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Hepatocelularni i kolorektalni karcinom se sve ćešće javlja i ima visoku stopu smrtnosti. Postojanje aktivnih mehanizama hemorezistencije otežava korisnost hemoterapije. Upravo je pojava hemorezistencije podstakla opsežna istraživanja za razvoj novih terapeutskih agenasa i strategija za lečenje ovih vrsta karcinoma. Prirodni proizvodi biljnog porekla (sekundarni metaboliti) su bogat izvor potencijalnih lekova. Zbog toga smo u istraživanju usmerili pažnju na etil-acetatni ekstrakt biljke Frangula alnus (FA) i njegovu dominantnu komponentu emodin (E) i ispitali njihovo antikancerski potencijal na ćelijama hepatocelularnog (HepG2) i kolorektalnog (HCT116) karcinoma i citotoksičnost na normalnim MRC -5 ćelijama. Pokazan je snažan antioksidativni potencijal ekstrakta (DPPH i TBA test). Citotoksičnost ispitana MTT testom pokazala je jaku toksičnost obe supstance na ćelije HepG2 i HCT116, ali bez uticaja na MRC-5 ćelije. Dalje je analiza ćelijskog ciklusa pokazala da su i FA i E izazvali zaustavljanje u G1 fazi i blago nakupljanje ćelija u G2/M fazi. Pored toga, obe supstance su uvele ćelije u apoptozu i nekrozu i uticale su na mitohondrijalni membranski potencijal. Dalje, izazvali su značajan genotoksični efekat u kometnom testu na svim ćelijskim linijama. Može se zaključiti da su FA i E dobri kandidati za nove antikancerogene agense prirodnog porekla. Međutim, neophodna su dodatna istraživanja, posebno kada je u pitanju citotoksična aktivnost i bezbednost primene ovih supstanci., Hepatocellular and colorectal carcinoma is swiftly increasing alongside a high mortality rate. The existence of active mechanisms of chemoresistance hampers the usefulness of chemotherapy. The emergence of chemoresistance encouraged extensive research to develop new therapeutic agents and strategies for the treatment of these types of cancer. Natural products of plant origin (secondary metabolites) have been the most successful source of potential drug leads. Therefore, we turned our attention to the Frangula alnus ethyl-acetate extract (FA) and its dominant constituent emodin (E) and explored them for their anticancer potential on hepatocellular (HepG2) and colorectal (HCT116) carcinoma cells, and cytotoxicity on normal MRC-5 cells. Strong antioxidant potential of the extract was demonstrated (DPPH and TBA test). Cytotoxicity investigated by the MTT assay showed strong cell toxicity of both substances on HepG2 and HCT116 cells, but without affecting MRC-5 cells. Next, the analysis of cell cycles exhibited that both FA and E induced arrest in the G1 phase and slight accumulation of cells in the G2/M phase. In addition, both substances introduced cells into apoptosis and necrosis and modulated mitochondrial membrane potential. Further on, they caused significant genotoxic effect in comet assay applied in all cell lines. It can be concluded that FA and E are good candidates for new anticancer agents of natural origin. However, additional studies are necessary, especially when it comes to the cytotoxic activity and safety of the application of these substances.

Pomegranate peel extract (PoPEx) has been shown to have antioxidant and anti-inflammatory properties, but its effect on the adaptive immune system has not been sufficiently investigated. In this study, the treatment of human peripheral blood mononuclear cells (PBMC) with PoPEx (range 6.25-400 mu g/mL) resulted in cytotoxicity at concentrations of 100 mu g/mL and higher, due to the induction of apoptosis and oxidative stress, whereas autophagy was reduced. At non-cytotoxic concentrations, the opposite effect on these processes was observed simultaneously with the inhibition of PHA-induced PBMC proliferation and a significant decrease in the expression of CD4. PoPEx differently modulated the expression of activation markers (CD69, CD25, ICOS) and PD1 (inhibitory marker), depending on the dose and T-cell subsets. PoPEx (starting from 12.5 mu g/mL) suppressed the production of Th1 (IFN-gamma), Th17 (IL-17A, IL-17F, and IL-22), Th9 (IL-9), and proinflammatory cytokines (TNF-alpha and IL-6) in culture supernatants. Lower concentrations upregulated Th2 (IL-5 and IL-13) and Treg (IL-10) responses as well as CD4+CD25hiFoxp3+ cell frequency. Higher concentrations of PoPEx increased the frequency of IL-10- and TGF-beta-producing T-cells (much higher in the CD4+ subset). In conclusion, our study suggested for the first time complex immunoregulatory effects of PoPEx on T cells, which could assist in the suppression of chronic inflammatory and autoimmune diseases.

Over-activated myeloid cells and disturbance in gut microbiota composition are critical factors contributing to the pathogenesis of Multiple Sclerosis (MS). Myeloid-derived suppressor cells (MDSCs) emerged as promising regulators of chronic inflammatory diseases, including autoimmune diseases. However, it remained unclear whether MDSCs display any therapeutic potential in MS, and how this therapy modulates gut microbiota composition. Here, we assessed the potential of in vitro generated bone marrow-derived MDSCs to ameliorate experimental autoimmune encephalomyelitis (EAE) in Dark Agouti rats and investigated how their application associates with the changes in gut microbiota composition. MDSCs differentiated with prostaglandin (PG)E2 (MDSC-PGE2) and control MDSCs (differentiated without PGE2) displayed strong immunosuppressive properties in vitro, but only MDSC-PGE2 significantly ameliorated EAE symptoms. This effect correlated with a reduced infiltration of Th17 and IFN-gamma-producing NK cells, and an increased proportion of regulatory T cells in the CNS and spleen. Importantly, both MDSCs and MDSC-PGE2 prevented EAE-induced reduction of gut microbiota diversity, but only MDSC-PGE2 prevented the extensive alterations in gut microbiota composition following their early migration into Payer's patches and mesenteric lymph nodes. This phenomenon was related to the significant enrichment of gut microbial taxa with potential immunoregulatory properties, as well as higher levels of butyrate, propionate, and putrescine in feces. This study provides new insights into the host-microbiota interactions in EAE, suggesting that activated MDSCs could be potentially used as an efficient therapy for acute phases of MS. Considering a significant association between the efficacy of MDSC-PGE2 and gut microbiota composition, our findings also provide a rationale for further exploring the specific microbial metabolites in MS therapy.

