Miodrag Čolić

Immunomodulatory properties of mesenchymal stem cells derived from dental pulp and dental follicle are susceptible to activation by toll-like receptor agonists.

Adult mesenchymal stem cells (MSCs) have recently become a potent tool in regenerative medicine. Due to certain shortcomings of obtaining bone marrow MSCs, alternate sources of MSCs have been sought. In this work, we studied MSCs from dental pulp (DP-MSCs) and dental follicle (DF-MSCs), isolated from the same tooth/donor, to define differences in their phenotypic properties, differentiation potential, and immunomodulatory activities. Both cell types showed colony-forming ability and expressed typical MSCs markers, but differed in the levels of their expression. DF-MSCs proliferated faster, contained cells larger in diameter, exhibited a higher potential to form adipocytes and a lower potential to form chondrocytes and osteoblasts, compared with DP-MSCs. In contrast to DF-MSCs, DP-MSCs produced the transforming growth factor (TGF)-β and suppressed proliferation of peripheral blood mononuclear cells, which could be neutralized with anti-TGF-β antibody. The treatment with toll-like receptor 3 (TLR3) agonist augmented the suppressive potential of both cell types and potentiated TGF-β and interleukin-6 secretions by these cells. TLR4 agonist augmented the suppressive potential of DF-MSCs and increased TGF-β production, but abrogated the immunosuppressive activity of DP-MSCs by inhibiting TGF-β production and the expression of indolamine-2,3-dioxygenase-1. Some of these effects correlated with the higher expression of TLR3 and TLR4 by DP-MSCs compared with DF-MSCs. When transplanted in imunocompetent xenogenic host, both cell types induced formation of granulomatous tissue. In conclusion, our results suggest that dental MSCs are functionally different and each of these functions should be further explored in vivo before their specific biomedical applications.

Proinflammatory and immunoregulatory mechanisms in periapical lesions

Proinflammatory and immunoregulatory cytokines are important for the pathogenesis of periapical lesions. However, little is known about how their functions are balanced and controlled at different phases of lesion development. The aim of this study was to examine the relationship between the production of Th1, Th2, Th17 and T regulatory cell (T reg) cytokines by human periapical lesion mononuclear cells (PL-MNC) in culture and their correlation with cellular composition and clinical presentation of the lesions. We show that symptomatic lesions are characterized by the infiltration of neutrophils, high production of IL-17, positive correlation between IL-17 and IFN-gamma, but not between IL-17 and IL-23 production. Most IL-17(+) cells coexpressed IFN-gamma. Asymptomatic lesions were phenotypically heterogeneous. The lesions with the predominance of T cells over B cells/plasma cells expressed higher levels of IFN-gamma which correlated with higher production of IL-12 and the frequency of macrophages. In contrast, in most B-type lesions higher levels of IL-5 and TGF-beta were observed, as well as positive correlation between the production of TGF-beta and IL-10. The addition of Th cytokines in PL-MNC cultures confirmed that Th1, Th2 and Th17 cytokines are mutually antagonistic, except that IL-17, unexpectedly, augmented the production of IFN-gamma. IL-10 and TGF-beta inhibited the production of both Th1 and Th17 cytokines. Dendritic cells (DCs) from periapical lesions, composed of immature (CD83(-)), and mature (CD83(+)) myeloid type DCs and plasmacytoid (BDCA2(+)) DCs produced higher levels of IL-12 and IL-23 but lower levels of IL-10 and TNF-alpha than monocyte (Mo) -derived DCs. IL-23 stimulated the production of IL-17 by PL-MNC, whereas the secretion of IFN-gamma was enhanced by both IL-12 and IL-23. Cumulatively, these results suggest that: (1) Th1 immune response is most probably important for all stages of periapical lesion development; (2) Th2 and immunoregulatory cytokines are more significant for advanced types of lesions with the predominance of B cells/plasma cells; (3) Th17 immune response seems to play a dominant role in exacerbating inflammation.

Production of proinflammatory and immunoregulatory cytokines by inflammatory cells from periapical lesions in culture

BACKGROUND The role of cytokines in pathogenesis of periapical lesions is not well understood. The aim of this study was to study the correlation between proinflammatory and immunoregulatory cytokines in periapical lesions and their relationship with cellular composition and clinical presentation. METHODS Inflammatory cells were isolated from 67 human periapical lesions and cultivated for 24 h. The levels of proinflammatory cytokines: interleukin-1 beta (IL-1beta), IL-6, IL-8 and tumour necrosis factor alpha (TNF-alpha) and immunoregulatory cytokines: transforming growth factor-beta (TGF-beta) and IL-10 were determined in culture supernatants using a fluorescent bead immunoassay or ELISA. The phenotype of cells was analysed by immunocytochemistry. RESULTS Inflammatory cells from symptomatic lesions which contained higher proportion of granulocytes, secreted higher levels of IL-1, IL-6 and IL-8 compared with asymptomatic lesions. Large-size lesions contained lower percentages of mononuclear phagocytes, higher percentages of CD8(+) T cells and produced higher levels of TNF-alpha, IL-6 and IL-10 compared with small-size lesions. There were negative correlations between the concentrations of TGF-beta and proinflammatory cytokines. TGF-beta, added to cultures, downregulated the levels of proinflammatory cytokines more strongly than IL-10, independently of clinical presentation of the lesions. By contrast, exogenous IL-10 was mainly immunosuppressive in cultures of asymptomatic lesions. CONCLUSION Symptomatic lesions are characterized by higher production of proinflammatory cytokines. Immunoregulatory cytokines are more important for suppression of inflammation in asymptomatic lesions and in this context the effect of TGF-beta is more potent and different from IL-10.

