Прегледај по Аутор "Čolić, Miodrag"

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    Anti-inflammatory effect of amalgam on periapical lesion cells in culture
    (Ministry of Defance, Serbia, 2021) Eraković, Mile; Duka, Miloš; Bekić, Marina; Milanović, Marijana; Tomić, Sergej; Vučević, Dragana; Čolić, Miodrag
    Background/Aim. Amalgam h as b een u sed f or y ears in dentistry, but the controversy on its adverse effects, both on local oral/dental tissues and systemic health, still exists. When used for retrograde filling in apical surgery, amalgam comes in close contact with the periapical tissue, and it is sometimes responsible for the induction of periapical lesion (PL) or its exacerbation. Therefore, the aim of the study was to examine the effect of amalgam on cytotoxicity and production of pro-inflammatory cytokine by cells isolated from PL. Methods. Conditioned medium from freshly prepared amalgam (ACM) was performed according to the ISO 10993-12 by incubating the alloy in RPMI medium (0.2 g/mL) for 3 days at 37°C. Cells were isolated from 20 human PLs after apicoectomy by collagenase/DNA-ase digestion and cultured with different dilutions of ACM. Cytotoxicity was determined by MTT assay (n = 7 cultures) and apoptosis/necrosis assays (n = 8 cultures), whereas cytokine production was measured by a Flow Cytomix Microbeads Assay (n = 8 cultures). Results. Undiluted (100%) and 75% ACM was cytotoxic due to induction of apoptosis of PL cells. Non-cytotoxic concentrations of ACM (50% and 25%) inhibited the production of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, and IL-8), concentrationdependently. Conclusion. For t he f irst t ime, o ur results showed an unexpected anti-inflammatory property of amalgam on PL cells, which could be beneficial for PL healing after apicoectomy
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    Antimicrobial properties of viscose yarns ring-spun with integrated amino-functionalized nanocellulose
    (Springer, 2021) Kokol, Vanja; Vivod, Vera; Peršin, Zdenka; Čolić, Miodrag; Kolar, Matjaž
    Bio-based, renewable and biodegradable products with multifunctional properties are also becoming basic trends in the textile sector. In this frame, cellulose nanofibrils (CNFs) have been surface modified with hexamethylenediamine/HMDA and used as an antimicrobial additive to a ring-spun viscose yarn. The CNF-HMDA suspension was first characterized in relation to its skin irritation potential, antimicrobial properties, and technical performance (dispersability and suspensability in different media) to optimize its sprayability on a viscose fiber sliver with the lowest sticking, thus to enable its spinning without flowing and tearing problems. The impact of CNF-HMDA content has been examined on the yarn‘s fineness, tensile strength, surface chemistry, wettability and antimicrobial properties. The yarn‘s antimicrobial properties were increasing with the content of CNF-HMDA, given a 99% reduction for S. aureus and C. albicans (log 1.6–2.1) in up to 3 h of exposure at minimum 33 mg/g, and for E. coli (log 0.69–2.95) at 100 mg/g of its addition, yielding 45–21% of bactericidal efficacy. Such an effect is related to homogeneously distributed CNF-HMDA when sprayed from a fast-evaporated bi-polar medium and using small (0.4 mm) nozzle opennings, thus giving a high positive charge (0.663 mmol/g) without affecting the yarn‘s tenacity and fineness, but improving its wettability. However, a non-ionic surfactant being used in the durability testing of functionalized yarn to 10-washing cycles, adheres onto it hydrophobically via the methylene chain of the HMDA, thus blocking its amino groups, and, as such, decreasing its antibacterial efficiency, which was slightly affected in the case when the washing was carried out without using it.
