The presence of Mott cells in the lymph nodes of rats with experimental autoimmune encephalomyelitis.

Mott cells are plasma cells that have multiple spherical Russell bodies packed in their cytoplasm. Russell bodies are dilated endoplasmic reticulum cisternae filled with aggregates of immunoglobulins that are neither secreted nor degraded. Mott cells were observed in our study by light and electron microscope in the lymph nodes of rats with experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Mott cells were detected on hematoxylin and eosin (HE)-stained lymph node sections as vacuolated cells with eccentrically positioned nuclei and large number of faint blue spherical inclusions in the cytoplasm. Electron microscopic investigation revealed the presence of Russell bodies of the "medusa" form inside Mott cells in lymph node ultra-thin sections of EAE animals. Mott cells expressed the plasma cell marker CD138 and either kappa or lambda immunoglobulin light chains, indicating their origin from polyclonally activated B cells. Finally, Mott cells were associated with active EAE, as they were not found in the lymph nodes of EAE-resistant Albino Oxford rats. The presence of Russell bodies implies an excessive production of immunoglobulins in EAE, thus further emphasizing the role of B cells, and among them Mott cells, in the pathogenesis of this animal model of multiple sclerosis.

Correlation Between Proteolytic Activity and Abdominal Aortic Aneurysm Wall Morphology with Intraluminal Thrombus Volume.

BACKGROUND: The aim of this study was to examine the influence of intraluminal thrombus (ILT) volume on the level of proteolytic activity and the content of abdominal aortic aneurysm (AAA) wall. METHODS: The research was designed as a cross-sectional study at the Clinic for Vascular and Endovascular Surgery, Clinical Center of Serbia in the period from April 2017 to February 2018. During this period, a total of 155 patients with asymptomatic AAA underwent open surgical treatment and 50 were included in the study based on inclusion and exclusion criteria. Before surgery, patients included in the study were examined by MRI. During the operation, samples of ILT and AAA wall were taken for biochemical analysis. RESULTS: A statistically significant correlation was found between the volume of the ILT and largest AAA diameter (ρ = 0.56; P < 0.001). The correlation of the ILT volume on the anterior wall and the concentration of MMP-9, MMP-2 and NE/ELA in the wall did not find statistical significance. Also, no statistically significant association was found between the volume of ILT and the concentration of ECM proteins (collagen type 3, elastin, proteoglycan) in the corresponding part of the wall. The association of ILT volume with MDA was also of no statistical significance. There was a positive statistical significance found in correlation of volume of ILT and catalase activity in the wall of AAA (ρ = 0.28, P = 0.049). CONCLUSIONS: The volume of ILT in the aneurysmal sac seemed not to affect the level of proteolytic activity and the content of the aneurysm wall. However, a positive correlation was found between the ILT and the catalase activity. The effect of ILT on the aneurysm wall and its role in the progression of aneurysmal disease should be examined in future studies.

Transcriptional block of AMPK-induced autophagy promotes glutamate excitotoxicity in nutrient-deprived SH-SY5Y neuroblastoma cells.

We investigated the role of autophagy, a controlled lysosomal degradation of cellular macromolecules and organelles, in glutamate excitotoxicity during nutrient deprivation in vitro. The incubation in low-glucose serum/amino acid-free cell culture medium synergized with glutamate in increasing AMP/ATP ratio and causing excitotoxic necrosis in SH-SY5Y human neuroblastoma cells. Glutamate suppressed starvation-triggered autophagy, as confirmed by diminished intracellular acidification, lower LC3 punctuation and LC3-I conversion to autophagosome-associated LC3-II, reduced expression of proautophagic beclin-1 and ATG5, increase of the selective autophagic target NBR1, and decreased number of autophagic vesicles. Similar results were observed in PC12 rat pheochromocytoma cells. Both glutamate-mediated excitotoxicity and autophagy inhibition in starved SH-SY5Y cells were reverted by NMDA antagonist memantine and mimicked by NMDA agonists D-aspartate and ibotenate. Glutamate reduced starvation-triggered phosphorylation of the energy sensor AMP-activated protein kinase (AMPK) without affecting the activity of mammalian target of rapamycin complex 1, a major negative regulator of autophagy. This was associated with reduced mRNA levels of autophagy transcriptional activators (FOXO3, ATF4) and molecules involved in autophagy initiation (ULK1, ATG13, FIP200), autophagosome nucleation/elongation (ATG14, beclin-1, ATG5), and autophagic cargo delivery to autophagosomes (SQSTM1). Glutamate-mediated transcriptional repression of autophagy was alleviated by overexpression of constitutively active AMPK. Genetic or pharmacological AMPK activation by AMPK overexpression or metformin, as well as genetic or pharmacological autophagy induction by TFEB overexpression or lithium chloride, reduced the sensitivity of nutrient-deprived SH-SY5Y cells to glutamate excitotoxicity. These data indicate that transcriptional inhibition of AMPK-dependent cytoprotective autophagy is involved in glutamate-mediated excitotoxicity during nutrient deprivation in vitro.

