Siiri Altraja

SabA is the H. pylori hemagglutinin and is polymorphic in binding to sialylated glycans

Adherence of Helicobacter pylori to inflamed gastric mucosa is dependent on the sialic acid–binding adhesin (SabA) and cognate sialylated/fucosylated glycans on the host cell surface. By in situ hybridization, H. pylori bacteria were observed in close association with erythrocytes in capillaries and post-capillary venules of the lamina propria of gastric mucosa in both infected humans and Rhesus monkeys. In vivo adherence of H. pylori to erythrocytes may require molecular mechanisms similar to the sialic acid–dependent in vitro agglutination of erythrocytes (i.e., sialic acid–dependent hemagglutination). In this context, the SabA adhesin was identified as the sialic acid–dependent hemagglutinin based on sialidase-sensitive hemagglutination, binding assays with sialylated glycoconjugates, and analysis of a series of isogenic sabA deletion mutants. The topographic presentation of binding sites for SabA on the erythrocyte membrane was mapped to gangliosides with extended core chains. However, receptor mapping revealed that the NeuAcα2–3Gal-disaccharide constitutes the minimal sialylated binding epitope required for SabA binding. Furthermore, clinical isolates demonstrated polymorphism in sialyl binding and complementation analysis of sabA mutants demonstrated that polymorphism in sialyl binding is an inherent property of the SabA protein itself. Gastric inflammation is associated with periodic changes in the composition of mucosal sialylation patterns. We suggest that dynamic adaptation in sialyl-binding properties during persistent infection specializes H. pylori both for individual variation in mucosal glycosylation and tropism for local areas of inflamed and/or dysplastic tissue.

Synthesis of tenascin and laminin beta2 chain in human bronchial epithelial cells is enhanced by cysteinyl leukotrienes via CysLT1 receptor

BackgroundCysteinyl leukotrienes (CysLTs) are key mediators of asthma, but their role in the genesis of airway remodeling is insufficiently understood. Recent evidence suggests that increased expression of tenascin (Tn) and laminin (Ln) β2 chain is indicative of the remodeling activity in asthma, but represents also an example of deposition of extracellular matrix, which affects the airway wall compliance. We tested the hypothesis that CysLTs affect production of Tn and Ln β2 chain by human bronchial epithelial cells and elucidated, which of the CysLT receptors, CysLT1 or CysLT2, mediate this effect.MethodsCultured BEAS-2B human bronchial epithelial cells were stimulated with leukotriene D4 (LTD4) and E4 (LTE4) and evaluated by immunocytochemistry, Western blotting, flow cytometry, and RT-PCR. CysLT receptors were differentially blocked with use of montelukast or BAY u9773.ResultsLTD4 and LTE4 significantly augmented the expression of Tn, whereas LTD4, distinctly from LTE4, was able to increase also the Ln β2 chain. Although the expression of CysLT2 prevailed over that of CysLT1, the up-regulation of Tn and Ln β2 chain by CysLTs was completely blocked by the CysLT1-selective antagonist montelukast with no difference between montelukast and the dual antagonist BAY u9773 for the inhibitory capacity.ConclusionThese findings suggest that the CysLT-induced up-regulation of Tn and Ln β2 chain, an important epithelium-linked aspect of airway remodeling, is mediated predominantly by the CysLT1 receptor. The results provide a novel aspect to support the use of CysLT1 receptor antagonists in the anti-remodeling treatment of asthma.

