Efekti aspirina na apoptozu neutrofilnih granulocita

Neutrophils are a part of the immune system, and they are involved in host defence against microorganisms. Neutrophil granulocytes have the shortest lifespan among leukocytes, which can be modulated by cytokines and pharmacological agents. The effect of aspirin on apoptosis of inflammatory granulocytes has not been studied in detail yet, and therefore was the chosen subject of this study. Inflammatory granulocytes have been isolated from polyvinyl sponges implanted under the skin of Albino Oxford (AO) rats. Inflammatory cells that were isolated 20 hours later were more than 95% neutrophil granulocytes. The cells were cultivated 24 h with different concentrations of aspirin ranging from 1 µM to 10 mM. After the cultivation period, apoptosis of neutrophils was assessed by morphological criteria, as well as by flow cytometry (after staining the cells with propidium iodide). We found that at concentrations from 0,1 mM to 2,5 mM aspirin inhibited apoptosis of granulocytes, but at 10 mM aspirin induced apoptosis of these cells.Neutrofilni granulociti su deo imunog sistema, uključenog u odbranu od mikroorganizama. Oni imaju najkraći životni vek među leukocitima, koji se može modulisati citokinima i farmakološkim agensima, a do sada nije ispitivan efekat aspirina na apoptozu inflamatornih granulocita. Zbog toga je u ovoj studiji ispitivan efekat aspirina na apoptozu inflamatornih neutrofilnih granulocita pacova. Inflamatorni granulociti su izolovani iz polivinilskih sunđera, potkožno implantiranih, pacovima Albino Oxford (AO) soja. Inflamatorne ćelije, izolovane 20 sati kasnije, najvećim delom (više od 95 %) predstavljaju neutrofilne granulocite. Ove ćelije su kultivisane 24 sata sa aspirinom u koncentracijama od 1 µM do 10 mM. Posle ovog perioda supernatanti su sakupljani i korišćeni za merenje koncentracije NO. Ćelije su bojene propidijum jodidom i apoptoza je analizirana na protočnom citofluorimetru, kao i pomoću morfoloških kriterijuma. Ustanovljeno je da u koncentracijama od 0,1 do 2,5 mM aspirin inhibira, a samo u visokim koncentracijama (10 mM) indukuje apoptozu ovih ćelija. Aspirinom indukovana apoptoza je bila u pozitivnoj korelaciji sa smanjenom produkcijom NO

Exogenous α-ketoglutarate Modulates Redox Metabolism and Functions of Human Dendritic Cells, Altering Their Capacity to Polarise T Cell Response

Alpha-ketoglutarate (αKG) emerged as a key regulator of energetic and redox metabolism in cells, affecting the immune response in various conditions. However, it remained unclear how the exogenous αKG modulates the functions of dendritic cells (DCs), key cells regulating T-cell response. Here we found that non-toxic doses of αKG display anti-inflammatory properties in human APC-T cell interaction models. In a model of monocyte-derived (mo)DCs, αKG impaired the differentiation, and the maturation of moDCs induced with lipopolysaccharide (LPS)/interferon (IFN)-γ, and decreased their capacity to induce Th1 cells. However, αKG also promoted IL-1β secretion by mature moDCs, despite inflammasome downregulation, potentiating their Th17 polarizing capacity. αKG induced the expression of anti-oxidative enzymes and hypoxia-induced factor (HIF)-1α in moDCs, activated Akt/FoxO1 pathway and increased autophagy flux, oxidative phosphorylation (OXPHOS) and glycolysis. This correlated with a higher capacity of immature αKG-moDCs to induce Th2 cells, and conventional regulatory T cells in an indolamine-dioxygenase (IDO)-1-dependent manner. Additionally, αKG increased moDCs’ capacity to induce non-conventional T regulatory (Tr)-1 and IL-10-producing CD8+T cells via up-regulated immunoglobulin-like transcript (ILT3) expression in OXPHOS-dependent manner. These results suggested that exogenous αKG-altered redox metabolism in moDCs contributed to their tolerogenic properties, which could be relevant for designing more efficient therapeutic approaches in DCs-mediated immunotherapies