Influence of preoperative statins and aspirin administration on biological and magnetic resonance imaging properties in patients with abdominal aortic aneurysm.

Background: Main objective of this study was to evaluate the influence of statins and/or acetylsalicylic acid on biochemical characteristics of abdominal aortic aneurysm (AAA) wall and intraluminal thrombus (ILT). Patients and methods: Fifty patients with asymptomatic infrarenal AAA were analyzed using magnetic resonance imaging on T1w sequence. Relative ILT signal intensity (SI) was determined as a ratio between ILT and psoas muscle SI. Samples containing the full ILT thickness and aneurysm wall were harvested from the anterior surface at the level of the maximal diameter. The concentration of enzymes such as matrix metalloproteinase (MMP) 9, MMP2 and neutrophil elastase (NE/ELA) were analyzed in ILT and AAA wall; while collagen type III, elastin and proteoglycan 4 were analyzed in harvested AAA wall. Oxidative stress in the AAA wall was assessed by catalase and malondialdehyde activity in tissue samples. Results: Relative ILT signal intensity (1.09 ± 0.41 vs 0.89 ± 0.21, p = 0.013) were higher in non-statin than in statin group. Patients who were taking aspirin had lower relative ILT area (0.89 ± 0.19 vs 1.13. ± 0.44, p = 0.016), and lower relative ILT signal intensity (0.85 [0.73-1.07] vs 1.01 [0.84-1.19], p = 0.021) compared to non-aspirin group. There were higher concentrations of elastin in AAA wall among patients taking both of aspirin and statins (1.21 [0.77-3.02] vs 0.78 (0.49-1.05) ng/ml, p = 0.044) than in patients who did not take both of these drugs. Conclusions: Relative ILT SI was lower in patients taking statin and aspirin. Combination of antiplatelet therapy and statins was associated with higher elastin concentrations in AAA wall.

Magnetic resonance imaging assessment of proteolytic enzyme concentrations and biologic properties of intraluminal thrombus in abdominal aortic aneurysms.

OBJECTIVE: The aim of the study was to determine whether magnetic resonance imaging (MRI) can be used in assessment of biologic activity of intraluminal thrombus (ILT) and proteolytic processes of the abdominal aortic aneurysm wall. METHODS: Using MRI, 50 patients with asymptomatic infrarenal abdominal aortic aneurysm were analyzed at the maximum aneurysm diameter on T1-weighted images in the arterial phase after administration of contrast material. Relative ILT signal intensity (SI) was determined as the ratio between ILT SI and psoas muscle SI. During surgery, the full thickness of the ILT and the adjacent part of the aneurysm wall were harvested at the maximal diameter for biochemical analysis. The concentrations of matrix metalloproteinase 9 and neutrophil elastase (NE/ELA) were analyzed in harvested thrombi, and the concentrations of collagen type III, elastin, and proteoglycans were analyzed in harvested aneurysm walls. RESULTS: A significant positive correlation was found between the NE/ELA concentration of the ILT and the relative SI (ρ = 0.309; P = .029). Furthermore, a negative correlation was observed between the elastin content of the aneurysm wall and the relative SI (ρ = -0.300; P = .034). No correlations were found between relative SI and concentration of matrix metalloproteinase 9, NE/ELA, collagen type III, or proteoglycan 4 in the aneurysm wall. CONCLUSIONS: These findings indicate a potential novel use of MRI in prediction of thrombus proteolytic enzyme concentrations and the extracellular matrix content of the aneurysm wall, thus providing additional information for the risk of potential aneurysm rupture.

