Ema Paulovičová

Currently, the incidence and prevalence of serious fungal infections is increasing, especially in immunosuppressed individuals. The co-administration of antibiotic and immunosuppressive therapies h...

Traditional medicinal herbs as Echinacea purpurea and Erigeron canadensis are recommended as a complementary supplementation for the treatment of diseases associated with immunological inflammation (e.g. common cold, coughs, bronchitis, upper respiratory infections, immunodeficiencies). This pathologic conditions are accompanied by the wide range of malfunctions or imbalances of the immune system, thus there is increased necessity for search of novel immunomodulation trends and immunopharmacologically active phytosubstances for effective pharmaco-immunomodulatory therapy. Anti-inflammatory immunobiological activity of polyphenolic polysaccharide-proteins of Echinacea purpurea and Erigeron canadensis are still not studied. Our results demonstrated the immunobiological effectivity of selected herbal polyphenolic polysaccharide-proteins isolated from flowers of medicinal plants Echinacea purpurea and Erigeron canadensis resulting into the significant immunostimulation of inflammatory TNF-α, IL-6, IL-1ß and IL-12 cytokines (p < 0.001). Both herbal polyphenolic polysaccharide-proteins triggered cell release of anti-inflammatory interleukin IL-10 (p < 0.001). Furthermore, the inductive cell release of growth factors M-CSF and GM-CSF has been demonstrated (p < 0.001). E. purpurea and E. canadensis polyphenolic polysaccharide-proteins accelerated the efficacy of cellular phagocytosis and free radical release, more pronounced with Erigeron treatment.

Poly(2-isopropenyl-2-oxazoline) (PIPOx) represents a functional polymer with high potential for drug delivery, tissue engineering, and immunomodulation. The immunomodulatory efficiency of the PIPOx formulation has been studied in vitro following splenic cells and RAW 264.7 macrophages exposition. The cell-specific immunomodulative effect on production of Th1, Th2, Th17, and Treg signature cytokines has been demonstrated. The impact on the functionality of PIPOx-sensitized RAW 264.7 macrophages was assessed by cell phagocytosis. Time- and concentration-dependent cell internalization and intracellular organelles colocalization of fluorescently labeled PIPOx has been examined. The in vitro results demonstrated the PIPOx bioavailability and the capability of triggering immune cell responses resulting in the induced production of cell-specific signature interleukins, important prerequisite properties for future potential biomedical applications.

Chitin, a polymer of β-(1→4)-linkedN-acetyl-d-glucosamine, is one of the main polysaccharide components of the fungal cell wall. Its N-deacetylated form, chitosan, is enzymatically produced in the cell wall by chitin deacetylases. It exerts immunomodulative, anti-inflammatory, anti-cancer, anti-bacterial, and anti-fungal activities with various medical applications. To study the immunobiological properties of chitosan oligosaccharides, we synthesized a series of β-(1→4)-linkedN-acetyl-d-glucosamine oligomers comprising 3, 5, and 7 monosaccharide units equipped with biotin tags. The key synthetic intermediate employed for oligosaccharide chain elongation, a disaccharide thioglycoside, was prepared by orthogonal glycosylation of a 4-OH thioglycoside acceptor with a glycosyl trichloroacetimidate bearing the temporary 4-O-tert-butyldimethylsilyl group. The use of silyl protection suppressed aglycon transfer and provided a high yield for the target disaccharide donor. Using synthesized chitosan oligomers, as well as previously obtained chitin counterparts, the immunobiological relationship between these synthetic oligosaccharides and RAW 264.7 cells was studiedin vitro. Evaluation of cell proliferation, phagocytosis, respiratory burst, and Th1, Th2, Th17, and Treg polarized cytokine expression demonstrated effective immune responsiveness and immunomodulation in RAW 264.7 cells exposed to chitin- and chitosan-derived oligosaccharides. Macrophage reactivity was accompanied by significant inductive dose- and structure-dependent protective Th1 and Th17 polarization, which was greater with exposure to chitosan- rather than chitin-derived oligosaccharides. Moreover, no antiproliferative or cytotoxic effects were observed, even following prolonged 48 h exposure. The obtained results demonstrate the potent immunobiological activity of these synthetically prepared chito-oligosaccharides.

The incidence and prevalence of serious fungal infections is rising, especially in immunosuppressed individuals. Moreover, co-administration of antibiotics and immunosuppressants has driven the emergence of new multidrug-resistant pathogens. The significant increase of multidrug-resistant pathogens, together with their ability to form biofilms, is associated with morbidity and mortality. Research on novel synthetically prepared immunomodulators as potential antifungal immunotherapeutics is of serious interest. Our study demonstrated the immunobiological activity of synthetically prepared biotinylated mannooligosaccharides mimicking Candida antigenic factors using RAW264.7 macrophages. Macrophage exposure to the set of eight structurally different mannooligosaccharides induced a release of Th1, Th2, Th17, and Treg cytokine signature patterns. The observed immune responses were tightly associated with structure, dose, exposure time, and selected signature cytokines. The viability/cytotoxicity of the mannooligosaccharide formulas was assessed based on cell proliferation. The structure-based immunomodulatory activity of the formulas was evaluated with respect to the length, branching and conformation of the various formulas. Glycoconjugate formulas with terminal β-mannosyl-units tended to be more potent in terms of Candida relevant cytokines IL-12 p70, IL-17, GM-CSF, IL-6, and TNFα induction and cell proliferation, and this tendency was associated with structural differences between the studied glycoconjugate formulas. The eight tested mannooligosaccharide conjugates can be considered potential in vitro immunomodulative agents suitable for in vitro Candida diagnostics or prospectively for subcellular anti-Candida vaccine design.