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    Fecal microbiota composition associates with the capacity of human peripheral blood monocytes to differentiate into immunogenic dendritic cells in vitro
    (2021) Radojević, Dušan; Tomić, Sergej; Mihajlović, Dušan; Tolinački, Maja; Pavlović, Bojan; Vučević, Dragana; Bojić, Svetlana; Golić, Nataša; Čolić, Miodrag; Đokić, Jelena
    Although promising for active immunization in cancer patients, dendritic cells (DCs) vaccines generated in vitro display high inter-individual variability in their immunogenicity, which mostly limits their therapeutic efficacy. Gut microbiota composition is a key emerging factor affecting individuals’ immune responses, but it is unknown how it affects the variability of donors’ precursor cells to differentiate into immunogenic DCs in vitro. By analyzing gut microbiota composition in 14 healthy donors, along with the phenotype and cytokines production by monocyte-derived DCs, we found significant correlations between immunogenic properties of DC and microbiota composition. Namely, donors who had higher α-diversity of gut microbiota and higher abundance of short-chain fatty acid (SCFAs) and SCFA-producing bacteria in feces, displayed lower expression of CD1a on immature (im)DC and higher expression of ILT-3, costimulatory molecules (CD86, CD40) proinflammatory cytokines (TNF-α, IL-6, IL-8) and IL-12p70/IL-10 ratio, all of which correlated with their lower maturation potential and immunogenicity upon stimulation with LPS/IFNγ, a well-known Th1 polarizing cocktail. In contrast, imDCs generated from donors with lower α-diversity and higher abundance of Bifidobacterium and Collinsella in feces displayed higher CD1a expression and higher potential to up-regulate CD86 and CD40, increase TNF-α, IL-6, IL-8 production, and IL-12p70/IL-10 ratio upon stimulation. These results emphasize the important role of gut microbiota on the capacity of donor precursor cells to differentiate into immunogenic DCs suitable for cancer therapy, which could be harnessed for improving the actual and future DC-based cancer therapies.
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    Fermentation characteristics of novel Coriolus versicolor and Lentinus edodes kombucha beverages and immunomodulatory potential of their polysaccharide extracts
    (Elsevier, 2021) Sknepnek, Aleksandra; Tomić, Sergej; Miletić, Dunja; Lević, Steva; Čolić, Miodrag; Nedović, Viktor; Nikšić, Miomir
    Medicinal mushrooms, Coriolus versicolor and Lentinus edodes are extremely attractive as nutraceuticals. Here we used fruiting bodies to prepare novel kombucha beverage. Microbiological, physicochemical and chemical properties were monitored for eleven days, while the immunological properties of kombucha polysaccharide extracts were determined in peripheral blood mononuclear cell (PBMC) cultures. FTIR analysis of polysaccharide extracts showed dominant presence of polysaccharides, in addition to phenols, lipids and proteins. C. versicolor kombucha extract displayed more complex polysaccharides, and a higher content of total polysaccharides, phenols and flavonoids compared to L. edodes kombucha extract. The extracts were not cytotoxic for PBMC in vitro up to 500 μg/ml, while immunomodulatory effects depended on their chemical compositions. The most prominent effect was on the reduction of Th2 cytokines and IL-10 in PBMC cultures. Based on these results, novel kombucha products could be recommended as functional beverages or nutraceuticals with potentially beneficial immunomodulatory effects in allergies.
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    Functionalization-dependent effects of cellulose nanofibrils on tolerogenic mechanisms of human dendritic cells
    (Dove Medical Press, 2018) Tomić, Sergej; Ilić, Nataša; Kokol, Vanja; Gruden-Movsesijan, Alisa; Mihajlović, Dušan; Bekić, Marina; Sofronić-Milosavljević, Ljiljana; Čolić, Miodrag; Vučević, Dragana
    Background: Cellulose nanofibrils (CNF) are attractive nanomaterials for various biomedical applications due to their excellent biocompatibility and biomimetic properties. However, their immunoregulatory properties are insufficiently investigated, especially in relation to their functionalization, which could cause problems during their clinical application. Methods: Using a model of human dendritic cells (DC), which have a central role in the regulation of immune response, we investigated how differentially functionalized CNF, ie, native (n) CNF, 2,2,6,6-tetramethylpiperidine 1-oxyl radical-oxidized (c) CNF, and 3-aminopropylphosphoric acid-functionalized (APAc) CNF, affect DC properties, their viability, morphology, differentiation and maturation potential, and the capacity to regulate T cell-mediated immune response. Results: Nontoxic doses of APAcCNF displayed the strongest inhibitory effects on DC differentiation, maturation, and T helper (Th) 1 and Th17 polarization capacity, followed by cCNF and nCNF, respectively. These results correlated with a specific pattern of regulatory cytokines production by APAcCNF-DC and their increased capacity to induce suppressive CD8+CD25+IL-10+ regulatory T cells in immunoglobulin-like transcript (ILT)-3- and ILT-4-dependent manner. In contrast, nCNF-DC induced predominantly suppressive CD4+CD25hiFoxP3hi regulatory T cells in indolamine 2,3-dioxygenase-1-dependent manner. Different tolerogenic properties of CNF correlated with their size and APA functionalization, as well as with different expression of CD209 and actin bundles at the place of contact with CNF. Conclusion: The capacity to induce different types of DC-mediated tolerogenic immune responses by functionalized CNF opens new perspectives for their application as well-tolerated nanomaterials in tissue engineering and novel platforms for the therapy of inflammatory T cell-mediated pathologies.
