Oxidative stress and activation of H2A.X in lung alveolar epithelial cells (A549) by nanoparticulate carbon black.

Fine airborne particulate matter enter the respiratory system, induce oxidative stress and initiate DNA damage. The aim of our study was the estimation of cell viability, oxidative stress, DNA damage, cell cycle alterations and activation of histone H2A.X. Experiments were done on lung alveolar epithelial (A549) cells grown for 24 h with 200 µg mL-1 coarse carbon black (CB), or nanoparticulate CB (NPCB). Neither CB nor glutathione depletion altered cell viability, growth rates, and H2A.X expression while NPCB decreased cell viability, increased oxidative stress and DNA damage. The cell cycle was blocked at G0/G1. NPCB but not CB increased expression and activation of H2A.X at mRNA and protein levels. Co-expression data point to γH2A.X as a major NPCB target, and show the interdependence of γH2A.X and oxidative stress. We conclude, that NPCB increases γ-H2A.X expression in A549 cells at mRNA and protein levels and stimulates H2A.X (Ser139), phosphorylation, associated with oxidative stress, the DNA damage response and G1 cell cycle arrest.

Co-expression of Hsp70 Protein and Autophagy Marker Protein LC3 in A549 Cells and THP1 Cells Exposed to Nanoparticles of Air Pollution.

The ability of air particulate matter (PM) to cause reactive oxygen species-driven protein damage is associated with both COPD and lung cancer, but the mechanisms are unsettled. In this study, we investigated the co-expression of Hsp70 and the autophagy marker protein LC3 in A549 cells (alveolar epithelial cell line) and THP-1 cells (monocyte/macrophage cells) grown in media supplemented with 100 µg/mL of four types of PM: carbon black (CB), urban dust (UD), nanoparticulate CB (NPCB), and nanoparticulate CB coated with benzo(a)pyrene (NPCB-BaP). Fluorescent monoclonal antibodies and flow cytometry were used to assess the expression and co-expression of HSP70 and LC3 proteins. Hsp70 expression was significantly increased by all PM, while LC3 was decreased by CB in A549 cells, unchanged by CB and UD in THP-1 cells and increased by NPCB and NPCB-BaP in both cell types. All PMs increased the Hsp70/LC3 ratio in binary scatterplots; the relationship was positive and linear, which may reflect chaperone-dependent autophagy. The UD was the only PM type that affected the slopes of the spatial trend lines and altered binary patterns of Hsp70/LC3 distribution in THP1 cells. These findings provide an insight into the molecular mechanisms regulating proteostasis in PM-exposed cells through the chaperone-autophagy system in the cytoplasm.

Cytotoxicity, Oxidative Stress, and Autophagy in Human Alveolar Epithelial Cell Line (A549 Cells) Exposed to Standardized Urban Dust.

Exposure to urban airborne particulate matter (PM) associates with adverse health effects, but the exact mechanisms remain unclear. In this study, we focused on cytotoxicity (MTT), oxidative stress (DCF/FC), DNA damage (PI/FC), necrosis/apoptosis (FC), and autophagy (LC3 expression; WB/FC) triggered by urban dust (UD) in naïve human alveolar epithelial A549 cells and in the cells with reduced glutathione (GSH). The A549 cells were grown in F12K/FCS media supplemented with coarse carbon black (CB; Huber990; 260 nm diameter; 200 µg·ml-1) or urban dust (UD; Standard Reference Materials; 200 µg·ml-1) for 24 h. To deplete intracellular glutathione (GSH), l-buthionine-(S,R)-sulfoximine (BSO; 100 mM; 24 h) was used. Pre-treatment with BSO depleted the cellular GSH by about 30%. A similar effect was noticed after UD. The CB was without any effects on the parameters tested, except for LC3 expression (autophagy) which increased by about twofold. However, UD decreased cell viability by about 27%, decreased cell proliferation in BSO pre-treated cells, increased ROS production, and increased both Hsp70 and LC3 proteins by about twofold, but most changes were unrelated to ROS-mediated GSH depletion. We conclude that urban dust-induced oxidative stress is important in PM toxicity, but other as yet unrecognized mechanisms are also involved.

