Inflammatory bowel disease (IBD) is a growing health problem, as incidence and prevalence of the disease are increasing, while treatment with biological agents is expensive and causes undesirable side effects. Therefore, the development of new treatments is a major scientific challenge. Chemokines and cytokines play a key role in the inflammatory response in the gastrointestinal tract, and hence represent a new target for the treatment of inflammatory diseases. The main aim of the doctoral dissertation was the development of lactic acid bacteria (LAB) Lactococcus lactis NZ9000 in Lactobacillus salivarius ATCC 11741 that would display on the surface appropriate binding proteins, which could achieve anti-inflammatory activity and improve the symptoms of inflammation. We have confirmed that the chemokine-binding proteins evasins from ticks are suitable for surface display on LAB Lactococcus lactis NZ9000 and Lactobacillus salivarius ATCC 11741. As an alternative approach to achieve anti-inflammatory activity in IBD, we successfully developed the delivery system for anti-inflammatory peptide bepecin (BPC-157) in fusion with a suitable surface carrier protein. Controlled release of bepecin was achieved with the help of intestinally located protease trypsin, or with the signal peptide. We also developed a system for monitoring and quantification of released bepecin. The effectiveness of both new approaches for the treatment of IBD was confirmed on cell models. The ability to bind chemokine CXCL8 was confirmed on a gut epithelial cell model, and the antioxidant activity of bepecin on a fibroblast cell model. In addition to the binding of chemokines, cytokine binding could also be used for the treatment of inflammatory bowel diseases. Therefore, we displayed on the surface of Lactococcus lactis NZ9000 non-immunoglobulin protein scaffolds prepared from the albumin-binding domain and capable of binding the p19 subunit of human IL-23 (ILP-binding proteins). By binding of p19, the interaction between IL-23 and its receptor could be prevented, thereby blocking IL-23-mediated signaling. Non-immunoglobulin scaffolds are an alternative to antibodies. We have identified binding proteins with anti-inflammatory activity suitable for surface display on LAB by reviewing their possible targets. In every case, an adequate amount of anti-inflammatory proteins or peptides at their site of action is important for effective anti-inflammatory activity. The amount of expressed proteins can be influenced by the optimization of the LAB host strain, whereby the inactivation or silencing of certain genes can increase the expression of recombinant proteins or reduce their degradation. The pre-developed plasmid pNZDual with two nisin promoters was used to develop the single-plasmid inducible system CRISPR-Cas9, which enables the inactivation or silencing of genes using CRISPR and CRISPRi technologies, and will be used to inactivate candidate genes in order to increase the expression of anti-inflammatory proteins. The desired anti-inflammatory activity of bacteria can be improved by introducing an appropriate delivery system, by which the bacteria are protected and delivered to the site of inflammation. Nanofibers are a more recent delivery system that could enable the incorporation of LAB and their delivery to the oral or vaginal mucosa, thus expanding the possibilities of treating inflammatory diseases. Within the doctoral thesis, the influence of process parameters of electrospinning, and parameters of the polymer solution were studied, and their effect on the morphology of nanofibers and the viability of the model LAB Lactobacillus plantarum ATCC 8014 assessed. The incorporation of probiotic bacteria into nanofibers enables us to simultaneously dry the bacteria, incorporate them with high efficiency, and prepare a patient friendly local delivery system with long a shelf life and fast release that enables protection and delivery of bacteria to the site of inflammation.
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