Quantification of oxidative stress markers in the blood sera following subacute administration of different oximes in rats.

Oxidative stress status, as a disruption of redox homeostasis, in the blood sera of Wistar rats caused by repeated application of selected acetylcholinesterase reactivators - asoxime, obidoxime, K027, K048, K074, and K075 were evaluated. Throughout this study, each oxime in a dose of 0.1 of LD50/kg im was given 2x/week for 4 weeks. Then, seven days after the last oximes' application, markers of lipid peroxidation (malondialdehyde, MDA), and protein oxidation (advanced oxidation protein products, AOPP), as well as the activity of antioxidant enzymes (catalase, CAT, superoxide dismutase, SOD, reduced glutathione, GSH, and oxidized glutathione, GSSG), were determined. Oxidative stress parameters, MDA and AOPP were significantly highest in the K048-, K074- and K075-treated groups (p < 0.001). The activity of CAT was significantly elevated in the obidoxime-treated group (p < 0.05), while treatment with K027, K048, and K074 induced high elevation in SOD levels (p < 0.01, p < 0.001). Interestingly, the activity of GSH in each oxime-treated group was significantly elevated. Unlike, treatment with obidoxime caused elevation in GSSG levels (p < 0.01). As a continuation of our previously published data, these results assure that applied oximes following subacute treatment ameliorated the oxidative status and further adverse systemic toxic effects in rats.

Oxidative stress status assessment of rats' brains injury following subacute exposure to K-oximes.

Oxidative stress status and morphological injuries in the brain of Wistar rats induced by repeated application of selected acetylcholinesterase reactivators - asoxime, obidoxime, K027, K048, K074, and K075 were evaluated. Each oxime in a dose of 0.1 of LD50/kg im was given 2x/week for 4 weeks. Markers of lipid peroxidation (malondialdehyde, MDA), and protein oxidation (advanced oxidation protein products, AOPP), as well as the activity of antioxidant enzymes (catalase, CAT, superoxide dismutase, SOD, glutathione reductase, GR, and glutathione peroxidase, GPx), were estimated in the brain tissue homogenates on day 35 of the study. Brain alterations were carefully quantified by semiquantitative grading scales - brain damage score (BDS). Oxidative stress parameters, MDA and AOPP were significantly highest in the asoxime-, obidoxime- and K075-treated groups (p < 0.001). The activity of SOD and CAT was significantly elevated in the obidoxime-, K048-, and K075-treated groups (p < 0.001). Besides, GR was markedly decreased in the obidoxime- and K074-treated groups (p < 0.01), while treatment with K048, K074 and K075 induced extremely high elevation in GPx levels (p < 0.001). In the same groups of rats, brain alterations associated with polymorphonuclear cell infiltrate were significantly more severe than those observed in animals receiving only asoxime or K027 (p < 0.001). The presented results confirmed that treatment with different oximes significantly improved the oxidative status and attenuated signs of inflammation in rats' brains. Presented results, together with our previously published data can help to predict likely adverse systemic toxic effects, and target organ systems, which are crucial for establishing risk categories, as well as in dose selection of K-oximes as drug candidates.

Gastrointestinal Helminths in Horses in Serbia and Various Factors Affecting the Prevalence.

PURPOSE: Data on gastrointestinal infections in horses in Southeastern Europe are limited; thus, this study aimed to add to the existing knowledge on this topic by reporting on the prevalence of intestinal parasites of horses in the territory of the Republic of Serbia. METHODS: In the period from April 2017 to December 2018, parasitological examination of 548 samples from horses of different breed, age and sex from four regions and 18 districts of the country was performed. Coprological diagnostic was done by using qualitative methods without concentration and qualitative methods with concentration of parasitic elements. Quantification of the obtained results was performed using semi-qualitative faecal egg count. RESULTS: Four helminthoses were detected in the examined samples: P. equorum (8.57%), O. equi (3.65%), strongylid eggs (71.17%) and Anoplocephala spp. (0.91%). The total prevalence of helminthoses was 77.19%. Monoinfections were significantly more present 70.07% compared to coinfections (7.12%). The highest prevalence of helminthoses was detected in free-ranging horses (93.10%-27/29), in autumn 86.67% (117/135) and winter 79.71% (165/207), in Sumadija and West Serbia region (100%), and in the youngest category (100%). Significant difference (p < 0.001) was detected in the prevalence of monoinfections by strongylids and O. equi and also coinfections by strongylid/P. equorum between horses of different age categories. CONCLUSION: Obtained results are of great contribution to clinical parasitology and pathology, especially from the aspect of animal health, welfare and preservation of horse population.

Theta burst stimulation influence the expression of BDNF in the spinal cord on the experimental autoimmune encephalomyelitis.