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    Harnessing immunomodulatory mechanisms of Trichinella spiralis to design novel nanomedical approaches for restoring self-tolerance in autoimmunity
    (Elsevier, 2021) Ilić, Nataša; Kosanović, Maja; Gruden-Movsesijan, Alisa; Glamočlija, Sofija; Sofronić-Milosavljević, Ljiljana; Čolić, Miodrag; Tomić, Sergej
    The rapid increase in the prevalence of autoimmune diseases in recent decades, especially in developed countries, coincided with improved living conditions and healthcare. Part of this increase could be ascribed to the lack of exposure to infectious agents like helminths that co-evolved with us and display potent immune regulatory actions. In this review we discussed many investigations, including our own, showing that Trichinella spiralis via its excretory-secretory products attenuate Th1/Th17 immunopathological response in autoimmunity and potentiate the protective Th2 and or regulatory T cell response, acting as an effective induction of tolerogenic dendritic cells (DCs), and probably mimicking the autoantigen in some diseases. A recent discovery of T. spiralis extracellular vesicles (TsEVs) suggested that inducing a complex regulation of the immune response requires simultaneous delivery of different signals in nano-sized packages. Indeed, different artificial nanomedical approaches discussed here suggested that co-delivery of multiple signals via nanoparticles is the most promising strategy for the treatment of autoimmune diseases. Although a long way is ahead of us before we could completely replicate natural nano-delivery systems which are both safe and potent in restoring self-tolerance, a clear path is being opened from a careful examination of parasite-host interactions
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    Immunological aspects of nanocellulose
    (Elsevier, 2020) Čolić, Miodrag; Tomić, Sergej; Bekić, Marina
    Cellulose is the most abundant natural polymer in the world. Nanoscale forms of cellulose, including cellulose nanofibers (CNF), cellulose nanocrystals (CNC) and bacterial nanocellulose (BC), are very attractive in industry, medicine and pharmacy. Biomedical applications of nanocellulose in tissue engineering, regenerative medicine, and controlled drug delivery are the most promising. Nanocellulose is considered a biocompatible nanomaterial and relatively safe for biomedical applications. However, more studies are needed to prove this hypothesis, especially those related to chronic exposure to nanocellulose. Besides toxicity, the response of the immune system is of particular importance in this sense. This paper provides a comprehensive and critical review of the current-state knowledge of the impact of nanocellulose on the immune system, especially on macrophages and dendritic cells (DC), as the central immunoregulatory cells, which has not been addressed in the literature sufficiently. Nanocellulose, especially CNC, can induce the inflammatory response upon the internalization by macrophages, but this reaction may be significantly modulated by introducing different functional groups on their surface. Our original results showed that nanocellulose has a potent immunotolerogenic potential. Native CNF potentiated the capacity of DC to induce conventional Tregs. When carboxyl groups were introduced on the CNF surface, the tolerogenic potential of DC was shifted towards the induction of regulatory CD8+ T cells, whereas the introduction of phosphonates on CNF surface potentiated DCs’ capacity to induce both regulatory CD8+ T cells and Type 1 regulatory (Tr-1) cells. These results are extremely important when considering the application of nanocellulose in vivo, especially for tissue regeneration and wound healing.