Gingiva-Derived Mesenchymal Stromal Cells (GMSCs) have been shown to play an important role in periodontitis. However, how P. gingivalis, one of the key etiological agents of the disease, affects healthy (H)- and periodontitis (P)-GMSCs is unknown. To address this problem, we established 10 H-GMSC and 12 P-GMSC lines. No significant differences in morphology, differentiation into chondroblasts and adipocytes, expression of characteristic MSCS markers, including pericyte antigens NG2 and PDGFR, were observed between H- and P-GMSC lines. However, proliferation, cell size and osteogenic potential were higher in P-GMSCs, in contrast to their lower ability to suppress mononuclear cell proliferation. P. gingivalis up-regulated the mRNA expression of IL-6, IL-8, MCP-1, GRO-alpha, RANTES, TLR-2, HIF-1 alpha, OPG, MMP-3, SDF-1, HGF and IP-10 in P-GMSCs, whereas only IL-6, MCP-1 and GRO-alpha were up-regulated in H-GMSCs. The expression of MCP-1, RANTES, IP-10 and HGF was significantly higher in P-GMSCs compared to H-GMSCs, but IDO1 was lower. No significant changes in the expression of TLR-3, TLR-4, TGF-beta, LAP, IGFBP4 and TIMP-1 were observed in both types of GMSCs. In conclusion, our results suggest that P-GMSCs retain their pro-inflammatory properties in culture, exhibit lower immunosuppressive potential than their healthy counterparts, and impaired regeneration-associated gene induction in culture. All these functions are potentiated significantly by P. gingivalis treatment.

The role of gut microbiota composition in efficacy of various immune-based therapies is increasingly recognized. Thus, the aim of our study was to investigate if the efficacy of myeloid-derived suppressor cells (MDSC)-Prostaglandin E2 (PGE2) therapy for multiple sclerosis (MS) correlates with gut microbiota composition and function. MDSC generated from bone marrow cells in the presence of PGE2 were applied to spinal cord homogenate/CFA-induced experimental autoimmune encephalomyelitis (EAE) in Dark Agouti (DA) rats, an animal model of MS. MDSC-PGE2 therapy resulted in a significant attenuation of EAE symptoms over 30 days of disease monitoring. These results correlated with lower percentage of proinflammatory interferon- gamma and interleukin-17 producing cells and higher percentage of anti-inflammatory IL-4 producing cells in spinal cord and spleen. Gut microbial composition were studied using amplicon(16S rRNA)-based metagenomic analyses of fecal samples collected prior to the induction of EAE and MDSC-PGE2 therapy application, and at the peak of the disease. The induction of EAE resulted in a decrease of microbiota diversity, whereas the MDSC-PGE2 therapy preserved the diversity in EAE-induced animals. The induction of EAE in control group associated with a higher relative abundance of Peptococcaceae, but the lower levels of Veillonellaceae and different groups of Prevotellaceae, known to produce immunosuppressive short chain fatty acid (SCFA), and Lactobacillus reuteri, known for its anti-inflammatory function. In contrast, there were no changes in levels of these immunoregulatory taxa in EAE-animals treated with MDSC-PGE2 therapy. Also, SCFA producing Ruminococcaceae, and Coriobacteriaceae, known to metabolize phytoestrogens to immunosuppressive metabolites were more abundant in EAE-animals treated with MDSC-PGE2 therapy. Predicted metabolic profiling obtained by PICRUSt2 revealed that pathways involved in biosynthesis of polyamines, metabolites known to contri

Widespread coronavirus disease (COVID)-19 is causing pneumonia, respiratory and multiorgan failure in susceptible individuals. Dysregulated immune response marks severe COVID-19, but the immunological mechanisms driving COVID-19 pathogenesis are still largely unknown, which is hampering the development of efficient treatments. Here we analyzed similar to 140 parameters of cellular and humoral immune response in peripheral blood of 41 COVID-19 patients and 16 age/gender-matched healthy donors by flow-cytometry, quantitative PCR, western blot and ELISA, followed by integrated correlation analyses with similar to 30 common clinical and laboratory parameters. We found that lymphocytopenia in severe COVID-19 patients (n=20) strongly affects T, NK and NKT cells, but not B cells and antibody production. Unlike increased activation of ICOS-1+ CD4+ T cells in mild COVID-19 patients (n=21), T cells in severe patients showed impaired activation, low IFN-gamma production and high functional exhaustion, which correlated with significantly down-regulated HLA-DR expression in monocytes, dendritic cells and B cells. The latter phenomenon was followed by lower interferon responsive factor (IRF)-8 and autophagy-related genes expressions, and the expansion of myeloid derived suppressor cells (MDSC). Intriguingly, PD-L1-, ILT-3-, and IDO-1-expressing monocytic MDSC were the dominant producers of IL-6 and IL-10, which correlated with the increased inflammation and accumulation of regulatory B and T cell subsets in severe COVID-19 patients. Overall, down-regulated IRF-8 and autophagy-related genes expression, and the expansion of MDSC subsets could play critical roles in dysregulating T cell response in COVID-19, which could have large implications in diagnostics and design of novel therapeutics for this disease.