Defect of Adaptation to Hypoxia in Patients With COPD Due to Reduction of Histone Deacetylase 7

BackgroundHypoxia inducible factor (HIF)-1 plays an important role in cellular adaptation to hypoxia by activating oxygen-regulated genes such as vascular endothelial growth factor (VEGF) and erythropoietin. Sputum VEGF levels are reported to be decreased in COPD, despite hypoxia. Here we show that patients with COPD fail to induce HIF-1α and VEGF under hypoxic condition because of a reduction in histone deacetylase (HDAC) 7.MethodsPeripheral blood mononuclear cells (PBMCs) were obtained from patients with moderate to severe COPD (n = 21), smokers without COPD (n = 12), and nonsmokers (n = 15). PBMCs were exposed to hypoxia (1% oxygen, 5% CO2, and 94% N2) for 24 h, and HIF-1α and HDAC7 protein expression in nuclear extracts were determined by sodium dodecyl sulfate poly acrylamide gel electrophoresis (SDS-PAGE)/Western blotting.ResultsHIF-1α was significantly induced by hypoxia in each group when compared with the normoxic condition (12-fold induction in nonsmokers, 24-fold induction in smokers without COPD, fourfold induction in COPD), but induction of HIF-1α under hypoxia was significantly lower in patients with COPD than in nonsmokers and smokers without COPD (P < .05 and P < .01, respectively). VEGF messenger RNA detected by quantitative real-time polymerase chain reaction was correlated with HIF-1α protein in nuclei (r = 0.79, P < .05), and HDAC7 protein expression was correlated with HIF-1α protein in nuclei (r = 0.46, P < .05). HDAC7 knockdown inhibited hypoxia-induced HIF-1α activity in U937 cells, and HIF-1α nuclear translocation and HIF-1α binding to the VEGF promoter in A549 cells.ConclusionsHDAC7 reduction in COPD causes a defect of HIF-1α induction response to hypoxia with impaired VEGF gene expression. This poor cellular adaptation might play a role in the pathogenesis of COPD

Melanoma Spheroids Grown Under Neural Crest Cell Conditions Are Highly Plastic Migratory/Invasive Tumor Cells Endowed with Immunomodulator Function

International audienceBACKGROUND: The aggressiveness of melanoma tumors is likely to rely on their well-recognized heterogeneity and plasticity. Melanoma comprises multi-subpopulations of cancer cells some of which may possess stem cell-like properties. Although useful, the sphere-formation assay to identify stem cell-like or tumor initiating cell subpopulations in melanoma has been challenged, and it is unclear if this model can predict a functional phenotype associated with aggressive tumor cells. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed the molecular and functional phenotypes of melanoma spheroids formed in neural crest cell medium. Whether from metastatic or advanced primary tumors, spheroid cells expressed melanoma-associated markers. They displayed higher capacity to differentiate along mesenchymal lineages and enhanced expression of SOX2, NANOG, KLF4, and/or OCT4 transcription factors, but not enhanced self-renewal or tumorigenicity when compared to their adherent counterparts. Gene expression profiling attributed a neural crest cell signature to these spheroids and indicated that a migratory/invasive and immune-function modulating program could be associated with these cells. In vitro assays confirmed that spheroids display enhanced migratory/invasive capacities. In immune activation assays, spheroid cells elicited a poorer allogenic response from immune cells and inhibited mitogen-dependent T cells activation and proliferation more efficiently than their adherent counterparts. Our findings reveal a novel immune-modulator function of melanoma spheroids and suggest specific roles for spheroids in invasion and in evasion of antitumor immunity. CONCLUSION/SIGNIFICANCE: The association of a more plastic, invasive and evasive, thus a more aggressive tumor phenotype with melanoma spheroids reveals a previously unrecognized aspect of tumor cells expanded as spheroid cultures. While of limited efficiency for melanoma initiating cell identification, our melanoma spheroid model predicted aggressive phenotype and suggested that aggressiveness and heterogeneity of melanoma tumors can be supported by subpopulations other than cancer stem cells. Therefore, it could be constructive to investigate melanoma aggressiveness, relevant to patients and clinical transferability

Effect of Hydrocortisone on Mortality and Organ Support in Patients With Severe COVID-19: The REMAP-CAP COVID-19 Corticosteroid Domain Randomized Clinical Trial.