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    Immunomodulatory Properties of Pomegranate Peel Extract in a Model of Human Peripheral Blood Mononuclear Cell Culture
    (MDPI, 2022) Čolić, Miodrag; Bekić, Marina; Tomić, Sergej; Đokić, Jelena; Radojević, Dušan; Ševikin, Katarina; Miljuš, Nataša; Marković, Milan; Škrbić, Ranko
    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 g/mL) resulted in cytotoxicity at concentrations of 100 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 g/mL) suppressed the production of Th1 (IFN-), Th17 (IL-17A, IL-17F, and IL-22), Th9 (IL-9), and proinflammatory cytokines (TNF- 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- -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.
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    Mesenchymal Stromal Cells from Healthy and Inflamed Human Gingiva Respond Differently to Porphyromonas gingivalis
    (MDPI, 2022) Bekić, Marina; Radanović, Marina; Đokić, Jelena; Tomić, Sergej; Eraković, Mile; Radojević, Dušan; Duka, Miloš; Marković, Dejan; Marković Milan; Ismaili, Bashkim; Bokonjić, Dejan; Čolić, Miodrag
    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- , RANTES, TLR-2, HIF-1 , OPG, MMP-3, SDF-1, HGF and IP-10 in P-GMSCs, whereas only IL-6, MCP-1 and GRO- 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- , 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.
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    Microstructure Characterisation and Identification of the Mechanical and Functional Properties of a New PMMA-ZnO Composite
    (MDPI, 2020) Rudolf, Rebeka; Popović, Danica; Tomić, Sergej; Bobovnik, Rajko; Lazić, Vojkan; Majerič, Peter; Anžel, Ivan; Čolić, Miodrag
    In this research work, we synthesised poly(methyl methacrylate) (PMMA) enriched with 2 wt.% zinc oxide nanoparticles (ZnO) through conventional heat polymerisation and characterised its microstructure. It was found that the distribution of ZnO nanoparticles was homogeneous through the volume of the PMMA. The mechanical testing of the PMMA-ZnO composite primarily included the determination of the compressive properties on real dentures, while density measurements were performed using a pycnometer. The testing of functional properties involved the identification of the colour of the new PMMA-ZnO composite, where pure PMMA acted as a control. In the second step, the PMMA-ZnO cytotoxicity assays were measured in vitro, which were shown to be similar to the control PMMA. Based on this, it could be concluded that the newly formed PMMA-ZnO composite did not induce direct or indirect cytotoxic e ects in L929 cell cultures; therefore, according to ISO/DIN 10993-5:2009, this composite was categorised as non-cytotoxic.
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    Periodontal Disease in Young Adults as a Risk Factor for Subclinical Atherosclerosis: A Clinical, Biochemical and Immunological Study
    (MDPI, 2023) Cicmil, Smiljka; Cicmil, Ana; Pavlic, Verica; Krunić, Jelena; Sladoje Puhalo, Dragana; Bokonjić, Dejan; Čolić, Miodrag
    Although a strong relationship between periodontal disease (PD) and atherosclerosis was shown in adults, little data are published in younger PD patients. Therefore, this study aimed to investigate and correlate clinical parameters of PD, pro- and immunoregulatory cytokines in gingival crevicular fluid (GCF) and serum, biochemical and hematological parameters associated with atherosclerosis risk, and carotid intima-media thickness (IMT) in our younger study participants (n = 78) (mean age 35.92 3.36 years) who were divided into two equal groups: subjects with and without PD. PD patients had higher values of IMT, hs-CRP, triglycerides, total cholesterol, and LDL; most proinflammatory and Th1/Th17-associated cytokines in GCF; and IL-8, IL-12, IL-18, and IL-17A in serum compared to subjects without PD. These cytokines in GCF positively correlated with most clinical periodontal parameters. Clinical periodontal parameters, TNF- and IL-8 in GCF and IL-17A, hs-CRP, and LDL in serum, had more significant predictive roles in developing subclinical atherosclerosis (IMT 0.75 mm) in comparison with other cytokines, fibrinogen, and other lipid status parameters. Hs-CRP correlated better with the proinflammatory cytokines than the parameters of lipid status. Except for serum IL-17A, there was no significant association of clinical and immunological PD parameters with lipid status. Overall, these results suggest that dyslipidemia and PD status seem to be independent risk factors for subclinical atherosclerosis in our younger PD population.
