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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. [Projects of the Serbian Ministry of Education, Science and Technological Development, Grant no. ON 175102 and Grant no. 172026]

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Background/Aim. The goal of modern clinical and experimental researches in the field of sepsis is to find one or more sensitive parameters that could predict the severity of sepsis and its outcome. In this study we investigated and compared the relationship of initial soluble Fas (sFas) plasma levels as well as Acute Physiology, Age and Chronic Health Evaluation II (APACHE II) score in 58 septic patients with severity and outcome of sepsis. Methods. The diagnosis and assessment of disease severity was performed on the same day, based on clinical and laboratory parameters. The blood samples were used for monitoring of laboratory standard parameters necessary for the diagnosis of sepsis, organ dysfunction and assessment of disease severity, as well as for determination of levels of sFas. According to consensus criteria, patients were divided into those with sepsis (n = 16), severe sepsis (n = 30) or septic shock (n = 12), those with (n = 26) and without (n = 32) multiple organ dysfunction syndrome (MODS), and survivors (n = 45) and non-survivors (n = 13). Results. Plasma sFas level (9.7 ± 10.1; 0-44.2 U/mL) was elevated in 54.4% of patients. All the patients with septic shock, 76.9% of the patients with MODS and 84.6% patients who died had elevated sFas level. We observed a strong positive correlation between sFas and APACHE II score (p < 0.001). The level of sFas was significantly higher in patients with septic shock compared to normotensive patients (p < 0.001), patients with MODS compared to those without MODS (p < 0.001) and survivors compared to nonsurvivors (p < 0.01). Conclusions. Our results suggest that initial sFas plasma levels in patients with sepsis correlated with the values of APACHE II score and separated very well the patients with septic shock versus the normotensive patients, the patients with and without MODS, and survivors versus non-survivors.

Background/Aim. Morbidity and mortality of continous ambulatory peritoneal dialysis (CAPD) patients is still very high. The aim of the study was to evaluate the effects of peritoneal dialysis (PD) solutions (standard vs biocompatible) on long-term patients’ and the techique survival. Methods. A total of 42 stable patients on CAPD participated in this crosssectional study. They were prospectively followed-up during the twelve years. Patients with severe anemia (Hb < 10 g/L) and malignant disease ware excluded. Twenty one (50%) patients were treated with the standard PD solutions (CAPDP- 1) while the other 21 (50%) were treated with biocompatible PD solutions [(lower level of glucose degradation products, lower concentration of Ca2+ and neutral pH (CAPDP-2)]. All patients were analyzed for a presence of vascular calcification, nutrition status, and parameters of inflammation after 2.5 ± 0.6 years of starting CAPD, and these variables considered in the analysis as risk factors. Results. The patients from the group CAPDP-2 compared to those from the group CAPDP-1 had lower level of high-sensitivity C-reactive protein (hs-CRP) (p = 0.003), and better nutritional status as confirmed by the mid-arm circumference (p = 0.015), and midarm muscle circumference (p = 0.002) and subjective global assessment (p = 0.000). Also, they had lower vascular calcifications as confirmed by intima media thickness (IMT) (p = 0.003), degree of carotid narrowing (p = 0.001) and calcified plaques of common carotid arteries (CCA) (p = 0.008). Kaplan- Meier analysis confirmed better survival of patients from the group CAPDP-2 than those from the group CAPDP-1 (1-, 5-, and 10-year patients survival rate was: 100%, 61.9% and 14.3% for the group CAPDP-1, and 100%, 85.7%, and 52.4% for the group CAPDP-2, respectively; p = 0.0345). The 1-, 5-, and 10-year technique survival rate was: 100%, 71.4%, and 38.1% for the group CAPDP-1, and 100%, 85.7%, and 76.2% for the group CAPDP-2, respectively; (p = 0.0719). Duration of dialysis, serum triglyceride and cardiovascular score (quantitative scoring system consisting of: ejection fraction (EF) of left ventricle < 50%; IMT > 1 mm; carotid narrowing degree > 50%, presence of carotid plaques in both common carotide, ischaemic heart disease, cerebrovascular event and peripheral vascular disease with or without amputation) were independent predictors of overall patient survival. Duration of dialysis was only independent predictor of overall technique survival. Conclusion. Although patients treated with biocompatible solutions showed significantly better survival, the role of biocompatibility of CAPD solutions in patients and technique survival have to be confirmed. Namely, multivariate analysis confirmed that duration of dialysis, serum triglyceride and cardiovascular score significantly predicted overall CAPD patients survival, while only duration of dialysis was found to be independent predictor of overall techique survival.

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. 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.

Background. In our previous experiments it was shown that R-MC46 monoclonal antibody (mAb), produced at our Institute, stimulated homotypic aggregation of rat granulocytes and production of proinflammatory cytokines. The aim of this study was to examine antigen expression and function, recognized by R-MC46 mAb on macrophages. Methods. The expression of R-MC46 antigen on thymic and peritoneal macrophages was investigated using immunocytochemistry and flow cytometry methods. Its biochemical characterization was performed by Western blot. The ability of R-MC46 mAb to modulate adhesion and phagocytosis by macrophages was studied by using co-culture experiments with autologous thymocytes. Results. R-MC46 mAb stained thymic macrophages more strongly than peritoneal macrophages. After in vivo treatment of peritoneal macrophages with Pristane, a significant up-regulation of the R-MC46 antigen expression was observed. Western blot analysis showed that the mAb recognized a low molecular weight antigen of about 5.5 kDa. R-MC46 mAb significantly enhanced binding and phagocytosis of thymocytes by both thymic and peritoneal macrophages. These processes were completely blocked by WT.3 (anti-CD18) mAb. The stimulation of binding thymocyte to macrophages was higher with the use of thymic macrophages,while the phagocytosis of these cells was higher in the presence of peritoneal macrophages. Conclusion. R-MC46 mAb recognized a new molecule expressed by rat macrophages. The antigen is most probably involved in β2 integrin-mediated adhesion and phagocytosis, as well as proinflammatory functions of macrophages.

Several laboratories have developed culture systems that allow the generation of large numbers of human dendritic cells (DC) from monocytes using granulocyte-macrophage colony stimulating factor (GM-CSF), and interleukin-4 (IL-4). In this work we provided evidence that GM-CSF (100 ng/ml) in combination with a low concentration of IL-4 (5 ng/ml) was efficient in the generation of immature, non-adherent, monocyte-derived DC as the same concentration of GM-CSF, and ten times higher concentration of IL-4 (50 ng/ml). This conclusion was based on the similar phenotype profile of DC such as the expression of CD1a, CD80, CD86, and HLA-DR, down-regulation of CD14, and the absence of CD83, as well as on their similar allostimulatory activity for T cells. A higher number of cells remained adherent in cultures with lower concentrations of IL-4 than in cultures with higher concentrations of the cytokine. However, most of these adherent cells down-regulated CD14 and stimulated the proliferation of alloreactive T cells. In contrast adherent cells cultivated with GM-CSF alone were predominantly macrophages as judged by the expression of CD14 and the inefficiency to stimulate alloreactive T cells. DC generated in the presence of lower concentrations of IL-4 had higher proapoptotic potential for the Jurkat cell line than DC differentiated with higher concentrations of IL-4, suggesting their stronger cytotoxic, anti-tumor effect.