Importance: Evidence regarding corticosteroid use for severe coronavirus disease 2019 (COVID-19) is limited. Objective: To determine whether hydrocortisone improves outcome for patients with severe COVID-19. Design, Setting, and Participants: An ongoing adaptive platform trial testing multiple interventions within multiple therapeutic domains, for example, antiviral agents, corticosteroids, or immunoglobulin. Between March 9 and June 17, 2020, 614 adult patients with suspected or confirmed COVID-19 were enrolled and randomized within at least 1 domain following admission to an intensive care unit (ICU) for respiratory or cardiovascular organ support at 121 sites in 8 countries. Of these, 403 were randomized to open-label interventions within the corticosteroid domain. The domain was halted after results from another trial were released. Follow-up ended August 12, 2020. Interventions: The corticosteroid domain randomized participants to a fixed 7-day course of intravenous hydrocortisone (50 mg or 100 mg every 6 hours) (n = 143), a shock-dependent course (50 mg every 6 hours when shock was clinically evident) (n = 152), or no hydrocortisone (n = 108). Main Outcomes and Measures: The primary end point was organ support-free days (days alive and free of ICU-based respiratory or cardiovascular support) within 21 days, where patients who died were assigned -1 day. The primary analysis was a bayesian cumulative logistic model that included all patients enrolled with severe COVID-19, adjusting for age, sex, site, region, time, assignment to interventions within other domains, and domain and intervention eligibility. Superiority was defined as the posterior probability of an odds ratio greater than 1 (threshold for trial conclusion of superiority >99%). Results: After excluding 19 participants who withdrew consent, there were 384 patients (mean age, 60 years; 29% female) randomized to the fixed-dose (n = 137), shock-dependent (n = 146), and no (n = 101) hydrocortisone groups; 379 (99%) completed the study and were included in the analysis. The mean age for the 3 groups ranged between 59.5 and 60.4 years; most patients were male (range, 70.6%-71.5%); mean body mass index ranged between 29.7 and 30.9; and patients receiving mechanical ventilation ranged between 50.0% and 63.5%. For the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively, the median organ support-free days were 0 (IQR, -1 to 15), 0 (IQR, -1 to 13), and 0 (-1 to 11) days (composed of 30%, 26%, and 33% mortality rates and 11.5, 9.5, and 6 median organ support-free days among survivors). The median adjusted odds ratio and bayesian probability of superiority were 1.43 (95% credible interval, 0.91-2.27) and 93% for fixed-dose hydrocortisone, respectively, and were 1.22 (95% credible interval, 0.76-1.94) and 80% for shock-dependent hydrocortisone compared with no hydrocortisone. Serious adverse events were reported in 4 (3%), 5 (3%), and 1 (1%) patients in the fixed-dose, shock-dependent, and no hydrocortisone groups, respectively. Conclusions and Relevance: Among patients with severe COVID-19, treatment with a 7-day fixed-dose course of hydrocortisone or shock-dependent dosing of hydrocortisone, compared with no hydrocortisone, resulted in 93% and 80% probabilities of superiority with regard to the odds of improvement in organ support-free days within 21 days. However, the trial was stopped early and no treatment strategy met prespecified criteria for statistical superiority, precluding definitive conclusions. Trial Registration: ClinicalTrials.gov Identifier: NCT02735707

Early mobilisation in critically ill COVID-19 patients: a subanalysis of the ESICM-initiated UNITE-COVID observational study

