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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 largesize 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. [Project of the Serbian Ministry of Education, Science and Technological Development, Grant no. OI 175102]

Introduction: Interleukin-8 (IL-8) is an important mediator of inflammation. However, little is known about its production in chronic dental periapical lesions and this was the main aim of this work. Methods: Inflammatory cells were isolated from clinically different periapical lesions and analyzed by morphological criteria. The mononuclear cells were isolated, phenotypically analyzed by immunocytochemistry and cultivated in vitro. IL-8 was measured in culture supernatants of these periapical lesion mononuclear cells (PL-MNC) using a microbeads fluorescence assay. Results: We found a relatively high production of IL-8 in 19 out of 21 periapical lesions included in the study. The level of IL-8 and the proportion of neutrophil granulocytes were significantly higher in the group of symptomatic lesions, compared to the asymptomatic lesions, but there was no statistically significant correlation between these parameters. According to the predominance of CD3+ T cells and Ig+/CD19+ B cells and plasma cells, lesions were divided into T-type and B-type lesions, respectively. The levels of IL-8 were significantly higher in the culture supernatants of PL-MNC in the T-type lesions and were positively correlated with the proportion of macrophages/dendritic cells (CD11c+ cells) and CD4+ T cells. Such a correlation was not shown in B-type lesions. Conclusion: These results suggest that PL-MNC are a significant source of IL-8, which is probably an important chemokine for the migration and function of different cell types at the site of chronic inflammation. [ABSTRACT FROM AUTHOR]

Background/Aim. Ligation of a Toll-like receptor (TLR) by specific TLR agonists is a powerful tool for maturation induction of monocyte-derived dendritic cells (MoDCs). Studies so far have shown that the treatment of dendritic cells (DCs) with a TLR3 ligand, polyinosinic-polycytidylic acid [Poly(I:C)], may be an appropriate activation agent for obtaining mature MoDCs, competent to prime effective immune responses. However, little is known about how subsequent interaction of MoDCs with T cell-derived stimuli, such as CD40 or interferon-γ (IFN-γ), modulates MoDC functions. Therefore, this problem was the main objective of this study. Methods. Immature MoDCs were prepared by cultivation of monocytes from peripheral blood mononuclear cells with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin (IL)-4 for 5 days. After that, maturation was induced by the treatment of these cells with Poly(I:C) for 2 days. At day 6, immature MoDCs and Poly(I:C)-activated MoDCs were incubated either with CD40 ligand (L)-transfected J558 cells or IFN-γ for additional 24 hours. Cytokine production was measured by ELISA and FlowCytomix Human T helper Th1/Th2 11plex. Allostimulatory capability of MoDCs was tested using an allogeneic mixed leukocyte reaction (MLR) assay. Results. Immature MoDCs showed a moderate potential for stimulation of proliferation of CD4+ T cells, which was enhanced by the treatment with Poly(I:C). Ligation of CD40 or treatment with IFN-γ of immature or Poly(I:C)-treated MoDCs significantly up-regulated their allostimulatory activity. MoDCs matured in the presence of Poly(I:C) up-regulated the production of IL- 12 and IL-10, which was followed by increased levels of IFN- γ and decreased levels of IL-5 in co-cultures with allogeneic CD4+ T cells. Ligation of CD40 on immature MoDCs upregulated the production of IL-12 and IL-23 which was accompanied by increased secretion of IFN-γ in co-culture. Stimulation of CD40 on Poly(I:C)-treated MoDCs significantly enhanced the production of IL-12, IL-23 and IL-10. However, such treated MoDCs decreased the production of IFN-γ and IL-10 and up-regulated the secretion of IL-17. Immature MoDCs treated with IFN-γ up-regulated IL-12, but lowered the production of IL-5 and IL-17 by CD4+ T cells. Treatment of Poly(I:C)-activated MoDCs with IFN-γ down-regulated the production of IL-12 and up-regulated IL- 10 by these cells and increased/decreased the levels of IL-10/ IFN-γ, respectively, in co-culture with CD4+ T cells. Conclusion. Treatment with Poly(I:C) or ligation of CD40 on immature MoDCs induces maturation of these cells into a phenotype that supports Th1 response. Activation of CD40 on Poly(I:C)-treated MoDCs shifts the immune response towards Th17. Treatment of immature MoDCs with IFN-γ down-regulated Th2 and Th17 responses. However, addition of IFN-γ to Poly(I:C)-activated MoDCs down-regulated Th1 response and promote T regulatory mechanisms. Each of these results may have functional and therapeutic implications.