Effects of Sideritis scardica Extract on Glucose Tolerance, Triglyceride Levels and Markers of Oxidative Stress in Ovariectomized Rats.

Menopause is characterized by deep metabolic disturbances, including decreased insulin sensitivity, adiposity, and changes in lipid profiles. Estrogen replacement therapy can partially reverse these changes, and while it is safe in most healthy postmenopausal women, there are still existing concerns regarding an increased risk for breast and endometrial cancer as well as a risk for cardiovascular and thromboembolic disease. Therefore, certain natural compounds with positive metabolic effects may be considered as a possible alternative or adjunctive treatment in patients not willing to take estrogens or patients with contraindications for estrogens. The aim of this study was to investigate the influence of Sideritis scardica (mountain tea) extract on metabolic disturbances induced by ovariectomy in rats. The study included 24 rats divided into three groups: ovariectomized rats treated with 200 mg/kg S. scardica extract for 24 weeks (n = 8), ovariectomized non-treated (n = 8), and Sham-operated (n = 8) rats. Food intake, weight gain, body composition, fasting glucose levels, response to oral glucose challenge, liver glycogen content, catalase activity, thiol groups, and malondialdehyde concentrations as well as AMP-activated protein kinase activity in liver cells were studied. Ovariectomized rats treated with S. scardica extract had lower blood triglycerides, reduced fasting glucose levels, as well lower glucose peaks after oral glucose challenge, increased liver glycogen content, and significantly higher catalase activity and thiol group concentration than non-treated ovariectomized rats. The ability of S. scardica extract to attenuate metabolic disturbances associated with ovariectomy was associated with the activation of AMP-activated protein kinase in liver cells.

Autophagy suppression sensitizes glioma cells to IMP dehydrogenase inhibition-induced apoptotic death.

We investigated the role of autophagy, a process of controlled self-digestion, in the in vitro anticancer action of the inosine monophosphate dehydrogenase (IMPDH) inhibitor ribavirin. Ribavirin-triggered oxidative stress, caspase activation, and apoptotic death in U251 human glioma cells were associated with the induction of autophagy, as confirmed by intracellular acidification, appearance of autophagic vesicles, conversion of microtubule associated protein 1 light chain 3 (LC3)-I to autophagosome-associated LC3-II, and degradation of autophagic target p62/sequestosome 1. Ribavirin downregulated the activity of autophagy-inhibiting mammalian target of rapamycin complex 1 (mTORC1), as indicated by a decrease in phosphorylation of the mTORC1 substrate ribosomal p70S6 kinase and reduction of the mTORC1-activating Src/Akt signaling. Guanosine supplementation inhibited, while IMPDH inhibitor tiazofurin mimicked ribavirin-mediated autophagy induction, suggesting the involvement of IMPDH blockade in the observed effect. Autophagy suppression by ammonium chloride, bafilomycin A1, or RNA interference-mediated knockdown of LC3 sensitized glioma cells to ribavirin-induced apoptosis. Ribavirin also induced cytoprotective autophagy associated with Akt/mTORC1 inhibition in C6 rat glioma cells. Our data demonstrate that ribavirin-triggered Akt/mTORC1-dependent autophagy counteracts apoptotic death of glioma cells, indicating autophagy suppression as a plausible therapeutic strategy for sensitization of cancer cells to IMPDH inhibition.

Development of resistance to antiglioma agents in rat C6 cells caused collateral sensitivity to doxorubicin.

Chemoresistance is a severe limitation to glioblastoma (GBM) therapy and there is a strong need to understand the underlying mechanisms that determine its response to different chemotherapeutics. Therefore, we induced resistance in C6 rat glioma cell line, which considerably resembles the characteristics of human GBM. The resistant phenotype was developed by 3-bis (2-chloroethyl)-1-nitrosourea (BCNU), one of the most commonly used therapeutic drug in the course of GBM treatment. After confirmation of the cross-resistance to cisplatin (CPt) and temozolomide (TMZ) in newly established RC6 cell line, we examined cell death induction and DNA damage by these drugs. Resistance to apoptosis and deficiency in forming DNA double-strand breaks was followed by significant decrease in the mRNA expression of pro-apoptotic and anti-apoptotic genes. The development of drug resistance was associated with significant increase in reactive oxygen species (ROS) and decrease in oxidized to reduced gluthatione ratio in RC6 cell line indicating a reduced level of oxidative stress. The mRNA expression levels of manganese superoxid dismutase (MnSOD), inducible nitric oxide synthase (iNOS) and gluthatione peroxidase (GPx) were increased while hypoxia-inducible factor 1-α (HIF-1α) was decreased in RC6 compared to C6 cells. This was in line with obtained changes in ROS content and increased antioxidative capacity of RC6 cells. Importantly, RC6 cells demonstrated collateral sensitivity to doxorubicin (DOX). The analysis of this phenomenon revealed increased accumulation of DOX in RC6 cells due to their adaptation to high ROS content and acidification of cytoplasm. In conclusion, newly established RC6 rat glioma cell line could be used as a starting material for the development of allogenic animal model and preclinical evaluation of new antiglioma agents. Collateral sensitivity to DOX obtained after BCNU treatment may prompt new studies aimed to find efficient delivery of DOX to the glioma site in brain.