Cigarette Smoke-Induced Oxidative Stress and Autophagy in Human Alveolar Epithelial Cell Line (A549 Cells).

Chronic exposure to cigarette smoke (CS) causes structural and functional changes in the respiratory tract. It is a major risk factor for cardiovascular and systemic pulmonary diseases. The aim of this study was to investigate the effect of acute CS exposure (2 h) on oxidative stress, heat shock protein 70 (HSP70) expression, autophagy (LC3 expression), and oxidative stress (DCF fluorescence) in human alveolar epithelial cell line A549. Cell culture medium was conditioned with CS using commercial cigarettes, and A549 cells were grown in modified media for 2 h. In some experiments, A549 cells were pretreated with 100 µM of L-buthionine-sulfoximine (BSO) for 24 h to induce glutathione (GSH) depletion. In the cells grown in CS-conditioned medium, GSH was depleted by more than 30%, and reactive oxygen species were increased. Moreover, there was a considerable overexpression of HSP70 and a substantial accumulation of LC3. Similar changes were found when the cells were pretreated with BSO. We conclude that the short-term exposure of epithelial cells to CS increases oxidative stress that entails enhanced autophagy activity.

Sialidase Attenuates Epidermal Growth Factor Response and Abolishes Antiproliferative Effects of Erlotinib in A549Alveolar Epithelial Cells.

Erlotinib is a widely used, reversible tyrosine kinase inhibitor (TKI), targeting pro-proliferative signaling of epidermal growth factor receptor (EGFR). The drug is approved for the first-line treatment of patients with metastatic non-small cell lung cancer with EGFR mutations. Extracellular glycans can affect EGFR expression, dimerization, phosphorylation, and EGF binding. In this study we investigated the effects of EGF and erlotinib on the cell cycle of naive and sialidase (alpha-neuraminidase)-pretreated human A549 alveolar epithelial cells. A549 cells were labeled with propidium iodide, and fractions of cells in different phases of cycle were quantified by flow cytometry. We found that neither did desialilation nor EGF, as well as erlotinib treatment, increase the number of damaged cells (subG0/G1 cell fraction), while erlotinib did significantly increase the number of G0/G1 cells and decrease S + G2/M cell fractions. In naive cells, EGF increased proliferating cell numbers by more than 40%, and this effect was blocked by erlotinib. In desialylated cells, however, proliferation was significantly decreased by about 29%, and EGF and erlotinib did not exert significant effects. We conclude that changes in alveolar epithelial cell membrane glycosylation may affect function of growth-promoting receptors and erlotinib effectiveness.

Phagocytosis and Autophagy in THP-1 Cells Exposed to Urban Dust: Possible Role of LC3-Associated Phagocytosis and Canonical Autophagy.

Exposure to ambient particulate matter (PM) increases mortality and morbidity due to respiratory and cardiovascular diseases. The aim of this study was to assess the effect of standardized urban dust (UD) on phagocytosis and autophagy in a monocyte-macrophage cell line (THP-1 cells). The cells were grown for 24 h in the medium supplemented with 200 µg·mL-1 coarse carbon black (CB) or UD. In some experiments glutathione (GSH) was depleted in THP-1 cells by buthionine sulfoximine. The cells were double stained with green latex beads (phagocytosis) and with red autophagy marker (LC3) and were evaluated in a flow cytometer. In naïve THP-1 cells, about 61% of them were classified as "negative", while 39% were classified as "double-positive". Both GSH depletion and UD treatment produced three distinct subpopulations of cells on bivariate scatterplots. A new subpopulation of cells (about 24% of the total number) appeared, with a lower autophagy and phagocytosis, but with a higher autophagy/phagocytosis ratio, when compared to highly positive cells. CB affected, to some extent, phagocytosis without a substantial effect on autophagy. In conclusion, the research on distinct pathways of immune cell activation may be relevant to the diagnostics and therapy of PM-induced pneumotoxicity, inflammation, and tumorigenesis.

MicroRNA-9 and Cell Proliferation in Lipopolysaccharide and Dexamethasone-Treated Naïve and Desialylated A549 Cells Grown in Cigarette Smoke Conditioned Medium.