E-Cigarette Affects the Metabolome of Primary Normal Human Bronchial Epithelial Cells

E-cigarettes are widely believed to be safer than conventional cigarettes and have been even suggested as aids for smoking cessation. However, while reasonable with some regards, this judgment is not yet supported by adequate biomedical research data. Since bronchial epithelial cells are the immediate target of inhaled toxicants, we hypothesized that exposure to e-cigarettes may affect the metabolome of human bronchial epithelial cells (HBEC) and that the changes are, at least in part, induced by oxidant-driven mechanisms. Therefore, we evaluated the effect of e-cigarette liquid (ECL) on the metabolome of HBEC and examined the potency of antioxidants to protect the cells. We assessed the changes of the intracellular metabolome upon treatment with ECL in comparison of the effect of cigarette smoke condensate (CSC) with mass spectrometry and principal component analysis on air-liquid interface model of normal HBEC. Thereafter, we evaluated the capability of the novel antioxidant tetrapeptide O-methyl-l-tyrosinyl-γ-l-glutamyl-l-cysteinylglycine (UPF1) to attenuate the effect of ECL. ECL caused a significant shift in the metabolome that gradually gained its maximum by the 5th hour and receded by the 7th hour. A second alteration followed at the 13th hour. Treatment with CSC caused a significant initial shift already by the 1st hour. ECL, but not CSC, significantly increased the concentrations of arginine, histidine, and xanthine. ECL, in parallel with CSC, increased the content of adenosine diphosphate and decreased that of three lipid species from the phosphatidylcholine family. UPF1 partially counteracted the ECL-induced deviations, UPF1’s maximum effect occurred at the 5th hour. The data support our hypothesis that ECL profoundly alters the metabolome of HBEC in a manner, which is comparable and partially overlapping with the effect of CSC. Hence, our results do not support the concept of harmlessness of e-cigarettes.

Alterations of Bronchial Epithelial Metabolome by Cigarette Smoke Are Reversible by an Antioxidant, O-Methyl-l-Tyrosinyl-γ-l-Glutamyl-l-Cysteinylglycine

Human bronchial epithelial cells (HBECs) have first-line contact with harmful substances during smoking, and changes in their metabolism most likely represent a defining factor in coping with the stress and development of airway diseases. This study was designed to determine the dynamics of metabolome changes in HBECs treated with cigarette smoke condensate (CSC), and to test whether normal metabolism can be restored by synthetic antioxidants. Principal component analysis, based on untargeted mass spectra, indicated that treatment of CSC-exposed HBECs with O-methyl-L-tyrosinyl-γ-L-glutamyl-L-cysteinylglycine (UPF1) acted faster than did N-acetylcysteine to revert the effect of CSC. The maximum effect of 10 μg/ml CSC itself on HBEC cell line, BEAS-2B, metabolism was seen at 2 hours after treatment, with return to the baseline level by 7 hours. In primary HBECs, the initial maximum effect was seen at 1 hour after CSC exposure. Certain metabolites associated with redox pathways and energy production were affected by CSC. Subsequent restoration of their content by UPF1 supports the hypothetical protective capacity of UPF1 against the oxidative stress and increased energy demand, respectively. Furthermore, UPF1 up-regulated the contents of phospholipid species identified as phosphatidylcholines and phosphatidylethanolamines in the CSC-exposed HBECs, indicating possible suppression of inflammatory processes along with an increase in spermidine as an endogenous cytoprotector. In conclusion, with this dynamic metabolomics study, we characterize the durability of the CSC-induced metabolic changes in BEAS-2B line cells and primary HBECs, and demonstrate the ability of UPF1 to significantly accelerate the recovery of HBECs from CSC insult.

Proteome changes of human bronchial epithelial cells in response to pro-inflammatory mediator leukotriene E4 and pro-remodelling factor TGF-β1