Neuroprotective arylpiperazine dopaminergic/serotonergic ligands suppress experimental autoimmune encephalomyelitis in rats.

Arylpiperazine-based dopaminergic/serotonergic ligands exert neuroprotective activity. We examined the effect of arylpiperazine D2 /5-HT1A ligands, N-{4-[2-(4-phenyl-piperazin-1-yl)-ethyl}-phenyl]-picolinamide (6a) and N-{3-[2-(4-phenyl-piperazin-1-yl)-ethyl]-phenyl}-picolinamide (6b), in experimental autoimmune encephalomyelitis (EAE), a model of neuroinflammation. Both compounds (10 mg/kg i.p.) reduced EAE clinical signs in spinal cord homogenate-immunized Dark Agouti rats. Compound 6b was more efficient in delaying the disease onset and reducing the maximal clinical score, which correlated with its higher affinity for D2 and 5-HT1A receptors. The protection was retained if treatment was limited to the effector (from day 8 onwards), but not the induction phase (day 0-7) of EAE. Compound 6b reduced CNS immune infiltration and expression of mRNA encoding the proinflammatory cytokines tumor necrosis factor, IL-6, IL-1, and GM-CSF, TH 1 cytokine IFN-γ, TH 17 cytokine IL-17, as well as the signature transcription factors of TH 1 (T-bet) and TH 17 (RORγt) cells. Arylpiperazine treatment reduced apoptosis and increased the activation of anti-apoptotic mediators Akt and p70S6 kinase in the CNS of EAE animals. The in vitro treatment with 6b protected oligodendrocyte cell line OLN-93 and neuronal cell line PC12 from mitogen-activated normal T cells or myelin basic protein-activated encephalitogenic T cells. In conclusion, arylpiperazine dopaminergic/serotonergic ligands suppress EAE through a direct neuroprotective action and decrease in CNS inflammation. Arylpiperazine dopaminergic/serotonergic ligands reduce neurological symptoms of acute autoimmune encephalomyelitis in rats without affecting the activation of autoreactive immune response, through mechanisms involving a decrease in CNS immune infiltration, as well as direct protection of CNS from immune-mediated damage. These data indicate potential usefulness of arylpiperazine-based compounds in the treatment of neuroinflammatory disorders such as multiple sclerosis.

Antioxidative activity of diarylheptanoids from the bark of black alder (Alnus glutinosa) and their interaction with anticancer drugs.

Diarylheptanoids belong to polyphenols, a group of plant secondary metabolites with multiple biological properties. Many of them display antioxidative, cytotoxic, or anticancer actions and are increasingly recognized as potential therapeutic agents. The aim of this study was to evaluate antioxidant and cytoprotective activity of two diarylheptanoids: platyphylloside 5(S)-1,7-di(4-hydroxyphenyl)-3-heptanone-5-O-ß-D-glucopyranoside (1) and its newly discovered analog 5(S)-1,7-di(4-hydroxyphenyl)-5-O-ß-D-[6-(E-p-coumaroylglucopyranosyl)]heptane-3-one (2), both isolated from the bark of black alder (Alnus glutinosa). To that end, we have employed a cancer cell line (NCI-H460), normal human keratinocytes (HaCaT), and peripheral blood mononuclear cells. The effects on cell growth were assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay. Cell death was examined by annexin V/propidium iodide staining on a flow cytometer. Reactive oxygen species production was examined by dihydroethidium staining. Mitochondrial structure and doxorubicin localization were visualized by fluorescent microscopy. Gene expression of manganese superoxide dismutase and hypoxia-inducible factor-1α was determined by reverse transcription polymerase chain reaction. Diarylheptanoids antagonized the effects of either doxorubicin or cisplatin, significantly increasing their IC50 values in normal cells. Diarylheptanoid 1 induced the retention of doxorubicin in cytoplasm and reduced mitochondrial fragmentation associated with doxorubicin application. Diarylheptanoid 2 reduced the reactive oxygen species production induced by cisplatin. Both compounds increased the messenger ribonucleic acid expression of enzymes involved in reactive oxygen species elimination (manganese superoxide dismutase and hypoxia-inducible factor-1α). These results indicate that neutralization of reactive oxygen species is an important mechanism of diarylheptanoid action, although these compounds exert a considerable anticancer effect. Therefore, these compounds may serve as protectors of normal cells during chemotherapy without significantly diminishing the effect of the applied chemotherapeutic.