Shotgun metagenomics reveals gut microbiota features associated with the efficacy of myeloid derived suppressor cells in the prevention of neuroinflammation

Although genetic predisposition to Multiple Sclerosis (MS) may play an essential role in disease development, myeloid cell overactivation and gut microbiota dysbiosis are key contributors to MS pathogenesis. Myeloid-Derived Suppressor Cells (MDSC)s are immature myeloid cells with strong immunosuppressive functions which can be exploited in the treatment of autoimmune diseases. Considering the limited data on MDSCs application in MS therapy and their poorly studied effects on the gut microbiota, we have investigated the therapeutic potential of mice MDSC differentiated according to the standard protocol (MDSC) and modified with the addition of prostaglandin (PG) E2 (MDSC-PGE2) to ameliorate experimental autoimmune encephalomyelitis (EAE) induced with MOG35-55/CFA/PtX in C57BL/6 mice. Additionally, we analyzed the changes in gut microbiota features in control and MDSC-treated animals by using a shotgun metagenomics approach. In mice, PGE2-activated MDSC significantly inhibited the onset and clinical course of EAE. This effect correlated with increased IL-10, TGF-β, IL-4 production, and Arginase-1 level in MDSC-PGE2, as well as with reduced leukocyte infiltrates in the spinal cord. MDSC-PGE2 protective effect is also reflected in the maintenance of gut microbiota composition based on Kraken2/Bracken2 and LEfSe analysis. We observed an increase of MS-associated species Romboutsia ilealis in the control EAE group, while in both MDSC treatments the increase in relative abundances of Muribaculum gordoncarteri and Duncaniella dubiosis, associated with immunoregulatory properties, was observed. Microbial metabolic pathways profiling using Humann3 pipeline also reveals the increase in pathways involved in the production of potentially immunoregulatory metabolites in the MDSC-PGE2 group. In conclusion, we pointed to the significant association between the efficacy of MDSC-PGE2 treatment and gut microbiota features which can be further exploited in order to improve MDSC-based EAE therapy.Book of abstract: 4th Belgrade Bioinformatics Conference, June 19-23, 202

Citotoksičnost legure titana obložene hidroksiapatitom pomoću mlaza plazme

Background/Aim. The deposition of hydroxyapatite (HAP) on the surface of titanium (Ti) alloys enhances bioactivity and osseointegration of the alloys which are widely used as implant materials in dentistry and orthopaedic surgery. However, the stability of HAP and subsequent biocompatibility of such alloys depends on the coating technique. The aim of this work was to test the cytotoxicity of a Ti alloy (Ti6Al4V), coated with HAP by a new plasma deposition method. Methods. The Ti6Al4V samples prepared as discs, 10 mm in diameter and 2 mm in thickness, were coated with HAP (one or both sides of the alloy) by an innovative atmospheric plasma jet method. The cytotoxicity of uncoated and HAP coated Ti6Al4V samples was evaluated by examining the morphological changes and viability of L929 fibroblasts in direct contact with the test materials. Adequate negative (polystyrene) and positive (nickel) control discs of the same size were used. The indirect cytotoxicity was determined by cultivating L929 cells with conditioning medium (CM), prepared as extract of the test samples incubated in the complete Roswell Park Memorial Institute (RPMI) 1640 medium for cell cultures. The cytotoxic effect was evaluated based on the degree of metabolic activity, necrosis, apoptosis and proliferation of L929 cells, using the appropriate assays. Results. Uncoated and one side HAP coated Ti6Al4V alloys were classified as non-cytotoxic according to the current ISO 10993-5 criteria, whereas two sides HAP coated Ti6Al4V alloy samples were slightlymoderate cytotoxic. The cytotoxicity manifested as the inhibition of metabolic activity and proliferation of L929 cells as well as the induction of their apoptosis and necrosis was significantly reduced by conditioning of HAP/Ti6Al4V alloys for 24 hours. The cytotoxic effect of HAP/Ti6Al4V CM only partly decreased in the presence of nifelate, a calcium (Ca) channel blocker, suggesting that Ca ions were not the only responsible cytotoxic agent. Conclusion. The original HAP coating procedure by atmospheric plasma spraying with high energy input enables the production of the stable adhesive coatings on Ti6Al4V alloys. Their cytotoxicity, which depends on the quantity of HAP coating layer, could be significantly reduced up to the non-cytotoxic level by prior conditioning of the alloys in culture medium. Such a procedure, which removes leachable toxic components, could be useful before implantation of HAP coated alloys in vivo. © 2019, Inst. Sci. inf., Univ. Defence in Belgrade. All rights reserved

Vitamin B Complex Treatment Attenuates Local Inflammation after Peripheral Nerve Injury.