Background Early mobilisation (EM) is an intervention that may improve the outcome of critically ill patients. There is limited data on EM in COVID-19 patients and its use during the first pandemic wave. Methods This is a pre-planned subanalysis of the ESICM UNITE-COVID, an international multicenter observational study involving critically ill COVID-19 patients in the ICU between February 15th and May 15th, 2020. We analysed variables associated with the initiation of EM (within 72 h of ICU admission) and explored the impact of EM on mortality, ICU and hospital length of stay, as well as discharge location. Statistical analyses were done using (generalised) linear mixed-effect models and ANOVAs. Results Mobilisation data from 4190 patients from 280 ICUs in 45 countries were analysed. 1114 (26.6%) of these patients received mobilisation within 72 h after ICU admission; 3076 (73.4%) did not. In our analysis of factors associated with EM, mechanical ventilation at admission (OR 0.29; 95% CI 0.25, 0.35; p = 0.001), higher age (OR 0.99; 95% CI 0.98, 1.00; p ≤ 0.001), pre-existing asthma (OR 0.84; 95% CI 0.73, 0.98; p = 0.028), and pre-existing kidney disease (OR 0.84; 95% CI 0.71, 0.99; p = 0.036) were negatively associated with the initiation of EM. EM was associated with a higher chance of being discharged home (OR 1.31; 95% CI 1.08, 1.58; p = 0.007) but was not associated with length of stay in ICU (adj. difference 0.91 days; 95% CI − 0.47, 1.37, p = 0.34) and hospital (adj. difference 1.4 days; 95% CI − 0.62, 2.35, p = 0.24) or mortality (OR 0.88; 95% CI 0.7, 1.09, p = 0.24) when adjusted for covariates. Conclusions Our findings demonstrate that a quarter of COVID-19 patients received EM. There was no association found between EM in COVID-19 patients' ICU and hospital length of stay or mortality. However, EM in COVID-19 patients was associated with increased odds of being discharged home rather than to a care facility. Trial registration ClinicalTrials.gov: NCT04836065 (retrospectively registered April 8th 2021)

Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Mode of Glucocorticoid Actions in Airway Disease