Isolation, characterization, and in vitro cytotoxicity of new sesquiterpenoids from Achillea clavennae.

Further phytochemical investigation of the aerial parts of Achillea clavennae has resulted in the isolation of three new sesquiterpene lactones: two highly oxygenated germacranolides (1, 2) and the iso-seco-guaianolide 9(R)-acetoxy-3-O-methyl-iso-seco-tanapartholide (3). Eight known compounds were also found, of which 9α-acetoxycanin (5), sintenin (6), and oleanolic acid (7) were detected for the first time. The structures of the isolated compounds were elucidated by combined spectroscopic methods (1D and 2D NMR, HRESIMS, CIMS, and FTIR). While the predominant metabolite germacranolide sintenin (6) was not cytotoxic, the new iso-seco-guaianolide (3) displayed cytotoxicity comparable to that of cisplatin and the lactone apressin (4), inducing partly apoptotic death in human U251 and rat C6 glioma cell lines.

Endoplasmic reticulum stress response in immune cells contributes to experimental autoimmune encephalomyelitis pathogenesis in rats.

We examined the role of endoplasmic reticulum (ER) stress and the ensuing unfolded protein response (UPR) in the development of the central nervous system (CNS)-directed immune response in the rat model of experimental autoimmune encephalomyelitis (EAE). The induction of EAE with syngeneic spinal cord homogenate in complete Freund's adjuvant (CFA) caused a time-dependent increase in the expression of ER stress/UPR markers glucose-regulated protein 78 (GRP78), X-box-binding protein 1 (XBP1), C/EBP homologous protein (CHOP), and phosphorylated eukaryotic initiation factor 2α (eIF2α) in the draining lymph nodes of both EAE-susceptible Dark Agouti (DA) and EAE-resistant Albino Oxford (AO) rats. However, the increase in ER stress markers was more pronounced in AO rats. CFA alone also induced ER stress, but the effect was weaker and less sustained compared to full immunization. The ultrastructural analysis of DA lymph node tissue by electron microscopy revealed ER dilatation in lymphocytes, macrophages, and plasma cells, while immunoblot analysis of CD3-sorted lymph node cells demonstrated the increase in ER stress/UPR markers in both CD3+ (T cell) and CD3- (non-T) cell compartments. A positive correlation was observed between the levels of ER stress/UPR markers in the CNS-infiltrated mononuclear cells and the clinical activity of the disease. Finally, the reduction of EAE clinical signs by ER stress inhibitor ursodeoxycholic acid was associated with the decrease in the expression of mRNA encoding pro-inflammatory cytokines TNF and IL-1ß, and encephalitogenic T cell cytokines IFN-γ and IL-17. Collectively, our data indicate that ER stress response in immune cells might be an important pathogenetic factor and a valid therapeutic target in the inflammatory damage of the CNS.

The mechanisms of in vitro cytotoxicity of mountain tea, Sideritis scardica, against the C6 glioma cell line.