Peripheral nerve injury (PNI) leads to a series of cellular and molecular events necessary for axon regeneration and reinnervation of target tissues, among which inflammation is crucial for the orchestration of all these processes. Macrophage activation underlies the pathogenesis of PNI and is characterized by morphological/phenotype transformation from proinflammatory (M1) to an anti-inflammatory (M2) type with different functions in the inflammatory and reparative process. The aim of this study was to evaluate influence of the vitamin B (B1, B2, B3, B5, B6, and B12) complex on the process of neuroinflammation that is in part regulated by l-type CaV1.2 calcium channels. A controlled transection of the motor branch of the femoral peripheral nerve was used as an experimental model. Animals were sacrificed after 1, 3, 7, and 14 injections of vitamin B complex. Isolated nerves were used for immunofluorescence analysis. Treatment with vitamin B complex decreased expression of proinflammatory and increased expression of anti-inflammatory cytokines, thus contributing to the resolution of neuroinflammation. In parallel, B vitamins decreased the number of M1 macrophages that expressed the CaV1.2 channel, and increased the number of M2 macrophages that expressed this channel, suggesting their role in M1/M2 transition after PNI. In conclusion, B vitamins had the potential for treatment of neuroinflammation and neuroregeneration and thereby might be an effective therapy for PNI in humans

Myeloid derived suppressor cells-therapy attenuates experimental autoimmune encephalomyelitis and modulates gut microbiota composition

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 contribute to homeostasis of gastrointestinal mucosa, were enriched in MDSC-PGE2 treated animals. Considering these results, the modification of gut microbiota composition and function could further increase efficacy of MDSC-PGE-2 based therapy of autoimmune diseases.Book of Abstracts: Belgrade BioInformatics Conference 202

Immunomodulatory effect of xylazine, an alpha(2) adrenergic agonist, on rat spleen cells in culture

Xylazine is an adrenergic alpha (2) agonist, which is used in veterinary medicine as a sedative and anesthetic agent. In this work we found that xylazine administered in vivo at a dose of 2.5 mg/kg enhanced spleen cell proliferation and interleukin 2 (IL-2) production in cultures stimulated with concanavalin A (Con A), whereas doses of 10 and 25 mg/kg were inhibitory. A similar stimulatory (10 muM) and inhibitory (50-500 muM) effect on splenocyte proliferation and IL-2 production was observed in vitro. Clonidine. another alpha (2) adrenergic agonist, only had a stimulatory proliferative effect on splenocytes. Yohimbine, an alpha (2) adrenergic antagonist, abrogated the stimulatory action of both clonidine and xylazine, but not the suppressive proliferative activity of xylazine in vitro. The inhibited proliferation of splenocytes to Con A correlated with increased apoptosis of T cells. The apoptosis was not blocked by yohimbine or antibodies to Fas and Fas-L. N-Nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) synthase. enhanced proliferation of splenocytes to Con Al partly abrogated the inhibitory effect of xylazine in the proliferation assay. and, only at high concentration (1000 muM), partly suppressed apoptosis of lymphocytes. The enhancing effect of L-NAME on the Con A-induced proliferation of splenocytes correlated with decreased NO production. However, decreased NO production observed in cultures with xylazine was followed by both decreased lymphocyte proliferation and apoptosis. Cumulatively, these results suggest that the immunosuppressive properties of xylazine on splenocytes in vitro are due to increased apoptosis of lymphocytes, predominantly involve NO-independent pathways, and are probably independent of its action through alpha (2) adrenoreceptors