Background. Stent thrombosis is potentially lethal complication with huge economic burden. The role of insufficient response to antiplatelet therapy is still unclear reason for its occurrence. Case report. We presented 54-year-old man with recurrent stent thrombosis on the 4th, 9th and 12th day after the primary percutaneous coronary intervention in spite of double antiaggregation therapy (aspirin+ clopidogrel). All possible procedural causes were excluded and reimplantation of intracoronary stent was insufficient to resolve the problem, so four platelet tests were performed: flow cytometry, Platelet Function Analyzer-100 test, aggregometry, and determination of gene polymorphism for P2Y12 receptor (directly involved in the mechanism of thienopyridine), and GPIIbIIIa receptor (final receptor in aggregation). The patient was the carrier of the major haplotype H1H1 for P2Y12 receptor and minor A1A2 for GPIIbIIIa receptor. The results of all the performed tests showed insufficient antiplatelet effect of aspirin and sufficient response to thienopyridin (not to clopidogrel, but to ticlopidine). Conclusion. Performance of platelet function tests is necessary in the case of major adverse cardiac events especially stent thrombosis, after implantation of intracoronary stent.

Background. Dendritic cells (DC) have been used for immunotherapy of malignant tumors, different kinds of infections, and other clinical conditions. For that purpose, optimal conditions for the generation of functionally mature DC in vitro are required. Two different protocols for the induction of maturation of monocyte-derived DC (MDDC) were compared in this study. Methods. MDDC were generated in vitro by cultivating adherent monocytes of healthy volunteers with granulocyte macrophage-colony stimulating factor (GM-CSF) and interleukin 4 (IL-4) during 6-days period. The immature DC thus prepared were induced to mature using two protocols. DC were stimulated for 2 days with lipopolysaccharide (LPS), or with a cocktail of proinflammatory mediators (PM) containing IL-1b, IL-6, tumor necrosis factor α (TNFα), and prostaglandin E2 (PGE2), respectively. Phenotypic characteristics of MDDC and their endocytic activity were studied by flow cytometry. Allostimulatory activity of these cells was tested in the mixed leukocyte reaction (MLR), whereas the production of cytokines was determined by ELISA kits. Results. MDDC matured with PM (PM-DC) were predominantly non-adherent cells, while about 30% of LPS-matured DC were adherent cells. In comparison with LPS-DC, PM-DC expressed higher levels of CD86 and CD83, had lower endocytic activity, produced higher levels of IL-10 and lower levels of IL-12, and more strongly stimulated proliferation of allogeneic lymphocytes. Conclusion The protocol based on the combination of proinflammatory cytokines and PGE2 is better for the induction of maturation of human MDDC in vitro than the protocol using LPS alone.

There is a great interest in molecular markers that would help in the preoperative diagnosis of malignant thyroid nodules in cases of indeterminate fine-needle aspiration cytology. The aim of this study was to determine the diagnostic accuracy of HMGA2 gene expression in discriminating benign from malignant thyroid nodules. In this study, 237 preoperative thyroid fine-needle aspiration samples were analyzed prospectively for the expression of the HMGA2 gene by real-time reverse transcription polymerase chain reaction. The results were evaluated against the postoperative histopathologic diagnosis or definitive cytologic diagnosis in cases of nodular goiter and Hashimoto thyroiditis. Among 237 samples from patients with thyroid nodules that were analyzed, 231 were adequate for real-time reverse transcription polymerase chain reaction analysis. With a cutoff value of 8.71 for relative gene expression, HMGA2 was positive in 19 (16.4%) of 116 nodular goiter, 1 (2.6%) of 39 Hashimoto thyroiditis, 9 (28.1%) of 32 follicular adenoma, 0 (0%) of 5 Hurthle cell adenoma, 32 (88.9%) of 36 papillary carcinoma, and 3 (100%) of 3 follicular carcinoma samples. In discriminating between malignant and benign thyroid nodules, HMGA2 has shown specificity of 84.5%, sensitivity of 91.9%, positive predictive value of 53.1%, and negative predictive value of 98.2%. High sensitivity and negative predictive value of HMGA2 for preoperative detection of malignant thyroid nodules shown in this study indicate that it may have a role as an ancillary marker in cytology in the management of patients with thyroid nodules.