In this study we assessed microRNA-9 (miR-9) levels (RT-PCR) and cell proliferation (flow cytometry) in naïve and desialylated human alveolar epithelial cells (A549 cells), grown for 24 h in cigarette smoke-conditioned medium. Cells were additionally treated with lipopolysaccharide (LPS) and/or dexamethasone. Proliferation positively correlated with miR-9 levels in both naïve and desialylated cells. Cigarette smoke decreased miR-9 levels in both cell types by about three-fold but there was no significant correlation between both parameters. Dexamethasone was without substantial effect on cigarette smoke-induced changes in proliferation of naïve cells, but some normalization was observed in desialylated cells. Dexamethasone increased miR-9 levels in both cell types grown in cigarette smoke-medium but the effect was stronger in desialylated cells. LPS increased cell proliferation and miR-9 by more than six-fold only in naïve cells, while correlation coefficient for both parameters in cigarette smoke-LPS group was 0.41. Herein we identify miR-9 as the cigarette smoke (decrease) and LPS-responsive but dexamethasone-unresponsive microRNA. It is possible that increased miR-9 levels in naïve A549 cells treated with LPS may be related to the activation of Toll-like receptor 4. Moreover, differences in cell response (both miR-9 and proliferation) to dexamethasone in naïve and desialylated cells may point to non-genomic dexamethasone effects.

Intracellular and Extracellular Cytokines in A549 Cells and THP1 Cells Exposed to Cigarette Smoke.

Cigarette smoke (CS) activates inflammatory cells and increases cytokine levels producing local and systemic inflammation. To assess changes in intracellular and extracellular cytokine levels we used human epithelial (A549 cells) and monocyte (THP-1) cell lines grown for 24 h in cigarette smoke-conditioned media. Cytokines were assessed using immunostaining/flow cytometry and ELISA assay. In THP1cells, grown in CS-conditioned media, the intracellular interleukins IL-1ß, IL-6, and IL-10 increased by more than tenfold, while less significant increases were found in A549 cells. IL-1α and IL-1ß, but not IL-6 or IL-10, were increased in the culture media, while IL-2 was raised by about fivefold only in the culture medium of A549 cells. IL-4, IL-6, IL-8, IL-10, IL-12, and tumor necrosis factor alpha were undetectable, while only a slight increase was observed in extracellular IL-17A (by about 60 %) in the medium of A549 cells and by about 115 % in the medium of THP1 cells. The interferon gamma (IFNγ) was increased by about eightfold, but only in the medium of THP1 cells grown with CS. We conclude that IL-1 and INFγ are the key cytokines responsible for pro-inflammatory signaling in epithelial cells and monocytes, respectively, exposed to cigarette smoke.

Costs of Treatment of Chronic Obstructive Pulmonary Disease.

The aim of this study was to analyze direct costs of COPD therapy in relation with clinical course and stage of the disease. Sixty patients with moderate to severe COPD were included into the study. The average cost was taken from institutional data file and was also assessed from a social perspective. Results were presented as average costs per patient per year. Forty two percent of patients was classified as GOLD D category, while categories A, B, and C accounted for 8 %, 27 %, and 23 %, respectively. Approximately 65 % of patients had 2-3 degrees of dyspnea according to the Modified Medical Research Council Dyspnea Scale. About 60 % of patients underwent two or three exacerbations per year and those patients had one or two co-morbidities diagnosed. Treatment costs almost doubled with disease progression, mainly due to exacerbations. In patients in Group C and Group D with exacerbations the direct costs were several times higher than in group A or B and the difference increased with progression of the disease. In Groups A and B, the costs of treatment of stable disease or with exacerbation were comparable. We conclude that costs of treatment of COPD patients were highest in advanced disease and were strongly related to COPD exacerbations.

Crosstalk Between Co-cultured A549 Cells and THP1 Cells Exposed to Cigarette Smoke.

