Petar Milosavljevic

Regulatory T-cells in Periapical Lesions

CD4+CD25hiFoxp3+ regulatory T-cells (Tregs) are of crucial importance in regulating the immune response, including the control of any defense against infection. Their presence in periapical lesions has not been demonstrated, as yet. We hypothesized that Tregs infiltrate periapical lesions, where they inhibit T-cell proliferation. The aim of this study was to characterize Tregs in periapical lesions by confocal microscopy, flow cytometry, and functional assays. We showed that CD4+CD25hiFoxp3+ cells in periapical lesions expressed IL-10 and TGF-β. Their frequency was significantly higher than in peripheral blood and correlated with the levels of TGF-β and IL-10 in culture supernatants of periapical lesion mononuclear cells. Tregs inhibited the proliferation of responder T-cells in vitro, at least in part, by stimulating the production of IL-10. These findings suggest that CD4+CD25hiFoxp3+ cells in periapical lesions may play regulatory roles in controlling local immune/inflammatory processes.

Mesenchymal stem cells from periapical lesions modulate differentiation and functional properties of monocyte-derived dendritic cells

Immunoregulatory mechanisms within periapical lesions (PLs) are as of yet unexplored. Considering the crucial role of DCs in controlling the immune response within PLs, the immunomodulatory properties of mesenchymal stem cells (MSCs), and the colocalization of MSCs and DCs in situ, we wondered whether MSCs from PLs modulate the development and functions of DCs. Using a model of monocyte‐derived DCs, we showed that PL‐MSCs inhibited differentiation of DCs via soluble factors, of which IL‐6 had a minor effect, but did not impair their subsequent maturation induced by pro‐inflammatory cytokines. However, upon maturation such DCs favored the production of Th2/Th17 cytokines by allogenic CD4+ lymphocytes in coculture, compared with mature DCs differentiated without PL‐MSCs. PL‐MSC‐differentiated DCs, cultivated with pro‐inflammatory cytokines and PL‐MSCs, although phenotypically mature, exhibited poor allostimulatory activity, induced anergy, Th2 polarization, differentiation of suppressive CD4+CD25highCD39+ Treg‐cell subsets via IDO‐1‐, ILT‐3‐, and ILT‐4‐dependent mechanisms, and increased production of TGF‐β in the coculture. In contrast, DCs cultivated with PL‐MSCs only during maturation stimulated proliferation and Th1 polarization of CD4+ T cells in an IL‐12‐independent manner. In conclusion, PL‐MSCs significantly modulate the development and functions of DCs, depending on the phase of DCs development during which the interaction occurs.

Oxidative stress in rat kidneys due to 3,4-methylenedioxymetamphetamine (ecstasy) toxicity

Aim:  The mechanism of MDMA (3,4‐methylenedioxymethamphetamine)‐induced toxicity is believed to be, in part, due to enhanced oxidative stress. As MDMA is eliminated via the kidney, the aim of this study was to investigate whether MDMA created conditions of oxidative stress within rat kidney.

Inverse production of IL-6 and IL-10 by abdominal aortic aneurysm explant tissues in culture.

BACKGROUND Abdominal aortic aneurysm is considered an atherosclerosis-related disease, but the mechanisms underlying abdominal aortic aneurysm remain poorly defined. Despite the large number of cytokines identified in an aneurysm sample, the relative importance of particular cytokines in aneurysm formation is unknown. We have studied the production of interleukin-6 and interleukin-10 cytokines in plasma and cultures of abdominal aortic aneurysm explant samples obtained from patients subjected to elective surgery and their correlation with cellular composition. MATERIALS AND METHODS Inflammatory cells from the abdominal aortic aneurysm samples were phenotypically characterized using specific monoclonal antibodies (anti-CD3, -CD4, -CD8, -CD19, -CD38, -CD68, -HLA-DR) by means of immunocytochemistry staining. Production of interleukin-6 and interleukin-10 in culture supernatants of abdominal aortic aneurysm explant samples expanded in vitro for 24 h was measured by enzyme-linked immunosorbent assay. RESULTS We showed that the levels of interleukin-6 and interleukin-10 in supernatants of abdominal aortic aneurysm sample cultures were higher by 73 and 86 times compared to their levels in plasma, respectively. In individual abdominal aortic aneurysm explant cultures, a negative correlation between interleukin-6 and interleukin-10 production was observed. Such inverse correlation was not detected in plasma. Based on these results, we divided abdominal aortic aneurysm into two cytokine-producing groups and showed that the interleukin-6(hi)/interleukin-10(lo) group contained higher percentages of granulocytes, HLA-DR(+), and CD68(+) cells but lower percentages of lymphocytes and plasma cells compared to the interleukin-6(lo)/interleukin-10(hi) group. Exogenously added interleukin-10 suppresses the production of interleukin-6 by abdominal aortic aneurysm explants. CONCLUSION These results suggest that interleukin-6 and interleukin-10 may have a different role in the pathogenesis of abdominal aortic aneurysm.