Synthetic glucocorticoids are the most potent anti-inflammatory agents used to treat chronic inflammatory disease, such as asthma. However, a small number (&lt;5%) of asthmatic patients and almost all patients with chronic obstructive pulmonary disease (COPD) do not respond well, or at all, to glucocorticoid therapy. If the molecular mechanism of glucocorticoid insensitivity is uncovered, it may in turn provide insight into the key mechanism of glucocorticoid action and allow a rational way to implement treatment regimens that restore glucocorticoid sensitivity. Glucocorticoids exert their effects by binding to a cytoplasmic glucocorticoid receptor (GR), which is subjected to post-translational modifications. Receptor phosphorylation, acetylation, nitrosylation, ubiquitinylation, and other modifications influence hormone binding, nuclear translocation, and protein half-life. Analysis of GR interactions to other molecules, such as coactivators or corepressors, may explain the genetic specificity of GR action. Priming with inflammatory cytokine or oxidative/nitrative stress is a mechanism for the glucocorticoid resistance observed in chronic inflammatory airway disease via reduction of corepressors or GR modification. Therapies targeting these aspects of the GR activation pathway may reverse glucocorticoid resistance in patients with glucocorticoid-insensitive airway disease and some patients with other inflammatory diseases, such as rheumatoid arthritis and inflammatory bowel disease. KEYWORDS: glucocorticoid, inflammation, asthma, COPD, glucocorticoid receptor, glucocorticoid resistance INTRODUCTION Although glucocorticoids are the most potent anti-inflammatory agents for the treatment of chronic inflammatory disease, such as asthma, a small population of asthmatic patients and almost all patients with chronic obstructive pulmonary disease (COPD) show a poor response to glucocorticoids Ito et al.: Mode of Glucocorticoid Actions in Airway Disease TheScientificWorldJOURNAL (2006) 6, 1750-1769 THE MOLECULAR BASIS OF INFLAMMATION IN ASTHMA AND COPD Lower airways inflammation is a central feature of many lung diseases including asthma and COPD. These diseases always involve recruitment and activation of inflammatory cells with changes in the structural cells of the lung, though the specific characteristics of the inflammatory response and the site of inflammation differ from one disease to another. Inflammation in asthma is associated with increased airway hyperresponsiveness leading to recurrent episodes of wheezing, breathlessness, chest tightness, and coughing, particularly at night or in the early morning. The inflammation is present even in those with very mild asthma; T-lymphocytes of the T-helper (Th) type 2 phenotype, eosinophils, macrophages/monocytes, and mast cells infiltrate the airway wall. Airway inflammation is also amplified during exacerbation with an increase in eosinophils and sometimes neutrophils. These conditions are also characterized by an increased expression of components of the inflammatory cascade including chemokines, cytokines, growth factors, enzymes, noxious gas, reactive oxygen, receptors, and adhesion molecules COPD is a chronic inflammatory disease of the lower airways and lung, which is enhanced during exacerbations Increased inflammatory gene transcription seen in inflammatory airway disease is regulated by proinflammatory transcription factors, such as nuclear factor-κB (NF-κB) and activator protein-1 (AP-1). NF-κB is ubiquitously expressed and is able to not only control the induction of inflammatory genes in its own right, but also enhances the activity of other cell-and signal-specific transcription factors Alterations in the structure of chromatin are critical to the regulation of gene expression Ito et al.: Mode of Glucocorticoid Actions in Airway Disease TheScientificWorldJOURNAL (2006) 6, 1750-1769 1752 FIGURE 1. Molecular mechanism of gene activation and repression. Gene activation and repression are regulated by acetylation of core histones. Histone acetylation is mediated by coactivators that have intrinsic HAT activity, opening up the chromatin structure to allow binding of RNA polymerase II and transcription factors that were unable to bind DNA in the closed chromatin configuration. This is reversed by corepressors, which include histone deacetylases (HDACs) and other associated proteins that reverse this acetylation, thereby causing gene silencing. STATs, signal transduction activated transcription factors. transcription is, therefore, associated with an increase in histone acetylation, whereas hypoacetylation is correlated with reduced transcription or gene silencing, which is regulated by histone deacetylase (HDAC) MECHANISMS OF GLUCOCORTICOID RECEPTOR FUNCTION Glucocorticoids exert their effects by binding to a single 777-amino-acid glucocorticoid receptor (GR) that is localized to the cytoplasm of almost all cell types Ito et al.: Mode of Glucocorticoid Actions in Airway Disease TheScientificWorldJOURNAL (2006) 6, 1750-1769 1753 FIGURE 2. Structure of GRα and its post-translational modification. The N-terminus represents the constitutive transcriptional activation function (AF-1). The C-terminus contains the LBD, ligand-dependent activation function (AF-2), dimerization domain, and cofactor binding domains, all separated from the DBD by the hinge region (H). GR is post-translationally modified. Su (sumolyation), Ub (ubiquitinylation), PS (serine phosphorylation), PT (threonine phosphorylation), Ac (acetylation). HOW TO SWITCH OFF INFLAMMATORY GENES The major anti-inflammatory effects of glucocorticoids are thought to be due to repression of inflammatory and immune genes Second, the GR represses gene expression via DNA binding. After GR nuclear translocation, GR combines with another GR to form a dimer and it binds to consensus DNA sites termed glucocorticoid response elements (GREs, GGTACAnnnTGTTCT) in the regulating regions of corticosteroid-responsive genes. The GR dimer can bind to a GRE that overlaps the DNA binding site for a proinflammatory transcription factor or at the start site of transcription such as in the genes for IL-6 and osteocalcin, thus blocking gene expression Ito et al.: Mode of Glucocorticoid Actions in Airway Disease TheScientificWorldJOURNAL (2006) 6, 1750-1769 1754 FIGURE 3. How glucocorticoids turn off gene transcription. Cytokine, oxidative stress, growth factors, and lipopolysaccharides (LPS) stimulate activation of transcriptional factors, such as NF-κB. Activated NF-κB translocates to nuclei and recruits HAT, and then acetylates histones. Consequently, genes are switched on as shown in the left side. GR exists in the cytoplasm (center of