Cigarette smoke (CS) is considered as a major etiological factor in the pathogenesis of chronic obstructive pulmonary disease. In this study we used A549 cells and THP-1 cells grown for 24 h in monoculture or in co-culture in CS-conditioned media and changes in their proliferation, viability, acetylated histone H3 levels and expression of extracellular antigens CD14, HLA-DR, CD11a, and CD11b were assessed. CS was highly toxic to A549 cells but not to THP1 cells. In A549 cells, oxidative stress reached the highest values after 1 h of CS exposure and then decreased. In THP1 cells oxidative stress was lower and increased progressively with time. CS decreased proliferation of A549 and THP1 cells by about 80% and 21%, respectively. CS did not alter acetylated histone H3 levels in A549 cells, while in THP1 cells the levels were reduced by about 35%. CS significantly increased expression of CD14, HLA-DR, CD11a, and CD11b in THP1 cells. In co-culture, naïve or CS-pretreated THP1 cells significantly protected A549 cells against CS toxicity but had higher death rates. These results show that epithelial cells are more fragile to CS than monocytes and that CS-activated monocytes may protect epithelial cells against CS-induced cytotoxicity.

Optimization and kinetic characterization of recombinant 1,3-ß-glucanase production in Escherichia coli K-12 strain BL21/pETSD10 - a bioreactor scale study.

UNLABELLED: The Escherichia coli K-12 strain BL21/pETSD10 was used to produce recombinant endocellular 1,3-ß-glucanase. This enzyme is responsible for the hydrolysis of the glycosidic bond in specific polysaccharides with tracts of unsubstituted ß-1,3-linked glucosyl residues. Conditions for the overproduction were experimentally examined, and the optimal values of the process on a bioreactor scale were found by interpolation of the experimental data. Cell induction was preferred during log-phase with relatively high cell density at OD600 near 1·1 with 0·074 g l(-1) of Isopropyl ß-D-1-thiogalactopyranoside (IPTG). The higher concentration of IPTG favors high enzyme production but with an excess of ballast protein. 1,3-ß-glucanase production was favoured with moderate culture aeration (0·7-0·9 vvm) and moderate stirring (125-150 rev min(-1) ). The highest specific glucanase activity (252 U g(-1) ) was found during validated experiments carried out at aeration at 135 rev min(-1) and stirring at 0·8 vvm. Due to high-tonnage industrial applications (i.e. to hemicellulose hydrolysis), the enzymatic preparation did not need to be highly purified. After pretreatment (precipitation with ammonium sulphate and dialysis) of the crude preparation, the enzymatic protein was one of the three main proteins in the preparation. The reaction rate with respect to the substrate (CM-curdlan) was described by the first order reaction equation (k = 1·95 l h(-1 ) g(-1) ). Products formed in the reaction are composed of nine glucose units on average. In the reaction conditions, the preparation showed very good stability (t1/2 = 202 h). SIGNIFICANCE AND IMPACT OF THE STUDY: The results contribute to the knowledge of cultivation parameters of E. coli K-12 strain BL21/pETSD10 on a bioreactor scale to overproduce an enzyme degrading ß-1,3-glucans. The optimal values of protein concentration, specific activity and total glucanase activity as a function of aeration and stirring were evaluated by numerical analysis. The obtained values were validated as positive. The protein degrades some bonds in hemicellulose. Thus, the protein could be applied as one of the degrading components for hemicellulose.

Tregs and HLA-DR expression in sputum cells of COPD patients treated with tiotropium and formoterol.

Immune cells expressing the activation markers HLA-DR and regulatory T cells (Tregs) may be involved in the regulation of chronic inflammation in chronic obstructive pulmonary disease (COPD). In this study we analyzed native and activated cell profiles in sputum of 22 stable COPD patients receiving formoterol (F) or formoterol + tiotropium (F + T) for 3 months. Cells were isolated from induced sputum and were examined on Coulter flow cytometer using fluorescent antibodies specific for CD3, CD4, CD8, CD14, CD19, CD25, CD127, and HLA-DR antigens. Cell profiles and cell activation were assessed by analysis of HLA-DR, CD25, and CD127 co-expression in double-stained samples. Tregs were defined as CD4⁺CD25(high) CD127(low) cells. We found that the combined therapy significantly decreased the CD8⁺ cell number (p < 0.01). At baseline, HLA-DR was expressed in about 10 % of sputum T or B cells and a higher expression was found on monocytes. The HLA-DR expression on lymphocytes, but not monocytes, was significantly lower (p < 0.01) in patients treated with F + T. Fractions of activated [CD4⁺ CD25⁺] cells were also significantly lower in the combined therapy group, except for the subpopulation of CD4⁺CD25(high) CD127(low) cells which was not altered. We conclude that tiotropium in add-on therapy to formoterol affects Treg cell profiles and decreases HLA-DR expression in airway lymphocytes.