Repetitive transcranial magnetic stimulation (rTMS) induces changes in expression of proteins engaged in the activity of excitatory and inhibitory systems, restores these functions and suppresses the progression of disability in experimental autoimmune encephalitis (EAE). The structural type of TMS, the arrangement as theta burst stimulation (TBS) has been applied as intermittent TBS (iTBS) and continuous TBS (cTBS) protocols to female adult DA rats. The animals were randomly divided into experimental groups: control group (C), group treated with complete Freund's adjuvant (CFA), experimental autoimmune encephalomyelitis (EAE) group, group treated with iTBS post EAE immunization (EAE + iTBS), group treated with cTBS post EAE immunization (EAE + cTBS), group of healthy animals treated with iTBS or cTBS. Therapeutic protocols of iTBS or cTBS in all EAE groups of animals were performed starting from 14 days post immunization (dpi), for 10 days with time point decapitation at 24 dpi. After decapitation, spinal cords were analysed for BDNF and Ki67 expression. The results revealed reduced BDNF expression in the rat's spinal cord of EAE animals in the stage of remission, which was associated with increased Ki67 and GFAP expressions. Decreased Iba 1 and BDNF expression, contrary to increased Iba 1 and Ki67 expression, suggests clustered microglia in the resolution phase of EAE. Enhanced GABA expression in spinal cord sections indicates higher GABA metabolic turnover, and also GAD activity in astrocytes, or prominent activity of GABAergic neurons. Both TBS protocols induced advance BDNF expression; amongst iTBS application provoked elevating of BDNF and stabilizing of GFAP and Ki67 expressions.

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

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.

Extraordinary biological properties of a new calcium hydroxyapatite/poly(lactide-co-glycolide)-based scaffold confirmed by in vivo investigation.

This study examined the potential of a new porous calcium hydroxyapatite scaffold covered with poly (lactide-co-glycolide) (PLGA) as a bone substitute, identifying its advantages over Geistlich Bio-Oss®, considered the gold standard, in in vivo biofunctionality investigations. Structural and morphological properties of the new scaffold were analyzed by scanning electron and atomic force microscopy. The biofunctionality assays were performed on New Zealand white rabbits using new scaffold for filling full-thickness defects of critical size. The evaluated parameters were: the presence of macrophages, giant cells, monoocytes, plasma cells, granulocytes, neoangiogenesis, fibroplasia, and the percentage of mineralization. Parallel biofunctionality assays were performed using Geistlich Bio-Oss®. The appearance of bone defects 12 weeks after the new scaffold implantation showed the presence of a small number of typical immune response cells. Furthermore, significantly reduced number of capillary buds, low intensity of fibroplasia and high degree of mineralization in a lamellar pattern indicated that the inflammation process has been almost completely overcome and that the new bone formed was in the final phase of remodeling. All biofunctionality assays proved the new scaffold's suitability as a bone substitute for applications in maxillofacial surgery. It showed numerous biological advantages over Geistlich Bio-Oss® which was reflected mainly as a lower number of giant cells surrounding implanted material and higher degree of mineralization in new formed bone.

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

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.

Size-dependent effects of gold nanoparticles uptake on maturation and antitumor functions of human dendritic cells in vitro.

Gold nanoparticles (GNPs) are claimed as outstanding biomedical tools for cancer diagnostics and photo-thermal therapy, but without enough evidence on their potentially adverse immunological effects. Using a model of human dendritic cells (DCs), we showed that 10 nm- and 50 nm-sized GNPs (GNP10 and GNP50, respectively) were internalized predominantly via dynamin-dependent mechanisms, and they both impaired LPS-induced maturation and allostimulatory capacity of DCs, although the effect of GNP10 was more prominent. However, GNP10 inhibited LPS-induced production of IL-12p70 by DCs, and potentiated their Th2 polarization capacity, while GNP50 promoted Th17 polarization. Such effects of GNP10 correlated with a stronger inhibition of LPS-induced changes in Ca2+ oscillations, their higher number per DC, and more frequent extra-endosomal localization, as judged by live-cell imaging, proton, and electron microscopy, respectively. Even when released from heat-killed necrotic HEp-2 cells, GNP10 inhibited the necrotic tumor cell-induced maturation and functions of DCs, potentiated their Th2/Th17 polarization capacity, and thus, impaired the DCs' capacity to induce T cell-mediated anti-tumor cytotoxicity in vitro. Therefore, GNP10 could potentially induce more adverse DC-mediated immunological effects, compared to GNP50.

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(+) CD25(high) CD39(+) Treg-cell subsets via IDO-1-, ILT-3-, and ILT-4-dependent mechanisms, and increased production of TGF-ß 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.

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.

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. METHODS: Adult male Wistar rats were divided into three groups, control treatment (water), acute MDMA administration (single oral dose: 5, 10, 20 or 40 mg/kg body weight) and subacute MDMA administration (5, 10, or 20 mg/kg body weight per day during 14 days). Animals were sacrificed 8 h after the single oral MDMA administration in the acute MDMA administration group and after the last MDMA administration in the subacute MDMA administration group. Rectal temperature measurements, oxidative stress status parameters and histological examinations were performed. RESULTS: In all MDMA-administered rats, rectal temperature markedly increased peaking approximately 1 h after MDMA ingestion. Superoxide dismutase activity and thiobarbituric acid reactive substances increased after MDMA administration. Histological examinations of the kidney revealed dose-dependent disruption of tissue structure in subacute MDMA-administered rats. The latter was not observed in acute MDMA-administered rats.

Correlation between phenotypic characteristics of mononuclear cells isolated from human periapical lesions and their in vitro production of Th1 and Th2 cytokines.

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.

Differentiation of human dendritic cells from monocytes in vitro using granulocyte-macrophage colony stimulating factor and low concentration of interleukin-4.

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.