Sideritis scardica (mountain tea) is an endemic plant on the Balkan Peninsula traditionally used for treating different conditions, mainly of inflammatory nature. This study was aimed to examine the cytotoxic activity of different S. scardica extracts against the rat glioma C6 line and rat astrocytes in primary culture. The obtained data revealed that diethyl ether (extract 2) and ethyl acetate (extract 3) extracts of S. scardica exerted a cytotoxic effect on C6 rat glioma cells. Diethyl ether extract induced an increase in reactive oxygen species production, leading to apoptotic and autophagic cell death. Ethyl acetate extract induced G2 M cell cycle arrest and autophagy. None of the tested extracts was cytotoxic to rat astrocytes in primary culture. Cytotoxic effects of S. scardica extracts were, at least in part, mediated by their flavonoid constituents apigenin and luteolin that, when applied alone, induced cell cycle arrest, apoptosis, and autophagy.

P53 and survivin expression in renal cell carcinoma.

Objective: Mutation of p53 is detected in more than 50% of human cancers, expression of p53 has a potential prognostic value in patients with renal cell carcinoma (RCC). Survivin is a member of the inhibitor of apoptosis protein family, its overexpression is observed in many malignancies, including RCC. The aim of the study was to estimate a correlation between survivin and p53 expression in tumor samples and the histologic type of a tumor, tumor stage, tumor grade, and survival of patients. Materials and Methods: Tumor samples were collected from surgical specimens of 90 patients who underwent radical or partial nephrectomy for RCC between November 2017 and July 2020. Tumors were staged according to the UICC (The Union for International Cancer Control) TNM classification system and histopathologically graded according to Fuhrman nuclear grade system. Histopathological diagnosis was confirmed with standard light microscopic evaluation, using hematoxylin and eosin staining and standard p53 and survivin antibodies. Results: Positive p53 staining was observed in 36.7% of tumor specimens and 24.4% were survivin positive. There was a statistically significant correlation between p53 or survivin expression and histologic subtype of clear cell RCC as well as Type I and II of papillary RCC. There was a statistically significant correlation between p53 expression and tumor size, stage, and grade. The p53 or survivin expression was related to lower overall survival. Conclusion: The results of this study suggest that p53 overexpression and survivin positivity in RCC patients could be associated with poor prognosis. Thus, these proteins could be used as prognostic markers in RCC.

Proapoptotic and proautophagy effect of H1-receptor antagonist desloratadine in human glioblastoma cell lines.

Glioblastomas are aggressive and usually incurable high-grade gliomas without adequate treatment. In this study, we aimed to investigate the potential of desloratadine to induce apoptosis/autophagy as genetically regulated processes that can seal cancer cell fates. All experiments were performed on U251 human glioblastoma cell line and primary human glioblastoma cell culture. Cytotoxic effect of desloratadine was investigated using MTT and CV assays, while oxidative stress, apoptosis, and autophagy were detected by flow cytometry and immunoblot. Desloratadine treatment decreased cell viability of U251 human glioblastoma cell line and primary human glioblastoma cell culture (IC50 value 50 µM) by an increase of intracellular reactive oxygen species and caspase activity. Also, desloratadine decreased the expression of main autophagy repressor mTOR and its upstream activator Akt and increased the expression of AMPK. Desloratadine exerted dual cytotoxic effect inducing both apoptosis- and mTOR/AMPK-dependent cytotoxic autophagy in glioblastoma cells and primary glioblastoma cell culture.

Cyclohexyl analogues of ethylenediamine dipropanoic acid induce caspase-independent mitochondrial apoptosis in human leukemic cells.

We investigated the cytotoxicity of recently synthesized (S,S)-ethylendiamine-N,N'-di-2-(3-cyclohexyl)propanoic acid esters toward human leukemic cell lines and healthy blood mononuclear cells. Cell viability was assessed by acid phosphatase assay, apoptosis, and differentiation were analyzed by flow cytometry and electron microscopy, while intracellular localization of apoptosis-inducing factor (AIF) was determined by immunoblotting. It was demonstrated that methyl, ethyl, and n-propyl esters were toxic to HL-60, REH, MOLT-4, KG-1, JVM-2, and K-562 leukemic cell lines, while the nonesterified parental compound and n-butyl ester were devoid of cytotoxic action. The ethyl ester exhibited the highest cytotoxic activity (IC50 10.7 µM-45.4 µM), which was comparable to that of the prototypical anticancer drug cisplatin. The observed cytotoxic effect in HL-60 cells was associated with an increase in superoxide production and mitochondrial membrane depolarization, leading to apoptotic cell death characterized by phosphatidylserine externalization and DNA fragmentation in the absence of autophagic response. DNA fragmentation preceded caspase activation and followed AIF translocation from mitochondria to nucleus, which was indicative of caspase-independent apoptotic cell death. HL-60 cells treated with subtoxic concentration of the compound displayed morphological signs of granulocytic differentiation (nuclear indentations and presence of cytoplasmic primary granules), as well as an increased expression of differentiation markers CD11b and CD15. The cyclohexyl analogues of ethylenediamine dipropanoic acid were also toxic to peripheral blood mononuclear cells of both healthy controls and leukemic patients, the latter being more sensitive. Our data demonstrate that the toxicity of the investigated cyclohexyl compounds against leukemic cell lines is mediated by caspase-independent apoptosis associated with oxidative stress, mitochondrial dysfunction, and AIF translocation.