Inflammatory environment chronically activates bronchial epithelial cells to stimulate airway cells including epithelial cells themselves by secreting pro-inflammatory and regulatory factors. Proteomic approach is most relevant to screen the epithelial pathways following the inflammatory stimuli. We compared protein expression of the human bronchial epithelial cells exposed to leukotriene E(4) (LTE(4)) and transforming growth factor-beta(1) (TGF-beta(1)) with that of non-stimulated cells. The proteins were separated by 2-DE and the differentially expressed proteins were identified by MALDI-TOF MS and TOF/TOF tandem MS/MS. This approach allowed identification of 31 proteins, of which 26 corresponded to different proteins. beta-tubulin, significantly down-regulated by LTE(4), was confirmed as a ciliated cell marker beta-tubulin IV, whose decrease by LTE(4) was further corroborated by flow cytometry and RT-qPCR. This refers to a contribution of cysteinyl leukotrienes to epithelial remodelling and initiation of epithelial-mesenchymal transition in conducting airways. Of the affected proteins by TGF-beta(1), clinically most relevant ones were up-regulated antioxidant enzyme superoxide dismutase 1, pro-fibrotic enzyme protein disulfide-isomerase and heat shock 70 kDa protein 9B. The changed protein profiles from this study add novel aspects to improve our understanding of the airway pathobiology, provide hints for further directed airway research and may contribute to selecting targets for future therapeutics.

Cigarette smoke-induced differential regulation of glutathione metabolism in bronchial epithelial cells is balanced by an antioxidant tetrapeptide UPF1

Airway epithelium is a principal target for inhaled oxidants like cigarette smoke, which induce epithelial injury and thereby provoke pathogenesis of chronic airway diseases. Alterations in airway epithelial glutathione (GSH) metabolism are central in causing a loss of reducing environment, however, data are scarce on epithelial cells from larger bronchi. We showed a transient depletion of intracellular GSH in human bronchial epithelial cells after exposure to cigarette smoke condensate (CSC), which later followed by a prolonged elevation. Of the GSH-regulating enzymes, CSC increased mRNA expression of both catalytic (GCLC) and modifier (GCLM) subunits of glutamate-cysteine ligase. UPF1, a tetrapeptide GSH analogue, 4-methoxy-L-tyrosinyl-γ-L-glutamyl-L-cysteinyl-glycine, known to possess a 50-fold higher hydroxyl radical scavenging efficiency than does GSH, normalized the intracellular GSH level in the human bronchial epithelial cells under oxidative stress caused by CSC. UPF1 restored the GCLM and GSH reductase mRNA levels, which were significantly augmented by CSC treatment, back to the level of untreated control cells, referring to a successful establishment of control by UPF1 upon the over-accumulation of GSH. Moreover, UPF1 showed a significantly more potent antioxidant capacity than did N-acetyl-L-cysteine (NAC) and, compared to NAC, demonstrated a better potential to assure the whole GSH homeostasis in human bronchial epithelial cells. The current study suggests that UPF1 is capable of maintaining intracellular GSH level under CSC-induced oxidative stress in bronchial epithelial cells via balanced control over GSH-regulating enzymes, reflecting an improved perception of cellular redox conditions and thereby warranting improved adjustment of GSH accumulation.

Phenotyping of Chronic Obstructive Pulmonary Disease Based on the Integration of Metabolomes and Clinical Characteristics

Apart from the refined management-oriented clinical stratification of chronic obstructive pulmonary disease (COPD), the molecular pathologies behind this highly prevalent disease have remained obscure. The aim of this study was the characterization of patients with COPD, based on the metabolomic profiling of peripheral blood and exhaled breath condensate (EBC) within the context of defined clinical and demographic variables. Mass-spectrometry-based targeted analysis of serum metabolites (mainly amino acids and lipid species), untargeted profiles of serum and EBC of patients with COPD of different clinical characteristics (n = 25) and control individuals (n = 21) were performed. From the combined clinical/demographic and metabolomics data, associations between clinical/demographic and metabolic parameters were searched and a de novo phenotyping for COPD was attempted. Adjoining the clinical parameters, sphingomyelins were the best to differentiate COPD patients from controls. Unsaturated fatty acid-containing lipids, ornithine metabolism and plasma protein composition-associated signals from the untargeted analysis differentiated the Global Initiative for COPD (GOLD) categories. Hierarchical clustering did not reveal a clinical-metabolomic stratification superior to the strata set by the GOLD consensus. We conclude that while metabolomics approaches are good for finding biomarkers and clarifying the mechanism of the disease, there are no distinct co-variate independent clinical-metabolic phenotypes.