Biomolecules2015, 5(3), 1399-1440; doi:10.3390/biom5031399 - published 2 July 2015 Show/Hide Abstract
Abstract: Inflammation is considered to play a central role in a diverse range of disease outcomes associated with exposure to various types of inhalable particulates. The initial mechanisms through which particles trigger cellular responses leading to activation of inflammatory responses are crucial to clarify in order to understand what physico-chemical characteristics govern the inflammogenic activity of particulate matter and why some particles are more harmful than others. Recent research suggests that molecular triggering mechanisms involved in activation of proinflammatory genes and onset of inflammatory reactions by particles or soluble particle components can be categorized into direct formation of reactive oxygen species (ROS) with subsequent oxidative stress, interaction with the lipid layer of cellular membranes, activation of cell surface receptors, and direct interactions with intracellular molecular targets. The present review focuses on the immediate effects and responses in cells exposed to particles and central down-stream signaling mechanisms involved in regulation of proinflammatory genes, with special emphasis on the role of oxidant and non-oxidant triggering mechanisms. Importantly, ROS act as a central second-messenger in a variety of signaling pathways. Even non-oxidant mediated triggering mechanisms are therefore also likely to activate downstream redox-regulated events.
Biomolecules2015, 5(3), 1386-1398; doi:10.3390/biom5031386 - published 1 July 2015 Show/Hide Abstract
Abstract: Interleukin-8 (IL-8) is a neutrophil chemokine that is encoded on the CXCL8 gene. Normally CXCL8 expression is repressed due to histone deacetylation, octamer-1 binding to the promoter and the inhibitory effect of nuclear factor-κB repressing factor (NRF). However, in response to a suitable stimulus, the human CXCL8 gene undergoes transcription due to its inducible promoter that is regulated by the transcription factors nuclear factor-κB (NF-κB), activating protein (AP-1), CAAT/enhancer-binding protein β (C/EBPβ, also known as NF-IL-6), C/EBP homologous protein (CHOP) and cAMP response element binding protein (CREB). CXCL8 mRNA is then stabilised by the activity of p38 mitogen-activated protein kinase (p38 MAPK). Cystic fibrosis (CF) lung disease is characterised by a neutrophil-dominated airway inflammatory response. A major factor contributing to the large number of neutrophils is the higher than normal levels of IL-8 that are present within the CF lung. Infection and inflammation, together with intrinsic alterations in CF airway cells are responsible for the abnormally high intrapulmonary levels of IL-8. Strategies to inhibit aberrantly high CXCL8 expression hold therapeutic potential for CF lung disease.
Biomolecules2015, 5(3), 1339-1385; doi:10.3390/biom5031339 - published 29 June 2015 Show/Hide Abstract
Abstract: Background: The quantitative, measurable detection of drinking is important for the successful treatment of alcohol misuse in transplantation of patients with alcohol disorders, people living with human immunodeficiency virus that need to adhere to medication, and special occupational hazard offenders, many of whom continually deny drinking. Their initial misconduct usually leads to medical problems associated with drinking, impulsive social behavior, and drunk driving. The accurate identification of alcohol consumption via biochemical tests contributes significantly to the monitoring of drinking behavior. Methods: A systematic review of the current methods used to measure biomarkers of alcohol consumption was conducted using PubMed and Google Scholar databases (2010–2015). The names of the tests have been identified. The methods and publications that correlate between the social instruments and the biochemical tests were further investigated. There is a clear need for assays standardization to ensure the use of these biochemical tests as routine biomarkers. Findings: Alcohol ingestion can be measured using a breath test. Because alcohol is rapidly eliminated from the circulation, the time for detection by this analysis is in the range of hours. Alcohol consumption can alternatively be detected by direct measurement of ethanol concentration in blood or urine. Several markers have been proposed to extend the interval and sensitivities of detection, including ethyl glucuronide and ethyl sulfate in urine, phosphatidylethanol in blood, and ethyl glucuronide and fatty acid ethyl esters in hair, among others. Moreover, there is a need to correlate the indirect biomarker carbohydrate deficient transferrin, which reflects longer lasting consumption of higher amounts of alcohol, with serum γ-glutamyl transpeptidase, another long term indirect biomarker that is routinely used and standardized in laboratory medicine.