Immunomodulatory Activity of IL-27 in Human Periapical Lesions

IL-27, a cytokine with pro-inflammatory and anti-inflammatory properties, is a new member of the IL-6/IL-12 family, whose function in periapical lesions is unknown. We hypothesized that the production of IL-27 and its effect depend upon the type of immune/inflammatory response and clinical presentation of periapical lesions. We tested this hypothesis by studying the expression and function of IL-27 in human periapical lesions, both in situ and in culture. Immunohistochemistry demonstrated the strongest expression of IL-27 by endothelial cells and mononuclear phagocytes. Its production by periapical lesion mononuclear cells (PL-MNC), especially in symptomatic lesions, was significantly higher compared with that in peripheral blood MNC and correlated with the frequency of CD14+ and CD3+ cells. Exogenous IL-27 stimulated Th1 and down-regulated Th17 cytokine production by PL-MNC from symptomatic lesions, but down-regulated Th1 and Th2 responses in asymptomatic lesions. These findings suggest that IL-27 is an immunomodulatory cytokine in periapical lesions, with complex biological effects.

Disulfiram moderately restores impaired hepatic redox status of rats subchronically exposed to cadmium

Abstract Examination of cadmium (Cd) toxicity and disulfiram (DSF) effect on liver was focused on oxidative stress (OS), bioelements status, morphological and functional changes. Male Wistar rats were intraperitoneally treated with 1 mg CdCl2/kg BW/day; orally with 178.5 mg DSF/kg BW/day for 1, 3, 10 and 21 days; and co-exposed from 22nd to 42nd day. The co-exposure nearly restored previously suppressed total superoxide dismutase (SOD), catalase (CAT) and increased glutathione peroxidase (GPx) activities; increased previously reduced glutathione reductase (GR) and total glutathione-S-transferase (GST) activities; reduced previously increased superoxide anion radical (O2·−) and malondialdehyde (MDA) levels; increased zinc (Zn) and iron (Fe), and decreased copper (Cu) (yet above control value), while magnesium (Mg) was not affected; and decreased serum alanine aminotransferases (ALT) levels. Histopathological examination showed signs of inflammation process as previously demonstrated by exposure to Cd. Overall, we ascertained partial liver redox status improvement, compared with the formerly Cd-induced impact. Graphical Abstract

Skin response to epicutaneous application of anticoagulant rodenticide warfarin is characterized by differential time- and dose-dependent changes in cell activity.

Abstract Context: Skin is the target of both acute and chronic exposure to warfarin, coumarin anticoagulant. Single exposure of rat skin to this agent induces early (24 h following epicutaneous administration) local response which might be part of inflammatory/reparatory homeostatic program or introduction to pathological events in exposed skin. Objective: To examine time-dependent changes in skin of rats exposed to epicutaneously applied warfarin. Materials and methods: The effect of low (10 μg) and high (100 μg) doses of warfarin on histologically evident changes of epidermis (epidermal thickness) and dermis (numbers of mesenchymal cells and dermal capillaries), skin cell proliferative activity (Ki67+ and PCNA+ cells) and apoptotic (TUNEL+) and necrotic (ultra structural appearance) cells was examined one, three and seven days after the application. Results: Both warfarin doses affected the majority of skin cell activity, but with differential time-course of skin epidermal and dermal cells state/activity. The occurrence of necrotic/apoptotic epidermal and dermal cells was noted the first day after the application and the activities which point to tissue reparation/remodeling were observed seven days after skin exposure to this agent. Discussion: The observed pattern of changes (early evidence of cell/tissue injury which was later followed by signs of cell activity characteristic for tissue reparation/remodeling) implied warfarin-induced toxicity in skin cells as stimulus for subsequent activities relevant for tissue homeostasis. Conclusion: The data presented provide new and additional information concerning skin responses to warfarin that gains access to this tissue.