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    Phosphonate-Modified Cellulose Nanocrystals Potentiate the Th1 Polarising Capacity of Monocyte-Derived Dendritic Cells via GABA-B Receptor
    (Dove Medical Press, 2022) Bekić, Marina; Vasiljević, Miloš; Stojanović, Dušica; Kokol, Vanja; Mihajlović, Dušan; Vučević, Dragana; Uskoković, Petar; Čolić, Miodrag; Tomić, Sergej
    Purpose: Phosphonates, like 3-AminoPropylphosphonic Acid (ApA), possess a great potential for the therapy of bone tumours, and their delivery via cellulose nanocrystals (CNCs) seems a promising approach for their increased efficacy in target tissues. However, the immunological effects of CNC-phosphonates have not been investigated thoroughly. The main aim was to examine how the modification of CNCs with phosphonate affects their immunomodulatory properties in human cells. Methods: Wood-based native (n) CNCs were modified via oxidation (ox-CNCs) and subsequent conjugation with ApA (ApA-CNCs). CNCs were characterised by atomic force microscopy (AFM) and nanoindentation. Cytotoxicity and immunomodulatory potential of CNCs were investigated in cultures of human peripheral blood mononuclear cells (PBMCs) and monocyte-derived dendritic cells (MoDCs)/T cells co-cultures by monitoring phenotype, cytokines production, allostimulatory and Th/Treg polarisation capacity. Results: AFM showed an increase in CNCs' thickens, elasticity modulus and hardness during the modification with ApA. When applied at non-toxic doses, nCNCs showed a tolerogenic potential upon internalisation by MoDCs, as judged by their increased capacity to up-regulate tolerogenic markers and induce regulatory T cells (Treg), especially when present during the differentiation of MoDCs. In contrast, ox- and ApA-CNCs induced oxidative stress and autophagy in MoDCs, which correlated with their stimulatory effect on the maturation of MoDCs, but also inhibition of MoDCs differentiation. ApA-CNC-treated MoDCs displayed the highest allostimulatory and Th1/CTL polarising activity in co-cultures with T cells. These effects of ApA-CNCs were mediated via GABA-B receptor-induced lowering of cAMP levels in MoDCs, and they could be blocked by GABA-B receptor inhibitor. Moreover, the Th1 polarising and allostimulatory capacity of MoDCs differentiated with ApA-CNC were largely preserved upon the maturation of MoDCs, whereas nCNC- and ox-CNC-differentiated MoDCs displayed an increased tolerogenic potential. Conclusion: The delivery of ApA via CNCs induces potent DC-mediated Th1 polarisation, which could be beneficial in their potential application in tumour therapy.