Anti-inflammatory, gastroprotective, and cytotoxic effects of Sideritis scardica extracts.

Sideritis scardica Griseb. (ironwort, mountain tea), an endemic plant of the Balkan Peninsula, has been used in traditional medicine in the treatment of gastrointestinal complaints, inflammation, and rheumatic disorders. This study aimed to evaluate its gastroprotective and anti-inflammatory activities. Besides, continuously increasing interest in assessing the role of the plant active constituents preventing the risk of cancer was a reason to make a detailed examination of the investigated ethanol, diethyl ether, ethyl acetate, and N-butanol extracts regarding cytotoxicity. Oral administration of the investigated extracts caused a dose-dependent anti-inflammatory effect in a model of carrageenan-induced rat paw edema. Gastroprotective activity of the extracts was investigated using an ethanol-induced acute stress ulcer in rats. The cytotoxic activity of plant extracts was assessed on PBMC, B16, and HL-60 cells and compared to the cytotoxicity of phenolic compounds identified in extracts. Apoptotic and necrotic cell death were analyzed by double staining with fluoresceinisothiocyanate (FITC)-conjugated annexin V and PI. The developed HPLC method enabled qualitative fingerprint analysis of phenolic compounds in the investigated extracts. Compared to the effect of the positive control, the anti-inflammatory drug indomethacine (4 mg/kg), which produced a 50 % decrease in inflammation, diethyl ether and N-butanol extracts exhibited about the same effect in doses of 200 and 100 mg/kg (53.6 and 48.7 %; 48.4 and 49.9 %, respectively). All investigated extracts produced dose-dependent gastroprotective activity with the efficacy comparable to that of the reference drug ranitidine. The diethyl ether extract showed significant dose-dependent cytotoxicity on B16 cells and HL-60 cells, decreasing cell growth to 51.3 % and 77.5 % of control, respectively, when used at 100 µg/mL. It seems that phenolic compounds (apigenin, luteolin, and their corresponding glycosides) are responsible for the diethyl ether extract cytotoxic effect. It also appears that induction of oxidative stress might be involved in its cytotoxicity, since B16 and HL-60 cells increased their ROS production in response to treatment with diethyl ether extract. Neither of the tested extracts nor any phenolic compounds showed significant cytotoxic effect to human PBMC. These results demonstrated the potent anti-inflammatory and gastroprotective activities, as well as the promising cytotoxicity.

Hallmarks of tumor-associated microglia response to experimental U87 human glioblastoma xenograft.

Glioblastoma (GBM) is one of the deadliest primary brain neoplasm, heavily infiltrated with tumor-associated microglia/macrophages (TAM), which has received a great deal of interest. Bearing in mind that the number of peripheral macrophages by the 14th day is negligible, in our study TAM were referred to as microglia. Here we evaluated histopathological characterization of TAM and kinetics of their infiltration in U87 orthotopic GBM, a commonly used model in preclinical research. To mimic different stages of GBM growth, we evaluated three-time points. Our data showed that the highest areal density of TAM was 7 days after GBM inoculation, with ability to proliferate early after initiation of GBM growth. The areal density of TAM within the tumor correlated with GBM growth and proliferation. Moreover, microglia underwent substantial morphological changes upon exposure to GBM cells. A transition from ramified morphology in peritumoral area to ameboid shape with larger soma and shortened, thick branches in the tumor core was observed. Higher areal fraction of blood vessels also correlated with the areal density of TAM. Given these pro-invasive features of microglia, this GBM model represents a good basis for further testing microglia as a target and new strategy to fight with.

Study of the venom proteome of Vipera ammodytes ammodytes (Linnaeus, 1758): A qualitative overview, biochemical and biological profiling.