Third, the GR dimer, through the binding to GRE, may suppress inflammation by increasing the synthesis of anti-inflammatory proteins, such as IL-10, MAPK phosphatase-1 (MKP-1), the inhibitor of NF-κB (IκB-α), annexin 1 (and annexin 1 peptides), glucocorticoid-induced leucine zipper (GILZ), and secretory lymphocyte protease inhibitor (SLPI) Fourth, glucocorticoids can increase the levels of cell ribonucleases and mRNA destabilizing proteins, thereby reducing the levels of mRNA Last, GR is also reported to act on the cell membrane nonspecifically or through membrane-bound GR. This is known as nongenomic effects of glucocorticoids, but it is not fully elucidated GLUCOCORTICOID RECEPTOR MULTICOMPLEXES WITH VARYING COFACTORS Ito et al.: Mode of Glucocorticoid Actions in Airway Disease TheScientificWorldJOURNAL (2006) 6, 1750-1769 1755 Activation of GR by glucocorticoids is a multistep process that involves hormone binding, hormonedependent structural transformation, translocation into the nucleus, and identification of target genes. There is increasing evidence that each step in this process is controlled by the interaction of GR with cofactors, such as a heat shock protein 90 (hsp90)-containing chaperone complex, kinases, high mobility group (HMG) proteins, mineralocorticoid receptor, and other transcription factors Components of the BRG1(SW1/SNF) complex, an ATP-dependent chromatin remodeling complex, are the well-known coactivators associated with GR CBP and p300 are histone acetylases and act as scaffold molecules that interact stably or transiently with a large number of transcription factors, including GR. CBP/p300 is recruited to both GR and other NRs either directly, through AF-1, or indirectly through coactivators interacting with AF-2 and this is involved in GR transactivation Other well-known GR-associated coactivators are the molecules in the p160 coactivator family Li et al. showed that upon ligand treatment, progesterone receptor (PR) interacted preferentially with SRC-1, which recruited CBP and significantly enhanced acetylation at K5 of histone H4 Very interestingly, the GR coactivators, SRC-1 and GRIP-1 (SRC-2), also act as corepressors of the GR Real corepressors are also recruited to the GR multicomplex, such as SMRT (silencing mediator for retinoid and thyroid-hormone receptors), NCoR (nuclear receptor corepressor), and receptor-interacting protein 140 (RIP140) Ito et al.: Mode of Glucocorticoid Actions in Airway Disease TheScientificWorldJOURNAL (2006) 6, 1750-1769 1756 recruitment HDAC2 is the most important corepressor to determine glucocorticoid insensitivity POST-TRANSLATIONAL MODIFICATION Phosphorylation GR is a phosphoprotein containing multiple potential sites for phosphorylation Eight phosphorylation sites (seven for serine, one for threonine) in mouse GR have been identified Bodwell et al. showed that GR response varies during the cell cycle, with cells being less sensitive to corticosteroids during G2/M, and in the G2/M period, GR was highly phosphorylated Acetylation HAT and HDAC, which are associated with GR, are also known as protein acetyltransferase/deacetylase as well as histone acetyltransferase/deacetylase. Previous studies have shown that both the estrogen receptor Ito et al.: Mode of Glucocorticoid Actions in Airway Disease TheScientificWorldJOURNAL (2006) 6, 1750-1769 1757 (ER) and the androgen receptor (AR) are acetylated within their hinge/LBDs and that this can modulate hormone-induced gene induction We previously reported that HDAC2 expression and activity is decreased in smokers[81] and patients with COPD Nitrosylation/Nitration Oxidative/nitrative stress is the key factor in pathogenesis of COPD and maybe in severe asthma Galigniana et al. showed that NO donors (S-nitroso-acetyl-DL-penicillamine, S-nitroso-DL-penicillamine, or S-nitroso-glutathione) decreased the number of ligand binding sites and K d for the binding of [ 3 H]-triamcinolone to immunoprecipitated GR from mouse L929 fibroblasts, without any change in GR protein levels Ito et al.: Mode of Glucocorticoid Actions in Airway Disease TheScientificWorldJOURNAL (2006) 6, 1750-1769 1758 FIGURE 4. GR acetylation. After ligand binding, GR is acetylated and dimerized. The dimer binds GRE and induces antiinflammatory factors where this binding likely causes side effects. On the other hand, HDAC2 is recruited to the acetylated GR and deacetylates it to allow binding to NF-κB, resulting in repression of NF-κB-dependent gene expression. Ubiquitinylation/Sumoylation The ubiquitin-proteasome-dependent protein degradation pathway (UPP) regulates the turnover of many transcription factors including steroid hormone receptors, such as the ER and PR. Glucocorticoid treatment induced a down-regulation of GR Wallace and Cidlowski indicated that the phosphorylation status of the glucocorticoid receptor plays a prominent role in receptor protein turnover and phosphorylation is a key signal for ubiquitination and proteasomal catabolism of GR Small ubiquitin-related modifier-1 (SUMO-1) is covalently attached to many cellular targets to regulate protein-protein and protein-DNA interactions, as well as localization and stability of the target protein. GR is reported to be post-translationally modified by SUMO-1 (sumoylated) in a ligand-enhanced fashion Ito et al.: Mode of Glucocorticoid Actions in Airway Disease TheScientificWorldJOURNAL (2006) 6, 1750-1769 1759 receptor&apos;s transactivation potential MOLECULAR MECHANISMS OF GLUCOCORTICOID RESISTANCE IN AIRWAY DISEASE As mentioned above, an important feature of severe asthma and COPD is glucocorticoid resistance Regarding COPD patients, several large studies suggest that long-term treatment with corticosteroids did not stop the inexorable decline of lung function. This is consistent with the demonstration that inhaled or oral corticosteroids fail to reduce inflammatory cell numbers, cytokines, chemokines, or proteases in induced sputum or bronchial biopsies of patients with COPD At a molecular level, resistance to the anti-inflammatory effects of glucocorticoids can be induced by several mechanisms. The reduction in corticosteroid responsiveness observed in cells from these subjects has been ascribed to reduced number of GR, altered affinity of the ligand for GR, reduced ability of the GR to bind to DNA, or increased expression of inflammatory transcription factors, such as AP-1, that compete for DNA binding Defects in GR Sequence and Pharmacokinetics Unlike familial glucocorticoid resistance where there is a mutation in the LBD of GR and a subsequent resetting of the basal cortisol level, corticosteroid-resistant (CR) patients have normal cortisol levels and are not addisonian It has