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    Plasma-Activated Medium Potentiates the Immunogenicity of Tumor Cell Lysates for Dendritic Cell-Based Cancer Vaccines
    (MDPI, 2021) Tomić, Sergej; Petrović, Anđelija; Puač, Nevena; Škoro, Nikola; Bekić, Marina; Petrović, Zoran Lj.; Čolić, Miodrag
    Autologous dendritic cells (DCs)-based vaccines are considered quite promising for cancer immunotherapy due to their exquisite potential to induce tumor antigen-specific cytotoxic T cells. However, a lack of efficient protocols for inducing immunogenic tumor antigens limits the efficacy of DC-based cancer vaccines. Here, we found that a plasma-activated medium (PAM) induces immunogenic cell death (ICD) in tumor cells but not in an immortalized L929 cell line or human peripheral blood mononuclear cells. PAM induced an accumulation of reactive oxygen species (ROS), autophagy, apoptosis, and necrosis in a concentration-dependent manner. The tumor lysates prepared after PAM treatment displayed increased immunogenicity in a model of DCs, compared to the lysates prepared by a standard freezing/thawing method. Mature DCs loaded with PAM lysates showed an increased maturation potential, as estimated by their increased expression of CD83, CD86, CD40, IL-12/IL-10 production, and attenuated PDL1 and ILT-4 expression, compared to the DCs treated with control tumor lysates. Moreover, in co-culture with allogeneic T cells, DCs loaded with PAM-lysates increased the proportion of cytotoxic IFN- + granzyme A+CD8+ T cells and IL-17A-producing T cells and preserved the Th1 response. In contrast, control tumor lysates-treated DCs increased the frequency of Th2 (CD4+IL-4+), CD4, and CD8 regulatory T cell subtypes, none of which was observed with DCs loaded with PAM-lysates. Cumulatively, these results suggest that the novel method for preparing immunogenic tumor lysates with PAM could be suitable for improved DC-based immunotherapy of cancer patients
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    Production of immunoregulatory cytokines in clinically asymptomatic periapical lesions depends on the lesions size
    (Ministry of Defance, Serbia, 2019) Duka, Miloš; Gazivoda, Dragan; Marković, Milan; Majstorović, Ivana; Tatomirović, Željka; Mihajlović, Dušan; Čolić, Miodrag
    Bacground/Aim. Development of periapical lesions (PLs) involves a complex cross-talk between pathogenic microor­ganisms from the root canal and host immune mechanisms. In these processes proinflammatory cytokines are involved in stimulation of inflammation and osteodestructive mecha­nisms, whereas anti-inflammatory cytokines, with the im­munoregulatory functions, have the opposite effects. How this balance is controlled is still the subject of numerous studies. The aim of this study was to examine whether the local production of interleukin (IL)-10, IL-27 and trans­forming growth factor (TGF)-β in human asymptomatic PLs depends on the lesion size and how levels of these im­munoregulatory cytokines correlate with the cellular compo­sition of PLs. Methods. The study was conducted on 30 PLs which were collected at the Clinic for Stomatology of the Military Medical Academy, Belgrade, Serbia. The PLs were divided according to their size into small- and large-size lesions (n = 12 and n = 18, respectively). The inflam­matory cells (PL-ICs) were isolated from PLs and cultivated for 24 hours in culture medium supplemented with phorbol myristate acetate and Ca2+ ionophore. Cytospins were proc­essed for immunocytology by using monoclonal antibodies to cell subsets. The levels of cytokines in culture super­natants were determined by the ELISA method. Statistical analysis was performed using the Student t-test and the Spearman’s correlation test. The values of p < 0.05 were considered to be significant. Results. The levels of IL-10 and TGF-β were significantly higher in the PL-ICs cultures of large-size lesions than in small ones (p < 0.01 and p < 0.05, respectively). In contrast, the levels of IL-27 were higher in the cultures of small-size lesions than in small ones (p < 0.05). Although the total number of PL-ICs and the proportion of mononuclear phagocytes were higher in the large-size PLs (p < 0.01 and p < 0.05, respectively), their main composition was not significantly different between the groups. The proportions of B cells/plasma cells (CD19/CD38+ cells), CD8+ T cells and CD14+ cells were significantly higher in the large-size PLs (p < 0.005; p < 0.05; p < 0.05, respectively). In contrast, the proportion of total T cells (CD3+ cells) was higher in the small-size lesions (p < 0.05). No statistically significant correlation was found between the levels of these cytokines and the composi­tion/phenotype of PL-ICs. Conclusion. This study sug­gests that IL-10, IL-27 and TGF-β may play different roles in suppression of unwanted immune/inflammatory reac­tions in asymptomatic PLs, depending on the extension of pathological process as judged by the size of lesions.