IL-17 and glutamate excitotoxicity in the pathogenesis of multiple sclerosis.

Immunoinflammatory‐mediated demyelination, the main pathological feature of multiple sclerosis (MS), is regularly accompanied by neurodegenerative processes, mostly in the form of axonal degeneration, which could be initiated by glutamate excitotoxicity. In the current study, the relationship between Th17‐mediated inflammatory and excitotoxic events was investigated during an active phase of MS. Cerebrospinal fluid (CSF) of patients with MS and control subjects was collected, and IL‐17A and glutamate levels were determined. IL‐17A level was significantly higher in patients with MS; whereas no statistically significant changes in glutamate concentrations were found. There was a direct correlation between IL‐17A and glutamate levels; IL‐17A levels were also associated with the neutrophil expansion in CSF and blood–brain barrier disruption. However, IL‐17A level and the number of neutrophils tended to fall with disease duration. The results suggest that Th17 cells might enhance and use glutamate excitotoxicity as an effector mechanism in the MS pathogenesis. Furthermore, Th17 immune response, as well as neutrophils, could be more important for MS onset rather than further disease development and progression, what could explain why some MS clinical trials, targeting Th17 cells in the later stage of the disease, failed to provide any clinical benefit.

Relationship between microstructure, cytotoxicity and corrosion properties of a Cu-Al-Ni shape memory alloy.

Cu-Al-Ni shape memory alloys (SMAs) have been investigated as materials for medical devices, but their biomedical application is still limited. The aim of this work was to compare the microstructure, corrosion and cytotoxicity in vitro of a Cu-Al-Ni SMA. Rapidly solidified (RS) thin ribbons, manufactured via melt spinning, were used for the tests. The control alloy was a permanent mould casting of the same composition, but without shape memory effect. The results show that RS ribbons are significantly more resistant to corrosion compared with the control alloy, as judged by the lesser release of Cu and Ni into the conditioning medium. These results correlate with the finding that RS ribbons were not cytotoxic to L929 mouse fibroblasts and rat thymocytes. In addition, the RS ribbon conditioning medium inhibited cellular proliferation and IL-2 production by activated rat splenocytes to a much lesser extent. The inhibitory effects were almost completely abolished by conditioning the RS ribbons in culture medium for 4 weeks. Microstructural analysis showed that RS ribbons are martensitic, with boron particles as a minor phase. In contrast, the control Cu-Al-Ni alloy had a complex multiphase microstructure. Examination of the alloy surfaces after conditioning by energy dispersive X-ray and Auger electron spectroscopy showed the formation of Cu and Al oxide layers and confirmed that the metals in RS ribbons are less susceptible to oxidation and corrosion compared with the control alloy. In conclusion, these results suggest that rapid solidification significantly improves the corrosion stability and biocompatibility in vitro of Cu-Al-Ni SMA ribbons.

Garlic extracts stimulate proliferation of rat lymphocytes in vitro by increasing IL-2 and IL-4 production.

Abstract Garlic components are known to modulate certain immune functions. However, mechanisms of their action are not sufficiently elucidated. This study was, therefore, undertaken to examine the effects of aqueous and ethanolic extracts prepared from a garlic powder sample on proliferation of rat spleen Iymphocytes in culture. Cells were stimulated with the combination of phorbol myristate acetate (PMA) and a Ca ionophore (A23187) or R73 monoclonal antibody (mAb) directed to the α β chain of T cell receptor. It has been shown that both extracts significantly stimulated proliferation of lymphocytes. The effect correlated with upregulation of the Interleukin 2 receptor a (IL-2Rα) expression and the increase in IL-2 production. Stimulation of IL-2 production by the extracts was higher in cultures with PMA / Ca ionophore than in cultures with R73 mAb. In contrast, both extracts stimulated production of IL-4 by splenocytes triggered by R73 mAb. The complete dependence of lymphocyte proliferation in cultures with R73 mAb and garlic extracts on IL-2 and IL-4 was demonstrated using neutralising mAbs to IL-2Rα and IL-4. These results suggest that the potentiating effect of garlic extracts on lymphocyte proliferation in vitro differs depending on specific stimulators of cell proliferation and probably on the type of responding cells.