previously been demonstrated, using whole cell binding assays, that no significant changes exist in monocyte and T-cell binding affinity (K d ), receptor density, and expression of the GR in patients with glucocorticoid-resistant asthma The mechanism of IL-2/IL-4, or IL-13 alone, induced defect of ligand binding characteristics have been explained in two ways. Leung and colleagues have associated these changes with an increased expression of the dominant negative isoform of GR, GRβ[94], although others have been unable to detect enhanced GRβ expression in PBMCs from these glucocorticoid-resistant patients Ito et al.: Mode of Glucocorticoid Actions in Airway Disease TheScientificWorldJOURNAL (2006) 6, 1750-1769 1760 Furthermore, Zhou et al. demonstrated at least 7 isoforms of GR by alternative splicing such as GRα, GRβ, GRγ, GR-P, GR-A, and the relative levels of these variants play a role in differential glucocorticoid-induced responsiveness GR Nuclear Translocation and GR/GRE Binding In one subgroup of severe patients, nuclear localization of GR in response to a high concentration (10 -6 M) of dexamethasone is impaired In a separate subgroup of severe asthma patients, GR nuclear translocation is normal, but dexamethasone cannot correctly stimulate histone H4 lysine (K)5 acetylation Cross-Talk with Other Transcription Factors Transcription factors, especially AP-1, were reported to be excessively activated in glucocorticoid-resistant asthma, in addition to a reduced ability of GR to interact and repress AP-1 activity Neutrophilic Inflammation Neutrophils have been implicated in the pathogenesis of many diseases including COPD, severe asthma, psoriasis, and a variety of collagen-vascular diseases Formation of 52-and 30-kD GR fragments due to proteolysis by neutrophil elastase (a 28-kD serine protease) is found in cytosol of leukemia cells Latent Viral Infection Ito et al.: Mode of Glucocorticoid Actions in Airway Disease TheScientificWorldJOURNAL (2006) 6, 1750-1769 1761 Epidemiologic studies have implicated childhood respiratory infection as an independent risk factor for the subsequent development of persistent asthma and COPD Reduced HDAC Activity in COPD and Severe Asthma We have demonstrated that in peripheral lung tissue and alveolar macrophages, there is a reduction in HDAC activity and HDAC2 expression in normal smokers, and a striking reduction in patients with COPD Oxidative Stress in COPD Exhaled markers of oxidative stress, such as 8-isoprostane and ethane, are increased in normal smokers with a much greater increase in patients with COPD, even when they have stopped smoking Ito et al.: Mode of Glucocorticoid Actions in Airway Disease TheScientificWorldJOURNAL (2006) 6, 1750-1769 1762 FIGURE 5. Proposed mechanism of glucocorticoid insensitivity in COPD patients. In normal alveolar macrophages, NF-κB and other transcription factors are activated following stimulation, and switch on HAT, leading to histone acetylation and, subsequently, to the transcription of genes encoding inflammatory proteins, such as TNF-α, IL-8, and MMP-9. Glucocorticoids reverse this by binding to GR and recruiting HDAC2. This reverses the histone acetylation induced by NF-κB and switches off the activated inflammatory genes. However, in COPD patients, cigarette smoke activates macrophages, but oxidative stress (acting through the formation of peroxynitrite) impairs the activity of HDAC2. This amplifies the inflammatory response to NF-κB activation, but also reduces the anti-inflammatory effect of glucocorticoids, as HDAC2 is now unable to reverse histone acetylation. This figure is adapted from Barnes et al. THERAPEUTIC IMPLICATION Inhaled glucocorticoids are now used as first-line therapy for the treatment of persistent asthma in adults and children in many countries, as they are the most effective treatments for asthma currently available Dissociated Corticosteroids There is an ongoing search for novel glucocorticoids that selectively transrepress without significant transactivation. The major task in developing these drugs is to dissociate the anti-inflammatory effects from the endocrine actions that are associated with side effects. Recently, a novel class of glucocorticoids has been described in which there is potent transrepression with relatively little transactivation. These &quot;dissociated&quot; glucocorticoids, including RU24858, RU40066, and ZK216348, have anti-inflammatory effects in vitro, although there is little or some separation of antiinflammatory effects and systemic side effects in vivo Ito et al.: Mode of Glucocorticoid Actions in Airway Disease TheScientificWorldJOURNAL (2006) 6, 1750-1769 1763 Restoration of Glucocorticoid Action As discussed above, oxidative stress is markedly increased in severe asthma and in patients with COPD, and can attenuate GR function via reduction of HDAC2 or excessive activation of transcriptional factors. Since oxidative and nitrative stress may drive the glucocorticoid insensitivity, antioxidants such as N-acetyl cysteine (NAC), fudostein, or inhibitors of inducible NO synthase (NOS2) should reverse glucocorticoid resistance. A couple of clinical studies with NAC in patients with COPD have produced variable results As well as HDAC reduction, defect of GR nuclear translocation is another important impairment of GR action. We recently showed that long-acting β 2 -agonist (LABA) is more effective in these patients. This combination therapy of LABA and glucocorticoid is established as a more effective therapy in asthma and also in COPD Nonsteroidal Anti-Inflammatory Treatments: Glucocorticoid-Sparing Therapy A variety of anti-inflammatory compounds are now being developed. Many of the anti-inflammatory effects of glucocorticoids appear to be mediated via inhibition of the transcriptional effects of NF-κB, and smallmolecule inhibitors of IKK (inhibitor of I-κB kinase-2) that activate NF-κB are in development CONCLUSION Current systemic and inhaled pharmacological treatment of severe asthma and COPD is unsatisfactory as it does not significantly influence the severity of the disease or its natural course apart from the treatment of exacerbations. The identification of an active resistance mechanism suggests that glucocorticoid resistance/insensitivity is potentially reversible, which would have enormous implications for the future therapy of chronic inflammatory diseases. Since oxidative and nitrative stress may inactivate HDAC2, interfere with the action of other HDACs, alter cofactor recruitment, or induce post-translational modification of GR, antioxidants, HDAC activators, and kinase inhibitors are likely to prove effective in restoring corticosteroid sensitivity in these diseases