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    Production of Soluble Receptor Activator of Nuclear Factor Kappa-Β Ligand and Osteoprotegerin by Apical Periodontitis Cells in Culture and Their Modulation by Cytokines
    (Hindawi, 2019) Duka, Miloš; Eraković, Mile; Dolićanin, Zana; Stefanović, Dara; Čolić, Miodrag
    RANKL, a bone-destructive cytokine, and OPG, its osteoprotective counterpart, are expressed in periapical lesions (PLs), which represent hystopatological manifestations of apical periodontitis. However, their regulation in PLs has not been elucidated yet. Therefore, our aim was to study the production of RANKL and OPG and their modulation by pro- and anti-inflammatory cytokines in PL cell cultures. Isolated PL cells were cultured alone or with addition of TNF-α, IFN-ϒ, IL-17, IL-4, IL-10, and IL- 33, respectively. The levels of RANKL and OPG in supernatants were measured by ELISA. The proportion of CD3+ (T cells) and CD19+/CD138+ (B cells/plasma cells) within isolated PLs was determined by immunocytochemistry. The levels of RANKL were higher in cultures of symptomatic PLs compared to asymptomatic PLs and PLs with the dominance of T cells (T-type lesions) over B cells/plasma cells (B-type lesions). A higher proportion of osteodestructive processes (RANKL/OPG ratio > 1.0) were detected in symptomatic PLs. The production of RANKL was upregulated by IFN-ϒ and IL-17 and higher concentrations of IL-33. IL-10 and lower concentrations of IL-33 augmented the production of OPG. The addition of either RANKL or anti-RANKL antibody to the cultures did not modify significantly the production of OPG. In conclusion, this original PL cell culture model suggests that increased bone destruction through upregulated production of RANKL could be associated with exacerbation of inflammation in PLs with the predominance of Th1 and Th17 responses and increased secretion of IL-33. In contrast, IL-10 and lower levels of IL-33, through upregulation of OPG, may suppress osteolytic processes.
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    Prostaglanin-E2 Potentiates the Suppressive Functions of Human Mononuclear Myeloid-Derived Suppressor Cells and Increases Their Capacity to Expand IL-10-Producing Regulatory T Cell Subsets
    (Frontiers, 2019) Tomić, Sergej; Joksimović, Bojan; Bekić, Marina; Vasiljević, Miloš; Milanović, Marijana; Čolić, Miodrag; Vučević, Dragana
    Myeloid-derived suppressor cells (MDSC) emerged as major factors driving the tumor progression due to numerous immunosuppressive mechanisms they possess. Prostaglandin (PG)E2 is shown critical for the induction of MDSC and their suppressive functions in vivo, but it is poorly understood how it affects the capacity of MDSC to induce different subsets of regulatory T cells (Treg). By using a novel protocol for the generation of mononuclear (M)-MDSC, we showed that PGE2 potentiates the GM-CSF/IL-6-dependent induction of CD33+CD11b+HLA-DR−CD14+ M-MDSC in vitro. PGE2 diminished the capacity of GM-CSF/IL-6 M-MDSC to produce proinflammatory cytokines upon activation and augmented their capacity to produce IL-27, IL-33, and TGF-b. These results correlated with an increased potential of GM-CSF/IL-6/PGE2 M-MDSC to suppress T cell proliferation, cells, and reduce the development of alloreactive Th17 and cytotoxic T cells. Interestingly, GM-CSF/IL-6/PGE2 M-MDSC displayed a lower capacity to induce TGF-b-producing FoxP3+ regulatory Treg compared to GM-CSF/IL-6 M-MDSC, as a consequence of reduced IDO-1 expression. In contrast, GM-CSF/IL-6/PGE2 M-MDSC potentiated IL-10 production by CD8+T, Th2, and particularly CD4+FoxP3− type 1 Treg, the latter of which depended on ILT3 and ILT4 expression. Cumulatively, PGE2 potentiated the suppressive phenotype and functions of GM-CSF/IL-6-induced M-MDSC and changed the mechanisms involved in Treg induction, which could be important for investigating new therapeutic strategies focused on MDSC-related effects in tumors and autoimmune diseases.
