obtainable non-invasively after full-term delivery, further supporting the translational potential of CHO-Ti-EVs.