Atorvastatin in combination with inhaled beclometasone modulates inflammatory sputum mediators in smokers with asthma

Background: Statins have pleiotropic immunomodulatory effects that may be beneficial in the treatment of asthma. We previously reported that treatment with atorvastatin improved asthma symptoms in smokers with asthma in the absence of a change in the concentration of a selection of sputum inflammatory mediators. Objective: To determine the effects of atorvastatin alone and in combination with inhaled corticosteroid on a range of sputum cytokines, chemokines and growth factors implicated in the pathogenesis of asthma, and their association with asthma control questionnaire (ACQ) and/or asthma quality of life questionnaire (AQLQ) scores. Methods: Sputum samples were analysed from a sub-group of 39 smokers with mild to moderate asthma recruited to a randomised controlled trial comparing atorvastatin (40mg/day) versus placebo for four weeks, followed by inhaled beclometasone (400μg/day) for a further four weeks. Induced sputum supernatant fluid was analysed (Luminex or biochemical analyses) for concentrations of 35 mediators. Results: Sputum mediator concentrations were not reduced by inhaled beclometasone alone. Atorvastatin significantly reduced sputum concentrations of CCL7, IL-12p70, sCD40L, FGF-2, CCL4, TGF-α and MMP-8 compared with placebo and, when combined with inhaled beclometasone, reduced sputum concentrations of MMP-8, IL-1β, IL-10, MMP-9, sCD40L, FGF-2, IL-7, G-CSF and CCL7 compared to ICS alone. Improvements in ACQ and/or AQLQ scores with atorvastatin and ICS were associated with decreases in G-CSF, IL-7, CCL2 and CXCL8. Conclusion: Short-term treatment with atorvastatin alone or in combination with inhaled beclometasone reduces several sputum cytokines, chemokines and growth factors concentrations unresponsive to inhaled corticosteroids alone, in smokers with asthma.</p