Inhaled corticosteroids increase siglec-5/14 expression in sputum cells of COPD patients.

Recent studies show that several Siglec receptors, such as Siglec-8 and Siglec-14, may be important therapeutic targets in asthma and COPD. Siglecs are a family of lectins belonging to the immunoglobulin superfamily and recognize sialic acid residues of glycoproteins. Most of Siglecs have intracellular immunoreceptor tyrosine-based inhibitory motifs (ITIM), implicating them in the suppression of immunoreceptor signaling. Siglec-5/14 may be involved in the negative regulation of innate immune responses. The aim of this study was to analyze Siglec-5/14 expression in induced sputum cells of COPD patients in the following treatment combinations: (1) a long-acting beta2-agonist, formoterol; (2) formoterol combined with a long-acting antimuscarinic agent, tiotropium; and (3) formoterol combined with an inhaled corticosteroid or formoterol combined with tiotropium and with an inhaled corticosteroid. Siglec expression was assessed in sputum cells by flow cytometry using a specific monoclonal antibody. Double staining of cells indicated that Siglec-5/14 is expressed in monocyte/macrophages and neutrophils, but not in lymphocytes. Siglec-5/14 expression was significantly higher in patients receiving combined therapy including inhaled corticosteroids compared with patients taking only formoterol or formoterol + tiotropium. Our results suggest that inhaled corticosteroids may exert beneficial or negative effects, depending on the patients' phenotype, through increased immunosuppressive Siglec-5 or immunoactivatory Siglec-14 receptors, respectively.

Histone acetylation and arachidonic acid cytotoxicity in HepG2 cells overexpressing CYP2E1.

The aim of this work was to assess the role of ethanol-derived acetate and acetate-mediated histone acetylation in arachidonic acid-induced stress in HepG2 cells and cells overexpressing CYP2E1. Cells were grown for 7 days with 1 mM sodium acetate or 100 mM ethanol; their acetylated histone proteins and histone deacetylase 2 expression was quantified using Western blot. Ethanol- or acetate-pretreated cells were also treated for 24 h with 60 µM arachidonic acid to induce oxidative stress. Cytotoxicity was estimated by lactate dehydrogenase release, 3-[4,5-dimethylthiazolyl-2] 2,5-diphenyltetrazolium bromide test, and by DNA damage, while oxidative stress was quantified using dichlorofluorescein diacetate. Cells grown with ethanol or acetate had increased acetylated histone H3 levels in both cell types and elevated acetylated histone H4 levels in cells overexpressing CYP2E1 but not in naïve cells. In cells overexpressing CYP2E1 grown with ethanol, expression of histone deacetylase 2 was reduced by about 40 %. Arachidonic acid altered cell proliferation and was cytotoxic mostly to cells engineered to overexpress CYP2E1 but both effects were significantly lower in cells pretreated with ethanol or acetate. Cytotoxicity was also significantly decreased by 4-methylpyrazole--a CYP2E1 inhibitor and by trichostatin--an inhibitor of histone deacetylases. In cells pretreated with acetate or ethanol, the oxidative stress induced by arachidonic acid was also significantly lower. Our data indicate that histone hyperacetylation may in some extent protect the cells against oxidative stress. It is possible that acetate may act as an antioxidant at histone level. This mechanism may be relevant to alcohol-induced liver injury.

Altered histone deacetylase activity and iNOS expression in cells isolated from induced sputum of COPD patients treated with tiotropium.