Introduction: Sodium salicylate (NaS) is a derivate of acetylsalicylic acid or aspirin, used as a nonsteroidal anti-inflammatory drug for centuries, for its analgesic and anti-inflammatory effects. It was found to modulate different signaling pathways, in a cell-specific way. Here, we explore the effect of NaS on cell growth and urokinase activity in MDA MB-231 breast cancer cells., Materials and Methods: We analyzed the effect of NaS treatment on cell growth by flow cytometry and viability test. The transwell migration assay was used to study the migratory response of the cells. The gene expression was analyzed by qRT-PCR on RNA level and by Western blot analysis on protein level. Urokinase activity was assessed by caseinolysis., Results: Sublethal concentrations of NaS decreased cell growth and inhibited urokinase activity. The latter was a consequence of decrease in urokinase expression and increase in expression of its inhibitors. Analysis of signaling molecules revealed activation of transforming growth factor-β signaling, increase in master transcription factors for epithelial-mesenchymal transition and changes in integrin expression., Conclusions: We propose that NaS causes partial cellular reprogramming through transforming growth factor-β signaling which, together with direct NaS influence, causes changes in expression in a set of genes involved in extracellular proteolysis. These data could be beneficial for the development of new therapeutic approaches in invasive breast cancer treatment., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The synthesis and whole body metabolism of L-arginine (Arg) are disturbed in renal diseases. Renal transplantation represents the best therapy in the end-stage of these diseases. In the present we compared alterations of plasma Arg and related compounds with renal excretory function in patients with end-stage renal disease, before and after kidney transplantation. Arg, asymmetric dimethylarginine (ADMA), citrulline (Cit), glutamine (Gln), ornithine (Orn), phenylalanine (Phe), tyrosine (Tyr), urea, creatinine, albumin, and nitrate were analyzed in patients before, immediately after (0-time) and 1, 2, 3, 7 and 14 days following living donors kidney transplantation. Healthy subjects were controls. Glomerular filtration rate (GFR) and amino acid molar ratios were calculated. Before transplantation creatinine, urea, Cit, Gln, ADMA, and nitrate were above, while GFR and Arg were below controls, confirming disturbed excretory and metabolic renal functions in patients with renal disease. Renal transplantation promptly normalized creatinine, urea, GFR, Cit, and nitrate. However, regardless of increased molar Phe/Tyr ratios, indicating increased net protein catabolism in peripheral tissues, low Arg and elevated ADMA concentrations persisted throughout the examined period. Alterations of other amino acids also suggest similarly disturbed Arg metabolism in patients after kidney transplantation. In conclusion, renal transplant promptly restored its excretory function, but increased net protein catabolism, disturbed Arg metabolism and endothelial dysfunction in entire body of these patients were not improved throughout the early period after the operation. That has to be considered in their therapy., (Copyright © 2014 Elsevier Inc. All rights reserved.)

DEC-205, a transmembrane receptor responsible for cross-presentation of apoptotic cell-derived antigens, is expressed by cortical thymic epithelial cells (TEC) and thymic dendritic cells (TDC) in humans and mice, but its function in T-cell development is still unclear. In this work we have studied for the first time the expression of DEC-205 in the rat thymus by HD83 monoclonal antibody (mAb) and immunohistochemistry, as well as the ability of this mAb to modulate thymocyte - TDC interactions in vitro. We showed the positivity of cortical TEC in situ, including thymic nurse cells (TNC) in suspension, and TDC, whereas subcapsular, perivascular and medullary TEC were negative. All examined DEC-205 positive and DEC-205 negative structures were MHC class II positive. HD83 mAb increased apoptosis of thymocytes in co-culture with TDC in vitro and the process was associated with increased binding of thymocytes to TDC in a rosette form. Since negative selection of thymocytes by clonal deletion (apoptosis) was mediated predominantly by TDC, our results suggest the possible indirect effect of the DEC-205 molecule in these mechanisms.