The response of human dendritic cells to co-ligation of pattern-recognition receptors

Dendritic cells (DCs) are key antigen-presenting cells that express a wide variety of pattern-recognition receptors (PRRs). Triggering of a single PRR, especially Toll-like receptors (TLRs) and C-type lectins, induces maturation of DCs, but cooperativity between multiple PRRs is needed in order to achieve an effective immune response. In this review, we summarize the published data related to the effect of individual and joint PRR agonists on DCs and Langerhans-like cells derived from monocytes (MoDCs and MoLCs, respectively). Our results demonstrate that MoDCs co-stimulated with TLR3/TLR7 and TLR3/Dectin-1 ligands induced superior T helper (Th)1 and Th17 immune responses, compared to effects of single agonists. The opposite outcome was observed after co-ligation of TLR3 and Langerin on MoLCs. These findings may be relevant to improve strategy for tumor immunotherapy.

Evaluation of the Immunomodulatory Activities of Royal Jelly Components In Vitro

In this work the effect of different components isolated from royal jelly (RJ) was studied using an in vitro rat T-cell proliferation assay. We found that lower concentrations of MEL 174 (final water extract of RJ) and MEL 147 (3-10-dihydroxydecanoic acid) stimulated T-cell proliferation, triggered by concanavalin A (Con-A) and the process was followed by an increase in the production of interleukin-2 (IL-2). Higher concentrations of MEL 174, MEL 247 (dry powder of RJ) and MEL 138 (trans-10-hydroxydec-2-enoic acid) inhibited T-cell proliferation. The inhibition of T-cell proliferation in the presence of MEL 174 was followed by a decrease in IL-2 production, which was partly abrogated by exogenous IL-2, a decrease in nitric oxide (NO) production and increased apoptosis. In conclusion, our results showed the complexity of biological activity of RJ and suggest that its water extract possesses the most potent immunomodulatory activity in vitro.

Optimization of a free separation of 30 free amino acids and peptides by capillary zone electrophoresis with indirect absorbance detection: a potential for quantification in physiological fluids

This report describes a rapid, single-run procedure, based on the optimization of capillary electrophoresis (CE) and indirect absorbance detection capabilities, which was developed for the separation and quantification of 30 underivatized physiological amino acids and peptides, usually present in biological fluids. p-Aminosalicylic acid buffered with sodium carbonate at pH 10.2+/-0.1 was used as the running electrolyte. Electrophoresis, carried out in a capillary (87 cm x 75 microm) at 15 kV potential (normal polarity), separated the examined compounds within 30 min. Limits of detection ranged from 1.93 to 20.08 micromol/l (median 6.71 micromol/l). The method was linear within the 50-200 micromol/l concentration range (r ranged from 0.684 to 0.989, median r=0.934). Within run migration times precision was good (median C.V.=0.7%). Less favorable within run peak area precision (median C.V.=6.6%) was obtained. The analytical procedure presented was successfully tested for separation and quantification of amino acids in physiological fluids, such as plasma or supernatant of macrophage cultures. Sample preparations require only a protein precipitation and dilution step.

Characterization and immunosuppressive properties of mesenchymal stem cells from periapical lesions.

AIM Mesenchymal stem cells (MSCs) isolated from healthy dental tissues are being investigated as an alternative source of MSCs for the treatment of damaged tissues and inflammatory diseases. Here we investigated whether MSCs from periapical lesions (PL-MSCs) also possess multi-lineage differentiation capacity and immunomodulatory properties. MATERIAL & METHODS PL-MSCs, isolated by collagenase/DNAse digestion from surgically extracted PLs, were compared with MSCs from non-inflamed dental pulp (DP-MSCs) and dental follicle (DF-MSCs) for their phenotype and multi-potent differentiation potential. The anti-inflammatory and immunomodulatory effects of PL-MSCs were studied in co-culture with peripheral blood mononuclear cells (PB-MNCs) and PL-inflammatory cells (PL-ICs). RESULTS PL-MSCs were characterized by typical MSCs phenotype, lower clonogenicity and self-renewal rate, compared to DF-MSCs and DP-MSCs. These cells possess the potential to differentiate into adipocyte-, osteoblast- and chondrocyte-like cells in vitro, which differs from that of DP-MSCs and DF-MSCs. PL-MSCs inhibited phytohemaglutinine-induced proliferation of PB-MNCs and production of IL-2, IFNγ and IL-5 in the co-culture, probably via TGF-β-dependent mechanisms. These cells also suppressed the production of IL-1β, IL-6, and TNF-α by PL-ICs via soluble mediators, whereas the suppression of IL-8 production required a direct cell-to-cell contact. CONCLUSION The differentiation potential of PL-MSCs and their immunosuppressive/anti-inflammatory properties could be beneficial for the treatment of chronic periodontal diseases.