Two main types of macrophages (Mφ) include inflammatory (M1) and anti-inflammatory (M2) macrophages. These cells can be obtained in vitro by polarization of monocytic cell lines using various stimuli. Since there is currently no consensus on the best method for the acquisition of reliable M1 and M2 macrophages from the THP-1 cell line, we decided to compare three different polarization protocols at the transcriptomic level. Whole transcriptomes of Mφ polarized according to the chosen protocols were analyzed using RNA-seq. Differential expression of genes and functional enrichment for gene ontology terms were assessed. Compared with other protocols, M1 macrophages polarized using PMA (61.3 ng/mL) and IFN-γ along with LPS had the highest expression of M1-associated regulatory genes and genes for M1 cytokines and chemokines. According to the GO enrichment analysis, genes involved in defensive and inflammatory processes were differentially expressed in these Mφ. However, all three chosen protocols which use Vit D₃, IL-13/IL-4, and IL-4, respectively, failed to promote the polarization of macrophages with a reliable M2 phenotype. Therefore, optimization or development of a new M2 polarization protocol is needed to achieve macrophages with a reliable anti-inflammatory phenotype.

Objectives: The aim of this study was to confirm the effectiveness of FMT on the basis of optimum composition of the faecal microbiota of the donor for support therapy in patients with ulcerative colitis, and to observe the viability of the microbiota in frozen and lyophilised administration forms of FMT under various storage conditions. Methods: The bacterial microbiota composition of the FMT samples was assessed using amplicon sequencing via next-generation sequencing (NGS) technology, conducted on the Illumina MiSeq platform. The BD FACS Canto flow cytometer was used to analyse the metabolic activity of FMT samples. Results: FMT analysis confirmed the presence of key butyrate-producing organisms, specifically highlighting species such as Bifidobacterium adolescentis, Faecalibacterium prausnitzi, Coprococcus catus, Eubacterium rectale, alongside contributions from genera Roseburia and Blautia. These organisms play a crucial role in maintaining intestinal health in humans. The viable microorganism counts were significantly higher (p < 0.001) in the frozen form of FMT (−70 °C) in comparison to lyophilised forms (−70 °C, 4 °C and 20 °C) throughout the storage period. Conclusion: The conducted NGS analyses allowed us to confirm the suitability of our FMT donor as a potential candidate for the target group of patients diagnosed with ulcerative colitis. From the point of view of optimum utilisation of FMT at its highest metabolic activity for the purpose of transplantation, its storage for a maximum of 2 months under specified conditions was confirmed as the most suitable for the frozen and all lyophilised FMT forms.

The aim of this pilot study was to comprehensively evaluate the gut microbiota, mycobiome, and metabolomic profile of six 4-month-old crossbred pigs (A–F) originating from the same litter and from a specific breeding facility intended for preclinical transplantation experiments, in order to assess their physiological uniformity and identify potential health-related risks prior to inclusion in a kidney transplantation study. The results demonstrated an overall high degree of microbial and metabolic uniformity among the animals, confirming the stability and suitability of the selected breeding source for experimental purposes. At the same time, several individual differences of potential clinical relevance were observed. Animals A, E, and F exhibited signs of microbial and metabolic imbalance, including reduced diversity, increased oxidative activity, and the presence of potentially pathogenic taxa (Porphyromonadaceae bacterium DJF B175, Aspergillus). In contrast, animals B, C, and D showed a balanced metabolic and microbial profile without pathological deviations. The obtained results highlight the importance of preoperative assessment of the gut bacteriome, mycobiome, and metabolome when selecting animals for transplantation experiments. Such a selective screening approach may contribute to the early identification of physiological deviations, reduction of interindividual variability, and increased reliability and translational potential of preclinical studies.