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    Sitagliptin Induces Tolerogenic Human Dendritic Cells
    (MDPI, 2023) Drakul, Marija; Tomić, Sergej; Bekić, Marina; Mihajlović, Dušan; Vasiljević, Miloš; Rakočević, Sara; Ðokić, Jelena; Popović, Nikola; Bokonjić, Dejan; Čolić, Miodrag
    Sitagliptin, an anti-diabetic drug, is a dipeptidyl peptidase (DPP)-4/CD26 inhibitor with additional anti-inflammatory and immunomodulatory properties. In this study, we investigated for the first time the effect of sitagliptin on the differentiation and functions of human dendritic cells generated from monocytes (MoDCs) for 4 days using the standard GM-CSF/IL-4 procedure. LPS/IFN-y treatment for an additional 24 h was used for maturation induction of MoDCs. Sitagliptin was added at the highest non-cytotoxic concentration (500 g/mL) either at the beginning (sita 0d protocol) or after MoDC differentiation (sita 4d protocol). Sitagliptin impaired differentiation and maturation of MoDCs as judged with the lower expression of CD40, CD83, CD86, NLRP3, and HLA-DR, retention of CD14 expression, and inhibited production of IL- , IL-12p70, IL-23, and IL-27. In contrast, the expression of CD26, tolerogenic DC markers (ILT4 and IDO1), and production of immunoregulatory cytokines (IL-10 and TGF- ) were increased. Generally, the sita 0d protocol was more efficient. Sitagliptin-treated MoDCs were poorer allostimulators of T-cells in MoDC/T-cell co-culture and inhibited Th1 and Th17 but augmented Th2 and Treg responses. Tolerogenic properties of sitagliptin-treated MoDCs were additionally confirmed by an increased frequency of CD4+CD25+CD127- FoxP3+ Tregs and Tr1 cells (CD4+IL-10+FoxP3-) in MoDC/T-cell co-culture. The differentiation of IL-10+ and TGF- + Tregs depended on the sitagliptin protocol used. A Western blot analysis showed that sitagliptin inhibited p65 expression of NF-kB and p38MAPK during the maturation of MoDCs. In conclusion, sitagliptin induces differentiation of tolerogenic DCs, and the effect is important when considering sitagliptin for treating autoimmune diseases and allotransplant rejection.
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    The Effect of Stabilisation Agents on the Immunomodulatory Properties of Gold Nanoparticles Obtained by Ultrasonic Spray Pyrolysis
    (MDPI, 2019) Bekić, Marina; Tomić, Serge; Rudolf, Rebeka; Milanović, Marijana; Vučević, Dragana; Anžel, Ivan; Čolić, Miodrag
    Gold nanoparticles (GNPs) have been investigated extensively as drug carriers in tumour immunotherapy in combinationwith photothermal therapy. For this purpose, GNPs should be stabilised in biological fluids. The goal of this study was to examine how stabilisation agents influence cytotoxicity andimmune response in vitro. SphericalGNPs, 20 nmin size, werepreparedbyultrasonic spraypyrolysis (USP). Three types of stabilising agents were used: sodium citrate (SC), polyvinyl-pyrrolidone (PVP), and poly-ethylene glycol (PEG). Pristine, non-stabilised GNPs were used as a control. The culture models were mouse L929 cells, B16F10 melanoma cells and human peripheral blood mononuclear cells (PBMNCs), obtained from healthy donors. Control SC- and PEG-GNPs were non-cytotoxic at concentrations (range 1–100 g/mL), in contrast to PVP-GNPs, which were cytotoxic at higher concentrations. Control GNPs inhibited the production of IFN-U slightly, and augmented the production of IL-10 by PHA-stimulated PBMNC cultures. PEG-GNPs inhibited the production of pro-inflammatory cytokines (IL-1, IL-6, IL-8, TNF- ) and Th1-related cytokines (IFN-U and IL-12p70), and increased the production of Th2 cytokines (IL-4 and IL-5). SC-PEG inhibited the production of IL-8 and IL-17A. In contrast, PVP-GNPs stimulated the production of pro-inflammatory cytokines, Th1 cytokines, and IL-17A, but also IL-10. When uptake of GNPs by monocytes/macrophages in PBMNC cultures was analysed, the ingestion of PEG- GNPs was significantly lower compared to SC- and PVP-GNPs. In conclusion, stabilisation agents modulate biocompatibility and immune response significantly, so their adequate choice for preparation of GNPs is an important factor when considering the use of GNPs for application in vivo.