Vipera ammodytes (Va), is the European venomous snake of the greatest medical importance. We analyzed whole venom proteome of the subspecies V. ammodytes ammodytes (Vaa) from Serbia for the first time using the shotgun proteomics approach and identified 99 proteins belonging to four enzymatic families: serine protease (SVSPs), L-amino acid oxidase (LAAOs), metalloproteinases (SVMPs), group II phospholipase (PLA2s), and five nonenzymatic families: cysteine-rich secretory proteins (CRISPs), C-type lectins (snaclecs), growth factors -nerve (NGFs) and vascular endothelium (VEGFs), and Kunitz-type protease inhibitors (SPIs). Considerable enzymatic activity of LAAO, SVSPs, and SVMPs and a high acidic PLA2 activity was measured implying potential of Vaa to produce haemotoxic, myotoxic, neuro and cardiotoxic effects. Moreover, significant antimicrobial activity of Vaa venom against Gram-negative (Klebsiella pneumoniae, Pseudomonas aeruginosa) and Gram-positive bacteria (Staphylococcus aureus) was found. The crude venom shows considerable potential cytotoxic activity on the C6 and HL60 and a moderate level of potency on B16 cell lines. HeLa cells showed the same sensitivity, while DU 145 and PC-3 are less sensitive than as normal cell line. Our data demonstrated a high complexity of Vaa and considerable enzymatic, antibacterial and cytotoxic activity, implying a great medical potential of Vaa venom as a promising source for new antibacterial and cytostatic agents.

Expression and functional activity of nucleoside transporters in human choroid plexus.

BACKGROUND: Human equilibrative nucleoside transporters (hENTs) 1-3 and human concentrative nucleoside transporters (hCNTs) 1-3 in the human choroid plexus (hCP) play a role in the homeostasis of adenosine and other naturally occurring nucleosides in the brain; in addition, hENT1, hENT2 and hCNT3 mediate membrane transport of nucleoside reverse transcriptase inhibitors that could be used to treat HIV infection, 3'-azido-3'-deoxythymidine, 2'3'-dideoxycytidine and 2'3'-dideoxyinosine. This study aimed to explore the expression levels and functional activities of hENTs 1-3 and hCNTs 1-3 in human choroid plexus. METHODS: Freshly-isolated pieces of lateral ventricle hCP, removed for various clinical reasons during neurosurgery, were obtained under Local Ethics Committee approval. Quantification of mRNAs that encoded hENTs and hCNTs was performed by the hydrolysis probes-based reverse transcription real time-polymerase chain reaction (RT-qPCR); for each gene of interest and for 18 S ribosomal RNA, which was an endogenous control, the efficiency of PCR reaction (E) and the quantification cycle (Cq) were calculated. The uptake of [(3)H]inosine by the choroid plexus pieces was investigated to explore the functional activity of hENTs and hCNTs in the hCP. RESULTS: RT-qPCR revealed that the mRNA encoding the intracellularly located transporter hENT3 was the most abundant, with E(-Cq )value being only about 40 fold less that the E(-Cq )value for 18 S ribosomal RNA; mRNAs encoding hENT1, hENT2 and hCNT3 were much less abundant than mRNA for the hENT3, while mRNAs encoding hCNT1 and hCNT2 were of very low abundance and not detectable. Uptake of [(3)H]inosine by the CP samples was linear and consisted of an Na(+)-dependent component, which was probably mediated by hCNT3, and Na(+)-independent component, mediated by hENTs. The latter component was not sensitive to inhibition by S-(4-nitrobenzyl)-6-thioinosine (NBMPR), when used at a concentration of 0.5 muM, a finding that excluded the involvement of hENT1, but it was very substantially inhibited by 10 muM NBMPR, a finding that suggested the involvement of hENT2 in uptake. CONCLUSION: Transcripts for hENT1-3 and hCNT3 were detected in human CP; mRNA for hENT3, an intracellularly located nucleoside transporter, was the most abundant. Human CP took up radiolabelled inosine by both concentrative and equilibrative processes. Concentrative uptake was probably mediated by hCNT3; the equilibrative uptake was mediated only by hENT2. The hENT1 transport activity was absent, which could suggest either that this protein was absent in the CP cells or that it was confined to the basolateral side of the CP epithelium.