Unlabelled: The development and progression of periapical dental lesions, mediated by the specific immune response, are poorly understood. In these processes, an interplay of different proinflammatory and immunoregulatory cytokines is of crucial importance., Objectives: To examine the activation of T helper 1 (Th1) and Th2 immune responses in 25 human periapical lesions based on the ex vivo production of interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) by mononuclear cells (PL-MNC)., Methods: The levels of IFN-gamma and IL-4 in culture supernatants of PL-MNC, determined by ELISA, were correlated with concentrations of these cytokines in cultures of control MNC from peripheral blood (PB-MNC), cellular composition of inflammatory cells and phenotypic characteristics of PL-MNC., Results: We detected high levels of IFN-gamma in all samples, after cell stimulation with phorbol myristate acetate and Ca(2+) ionophore, that were not statistically different from the levels of IFN-gamma in PB-MNC cultures. IL-4 was detected in 76% samples, but its concentrations were much lower than in PB-MNC samples. The levels of IFN-gamma were higher in cultures of PL-MNC isolated from periapical lesions with predominance of T cells (T-type lesions) and correlated positively with the proportion of antigen-presenting cells (macrophages and dendritic cells), CD4(+) T cells and IgG2(+) B cells/plasma cells. The levels of IL-4 correlated negatively with the proportion of macrophages, but positively with the number of mast cells and IgG4(+) cells. IL-18Ralpha, a stable marker of Th1 cells, was detected on a relatively small proportion of CD3(+) T cells and its expression correlated with the levels of IFN-gamma. However, the expression of ST2L, a stable Th2 cell marker, was not detected. The levels of Th1 and Th2 cytokines did not correlate with clinical characteristics of the lesions, defined by the presence of symptoms., Conclusion: Cumulatively, our results suggest the predominance of Th1 immune response in periapical lesions.

Dermatotoxic effects of epicutaneous application of a first-generation anticoagulant, warfarin (WF) were examined in rats. Selected parameters of skin activity were determined 24h following warfarin application, including metabolic viability of skin explants, some aspects of oxidative activity in skin tissue homogenates and inflammatory/immune relevant activity of epidermal cells from warfarin-treated skin. No changes in skin metabolic viability (MTT reduction) were noted ex vivo following WF application, suggesting the absence of immediate toxicity for skin. In contrast, increased formation of malondialdehyde (MDA), with a decrease in protein and non-protein thiols in homogenates of warfarin-treated skin was demonstrated, pointing to prooxidant activity in warfarin-treated skin. Increased costimulatory activity of epidermal cells isolated from warfarin-exposed skin in Con-A-stimulated T-cell activation/proliferation assay was noted, reflecting proinflammatory and immune-modulating capacity of warfarin for epidermis. No evident differences in skin histology between control and warfarin-treated skin were found at that time point, while striking changes in tissue integrity, cellularity and appearance 72 h following WF application were noted. The observed histological picture probably reflects a regenerative/inflammatory program related to oxidant/inflammation-type warfarin-evoked injury to the skin. Presented data demonstrate the potential of epicutaneously applied warfarin to modulate local skin activity in rats.

Subcutaneous implantation of polyvinyl sponges represents a suitable model for studying the mechanisms of acute and chronic inflammation, granulomatous foreign-body reaction, as well as wound healing. Using such a model in rats, we studied the phenotypic and functional characteristics of dendritic cells (DC). DC were purified from the sponge exudate using a combination of separation gradients, adherence to plastics, and immunomagnetic sorting. We have shown that the number of DC progressively increased in the sponges, reaching maximal values at day 10 after implantation, followed by their decrease thereafter. Inflammatory DC expressed MHC class II molecules and myeloid markers CD11b, CD11c, and CD68. A subset of DC expressed CD4, R-MC46, DEC-205, R-MC17, and CCR1. Compared to DC isolated in the early phase of inflammation (day 6 DC), DC in the late stage of inflammation (day 14 DC) had a lower capability to stimulate the proliferation of allogeneic lymphocytes and CD4(+) T cells. This finding correlated with the downregulation of CD80, CD86, and CD54 expression and the increased proportion of plasmacytoid MHC class II(+) His 24(+) His 48(+) DC. The suppression of allogeneic lymphocyte proliferation was abrogated by the treatment of DC with lipopolysaccharide. In addition, day 14 DC exerted tolerogenic capability in co-culture with allogenic CD4(+) T cells. These results correlated with the increased levels of IL-10 and TGF-beta in culture supernatants and the sponge exudate.