Chronic obstructive pulmonary disease (COPD) is the only major disease with increasing death rate. In COPD, progressive reduction in quality of life is closely related to the increasing limitation of airflow due to chronic bronchitis, cell hyperplasia, fibrosis, and irreversible lung damage. Signaling pathways involved in inflammatory processes in COPD and inflammatory response to therapy are unknown. Our aim was to isolate cells from induced sputum of COPD patients treated with formoterol or formoterol + tiotropium and assess enzymatic activity of histone deacetylases (HDACs) acetylated histone 4 (AcH4) and expression of inducible nitric oxide synthase (iNOS). HDACs are important in signal transduction and inflammation. iNOS is generating nitric oxide (NO) relevant to blood pressure regulation, inflammation and infections. Thirty stable COPD patients (21 males and 9 females, mean age 67 years) receiving 12 µg b.i.d. formoterol were assayed before and after 3 months add-on therapy consisting of 18 µg q.i.d. tiotropium. In all patients, spirometry, lung volumes, and DLCO were performed before and after tiotropium therapy and all patients were subjected to sputum induction. Sputum cells were isolated and processed to obtain cytosolic and nuclear fractions. HDAC activity was measured in nuclear fraction using colorimetric assay. Expression AcH4 and iNOS was quantified using Western blot. In patients receiving both drugs, FEV1 and lung volumes significantly improved compared with formoterol-only treated patients. Mean HDAC activity was slightly decreased (P < 0.05), while AcH4 levels and iNOS expression were significantly elevated in tiotropium-treated patients (increase by about 65 %; P < 0.01 and 77 %; P < 0.01 respectively). Our data show that beneficial effects of tiotropium in add-on therapy to formoterol may be related to altered histone signaling and increased iNOS expression.

Siglec-8 in induced sputum of COPD patients.

Chronic obstructive pulmonary disease (COPD) is related to infiltration and activation of inflammatory cells in airways and pulmonary tissue. In COPD, neutrophils are prominent, while eosinophilic influx is typical to asthma. Inflammatory cells express sialic acid-binding immunoglobulin like lectins called Siglecs, a family of innate immune receptors that are transmembrane I-type lectins binding sialic acid. One member of the Siglec family, Siglec-8, is expressed mostly in eosinophils and may be an important therapeutic target in asthma or COPD. The aim of our project was to quantify Siglec-8 expression in induced sputum cells of COPD patients treated with long-acting beta2-agonists (LABA) or combined with long-acting antimuscarinic agents (LAMA) - tiotropium bromide. Thirty stable COPD patients (21 males and 9 females, mean age 67 years) receiving 12 µg BID formoterol therapy were assessed before and after 3 months' add-on therapy consisting of 18 µg QID tiotropium. In all patients, spirometry, lung volumes, and DLCO were performed before and after therapy. The patients were subjected to sputum induction before and after therapy. Sputum cells were isolated and processed to obtain cell membranes. Siglec-8 protein expression was assessed using Western blot. In patients receiving tiotropium and formoterol, improved FEV1 and lung volumes were observed compared with formoterol-only treated patients. The mean Siglec-8 level was significantly higher in eosinophilic subgroup of COPD patients compared with non-eosinophilic patients before therapy 40,000 vs. 15,000 Adj. Vol. INT/mm(2). Our data show that Siglec-8 may be involved in COPD pathogenesis and may influence COPD phenotyping.

Tiotropium increases PPARγ and decreases CREB in cells isolated from induced sputum of COPD patients.

Chronic obstructive pulmonary disease (COPD) is characterized by progressive airflow limitation and chronic inflammation of airways and lung parenchyma. Our aim was to assess two important elements of intracellular signaling involved in regulation of inflammation in COPD in patients subjected to long-acting beta2-agonist or long-acting beta2-agonist plus long-acting antimuscarinic: peroxisome proliferator-activated receptor gamma (PPARγ) protein, which has antiinflammatory and immunomodulatory properties and cAMP response element binding protein (CREB) and activated (CREB-P) protein which has histone acetyltransferase activity and increases histone acetylation and transcriptional activation of chromatin. Twenty one stable COPD patients (18 males and 3 females, mean age 65 years) receiving 12 µg B.I.D formoterol were assayed before and after 3 month add-on therapy, consisting of 18 µg Q.D. tiotropium. In all patients, sputum induction, spirometry, lung volumes, and DLCO were performed before and after therapy. Sputum cells were isolated and processed to isolate cytosolic and nuclear fractions. PPARγ, CREB, or CREB-P proteins were quantified in subcellular fractions using Western blot. Tiotropium add-on therapy improved respiratory parameters: FEV1 and lung volumes. After therapy mean expression of PPARγ in cell nuclei was significantly increased by about 180%, while CREB and phosphorylated CREB levels in cytosol and nuclei were decreased by about 30%. Our data show that the mechanism whereby tiotropium reduces exacerbations may be associated not only with persistent increase in airway functions and reduced hyperinflation mediated by muscarinic receptors, but also with possible anti-inflammatory effects of the drug, involving increased PPARγ and decreased CREB signaling.