RV568, a narrow-spectrum kinase inhibitor with p38 MAPK-α and -γ selectivity, suppresses COPD inflammation

Novel anti-inflammatory approaches targeting chronically activated kinase pathways in chronic obstructive pulmonary disease (COPD) are needed. We evaluated RV568, a p38 mitogen-activated protein kinase-α and -γ and SRC family kinase inhibitor, in cellular and in vivo models relevant to COPD and examined its safety and efficacy in COPD patients.The anti-inflammatory activities of RV568 were tested in primary cultured monocytes, macrophages and bronchial epithelial cells and in vivo in lipopolysaccharide and cigarette smoke-exposed murine models. RV568 was evaluated in a 14-day trial in COPD patients.RV568 showed potent anti-inflammatory effects in monocytes and macrophages, which were often greater than those of corticosteroids or the p38 inhibitor Birb796. RV568 combined with corticosteroid had anti-inflammatory effects suggestive of a synergistic interaction in poly I:C-stimulated BEAS-2B cells and in the cigarette smoke model. In COPD patients, inhaled RV568 (50 µg and 100 µg) improved pre-bronchodilator forced expiratory volume in 1 s (69 mL and 48 mL respectively) and significantly reduced sputum malondialdehyde (p&lt;0.05) compared to placebo, although there were no changes in sputum cell counts. Adverse events during RV568 and placebo treatment were similar.RV568 shows potent anti-inflammatory effects on cell and animal models relevant to COPD. RV568 was well-tolerated and demonstrated a modest clinical benefit in a 14-day COPD clinical trial.</jats:p

Equipment and Procedure Description

This chapter collects the description of the main creep testing procedures in fibre-reinforced concrete (FRC) as well as the equipment used by each participant laboratory in the round-robin test (RRT). The description of each procedure was directly provided by the participants and follows the same structure for each participant where institution identification and contact information was included. Each participant summarised the most significant data such as specimen?s size, load configuration, parameters or environmental conditions in a quite useful table. Moreover, the participants included description and close-up pictures of the creep frames construction, the support boundary conditions, and the measurement devices used. Finally, the participants provided a complete description of the specific complete creep test procedure followed.Fil: Llano Torre, Aitor. Universidad Politécnica de Valencia; EspañaFil: Cavalaro, Sergio H. P.. University of Loughborough; Reino UnidoFil: Kusterle, Wolfgang. University of Applied Sciences; AlemaniaFil: Moro, Sandro. Master Builders Solutions; ItaliaFil: Zerbino, Raul Luis. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata; Argentina. Universidad Nacional de La Plata. Facultad de Ingeniería; ArgentinaFil: Gettu, Ravindra. Indian Institute of Technology Madras. Department of Civil Engineering; IndiaFil: Pauwels, Hans. Tohoku University. School of Engineering. Department of Architecture and Building Science; Japón. Bekaert; BélgicaFil: Nishiwaki, Tomoya. Tohoku University. School of Engineering. Department of Architecture and Building Science; JapónFil: Parmentier, Benoît. Belgium Building Research Institute; BélgicaFil: Buratti, Nicola. Universidad de Bologna; ItaliaFil: Toledo Filho, Romildo D.. Universidade Federal do Estado do Rio de Janeiro; BrasilFil: Charron, Jean-Philippe. Escuela Politécnica de Montreal; CanadáFil: Larive, Catherine. Centre d’Etudes des Tunnels; FranciaFil: Boshoff, William P.. University of Pretoria. Built Environment and Information Technology. Faculty of Engineering, ; SudáfricaFil: Bernard, E. Stefan. Technologies in Structural Engineering; AustraliaFil: Kompatscher, Michael. Versuchsstollen Hagerbach; Suiz