Biomolecules2015, 5(3), 1319-1338; doi:10.3390/biom5031319 - published 29 June 2015 Show/Hide Abstract
Abstract: Whereas mitochondria are well established as the source of ATP in oxidative phosphorylation (OXPHOS), it is debated if they are also the major cellular sources of reactive oxygen species (ROS). Here we describe the novel approach of combining high-resolution respirometry and fluorometric measurement of hydrogen peroxide (H2O2) production, applied to mitochondrial preparations (permeabilized cells, tissue homogenate, isolated mitochondria). The widely used H2O2 probe Amplex Red inhibited respiration in intact and permeabilized cells and should not be applied at concentrations above 10 µM. H2O2 fluxes were generally less than 1% of oxygen fluxes in physiological substrate and coupling states, specifically in permeabilized cells. H2O2 flux was consistently highest in the Complex II-linked LEAK state, reduced with CI&II-linked convergent electron flow and in mitochondria respiring at OXPHOS capacity, and were further diminished in uncoupled mitochondria respiring at electron transfer system capacity. Simultaneous measurement of mitochondrial respiration and H2O2 flux requires careful optimization of assay conditions and reveals information on mitochondrial function beyond separate analysis of ROS production.
Biomolecules2015, 5(3), 1302-1318; doi:10.3390/biom5031302 - published 29 June 2015 Show/Hide Abstract
Abstract: Appropriate innate and adaptive immune responses are essential for protection and resolution against chemical, physical or biological insults. Immune cell polarization is fundamental in orchestrating distinct phases of inflammation, specifically acute phase responses followed by resolution and tissue repair. Dysregulation of immune cell and inflammatory responses is a hallmark of multiple diseases encompassing atherosclerosis, rheumatoid arthritis, psoriasis and metabolic syndromes. A master transcriptional mediator of diverse inflammatory signaling and immune cell function is NF-κB, and altered control of this key regulator can lead to an effective switch from acute to chronic inflammatory responses. Members of the nuclear receptor (NR) superfamily of ligand-dependent transcription factors crosstalk with NF-κB to regulate immune cell function(s). Within the NR superfamily the NR4A1-3 orphan receptors have emerged as important regulators of immune cell polarization and NF-κB signaling. NR4A receptors modulate NF-κB activity in a dynamic fashion, either repressing or enhancing target gene expression leading to altered inflammatory outcome. Here we will discuss the pivotal role NR4A’s receptors play in orchestrating immune cell homeostasis through molecular crosstalk with NF-κB. Specifically, we will examine such NR4A/NF-κB interactions within the context of distinct cell phenotypes, including monocyte, macrophage, T cells, endothelial, and mesenchymal cells, which play a role in inflammation-associated disease. Finally, we review the therapeutic potential of altering NR4A/NF-κB interactions to limit hyper-inflammatory responses in vivo.
Biomolecules2015, 5(3), 1284-1301; doi:10.3390/biom5031284 - published 29 June 2015 Show/Hide Abstract
Abstract: Protein phosphatase 2A (PP2A) is one of the most abundant intracellular serine/threonine (Ser/Thr) phosphatases accounting for 1% of the total cellular protein content. PP2A is comprised of a heterodimeric core enzyme and a substrate-specific regulatory subunit. Potentially, at least seventy different compositions of PP2A exist because of variable regulatory subunit binding that accounts for various activity modulating numerous cell functions. Due to the constitutive phosphatase activity present inside cells, a sensitive assay is required to detect the changes of PP2A activity under various experimental conditions. We optimized a fluorescence assay (DIFMU assay) by combining it with prior anti-PP2A immunoprecipitation to quantify PP2A-specific phosphatase activity. It is also known that prior exposure to lipopolysaccharides (LPS) induces “immune tolerance” of the cells to subsequent stimulation. Herein we report that PP2A activity is upregulated in tolerized peritoneal macrophages, corresponding to decreased TNF-α secretion upon second LPS stimulation. We further examined the role of PP2A in the tolerance effect by using PP2ACαl°xl°x;lyM-Cre conditional knockout macrophages. We found that PP2A phosphatase activity cannot be further increased by tolerance. TNF-α secretion from tolerized PP2ACαl°xl°x;lyM-Cre macrophages is higher than tolerized control macrophages. Furthermore, we showed that the increased TNF-α secretion may be due to an epigenetic transcriptionally active signature on the promoter of TNF-α gene rather than regulation of the NFκB/IκB signaling pathway. These results suggest a role for increased PP2A activity in the regulation of immune tolerance.