Lipopolysaccharide changes sialylation pattern in the mouse central nervous system.

Sialylated glycoconjugates seem to play crucial role in the mechanisms that control the most important functions of the body. Sialylation is an important mechanism for the regulation of intercellular interactions that underlie neuronal plasticity as well as immune defense in the central nervous system (CNS). In this study, we analyzed the effect of lipopolysaccharide (LPS) on sialylation pattern in several regions of CNS. Additionally, we tested the effects of inflammatory stimulation on Siglec-F expression in microglial cells. Using lectin blotting with Maackia amurensis and Sambucus nigra agglutinins and immunostaining with antibody directed against PSA-NCAM we demonstrated altered expression of sialylated glycoconjugates differentially due to LPS-induced inflammation. We found that LPS caused significant increase of α2,3- and α2,6-linked sialic acids in the hippocampus and spinal cord. In the prefrontal cortex, the level of α2,3-linked sialic acids in selected glycoconjugates tended to be increased (p>0.05), while α2,6-linked sialic acids were reduced (p<0.05), while the expression of PSA-NCAM in all analyzed structures were significantly higher in comparison to the control group. The expression of Siglec-F in microglial cells stimulated with LPS remained unchanged. Given the significance of glycans in the brain biology we can conclude that sialic acids and their receptors Siglec may be crucial regulators of immune response in the CNS.

Intellectual functioning of childhood leukemia survivors--relation to Tau protein--a marker of white matter injury.

PURPOSE: Chemo- and radiotherapy used in acute lymphoblastic leukemia (ALL) can influence on brain functioning in the future. In a prospective study we analysed the cognitive functions of ALL survivors in relation to Tau protein as a marker of white matter injury. MATERIAL AND METHODS: Thirty-one survivors of childhood ALL (6.3 years after diagnosis); without the signs of CNS involvement, treated with chemotherapy alone, rested in first remission; underwent Intelligence tests- Wechsler Intelligence Scales (WISC-R, WAIS-R). Their results were analyzed in relation to the levels of Tau in cerebrospinal fluid (CSF) obtained during the treatment. RESULTS: The analysis showed that all survivors attained the average scores in intelligence tests. A negative correlation was found between methotrexate (MTX) doses and Freedom from Distractibility (FFD). Females had higher values of Performance Intelligence Quotient (PIQ) than males. A negative correlation was noted of Tau protein levels obtained from the last CSF with: Total and Verbal Intelligence Quotient, PIQ, Perceptual Organisation Index and FFD but not with Verbal Comprehension Index. CONCLUSION: Our results suggest the possibility of white matter injury during the treatment for ALL with chemotherapy alone. Elevated Tau protein level in CSF at the end of treatment might indicate future difficulties in neurocognitive functioning.

Cerebrospinal fluid IL-6, TNF-α and MCP-1 in children with acute lymphoblastic leukaemia during chemotherapy.

The aim of the study was to investigate the levels of cerebrospinal fluid (CSF) cytokines during chemotherapy of acute lymphoblastic leukaemia (ALL). Examination of 12 ALL child (6 boys and 6 girls) patients evidenced significant increases in interleukin-6 (IL-6) and monocyte chemotactic protein-1 (MCP-1) after induction treatment and significant increases in IL-6, tumour necrosis factor-α (TNF-α) and MCP-1 levels during the consolidation phase, as compared to their values at the time of diagnosis. There were no significant differences in CSF IL-6, TNF-α and MCP-1 concentrations after therapy. Our data suggest that standard ALL treatment may cause a subclinical inflammation and neurotoxicity.