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Introduction: The Activated Clotting Time (ACT) is commonly used to manage anticoagulation during cardiac surgery. The aim of this study was to compare the older manually operated Hemochron® Response and the automated Hemochron® Signature Elite. Methods: In this observational study the clinically relevant differences of both devices were investigated simultaneously, using duplicate measurements, in 29 patients who underwent a Coronary Artery Bypass Grafting (CABG) or Aortic Valve Replacement (AVR) in order to determine reliability, bias, and to detect which method has the lowest variation. Blood samples were obtained from the arterial line prior to surgery, after administration of 300 IU/kg heparin, 5 minutes after initiation of cardiopulmonary bypass and successively every 30 minutes, and after protamine administration. Results: A total of 202 measurements were performed. Of these 10 measurements were out of range in the Response and 9 in the Elite. About 27 single unstable magnet errors were seen in the Response versus no measurement errors in the Elite. No statistically significant differences between the Response (p = 0.22, Wilcoxon rank) and Elite (p = 0.064) duplicates were observed. The Response values were consistently higher during heparinization than the Elite measurements (p = 0.002, repeated measurements) with an average positive bias of around 56 seconds during heparinization (Bland-Altman). Overall, the coefficient of variation (CoV) increased during heparinization. Conclusion: The Elite was more reliable, but the variation was higher for the Elite than the Response. The observed positive bias in the Response compared to the Elite could affect heparin administration during surgery making the two systems not interchangeable.

Background Despite systemic anticoagulation and antithrombotic surface coating, oxygenator dysfunction remains one of most common technical complications of Extracorporeal membrane oxygenation (ECMO). Several parameters have been associated with an oxygenator exchange, but no guidelines for when to perform an exchange are published. An exchange, especially an emergency exchange, has a risk of complications. Therefore, a delicate balance between oxygenator dysfunction and the exchange of the oxygenator exists. This study aimed to identify risk factors and predictors for elective and emergency oxygenator exchanges. Methods This observational cohort study included all adult patients supported with veno-venous extracorporeal membrane oxygenation (V-V ECMO). We compared patients’ characteristics and laboratory values of patients with and without an oxygenator exchange and between an elective and emergency exchange, defined as an exchange outside office hours. Risk factors for an oxygenator exchange were identified with cox regression analyses, and risk factors for an emergency exchange were identified with logistic regression analyses. Results We included forty-five patients in the analyses. There were twenty-nine oxygenator exchanges in nineteen patients (42%). More than a third of the exchanges were emergency exchanges. Higher partial pressure of carbon dioxide (PaCO2), transmembrane pressure difference (ΔP), and hemoglobin (Hb) were associated with an oxygenator exchange. Lower lactate dehydrogenase (LDH) was the only risk factor for an emergency exchange. Conclusion Oxygenator exchange is frequent during V-V ECMO support. PaCO2, ΔP and Hb were associated with an oxygenator exchange and lower LDH with the risk of an emergency exchange.

The Activated Clotting Time (ACT) is commonly used to manage anticoagulation during cardiac surgery. The aim of this study was to compare the older manually operated HemochronIn this observational study the clinically relevant differences of both devices were investigated simultaneously, using duplicate measurements, in 29 patients who underwent a Coronary Artery Bypass Grafting (CABG) or Aortic Valve Replacement (AVR) in order to determine reliability, bias, and to detect which method has the lowest variation. Blood samples were obtained from the arterial line prior to surgery, after administration of 300 IU/kg heparin, 5 minutes after initiation of cardiopulmonary bypass and successively every 30 minutes, and after protamine administration.A total of 202 measurements were performed. Of these 10 measurements were out of range in the Response and 9 in the Elite. About 27 single unstable magnet errors were seen in the Response versus no measurement errors in the Elite. No statistically significant differences between the Response (p = 0.22, Wilcoxon rank) and Elite (p = 0.064) duplicates were observed. The Response values were consistently higher during heparinization than the Elite measurements (p = 0.002, repeated measurements) with an average positive bias of around 56 seconds during heparinization (Bland-Altman). Overall, the coefficient of variation (CoV) increased during heparinization.The Elite was more reliable, but the variation was higher for the Elite than the Response. The observed positive bias in the Response compared to the Elite could affect heparin administration during surgery making the two systems not interchangeable.

mtDNA variations often result in bioenergetic dysfunction inducing a metabolic switch toward glycolysis resulting in an unbalanced pH homeostasis. In hypoxic cells, expression of the tumor-associated carbonic anhydrase IX (CAIX) is enhanced to maintain cellular pH homeostasis. We hypothesized that cells with a dysfunctional oxidative phosphorylation machinery display elevated CAIX expression levels. Increased glycolysis was observed for cytoplasmic 143B mutant hybrid (m.3243A>G, >94.5%) cells (p 500 mm(3)) took longer for mutant hybrid xenografts, but growth rates were comparable with control tumors upon establishment. Previously, it has been shown that HIF-1 alpha is responsible for tumor establishment. In agreement, we found that HIF-1 alpha expression levels and the pimonidazole-positive hypoxic fraction were reduced for the mutant hybrid xenografts. Our results demonstrate that OXPHOS dysfunction leads to a decreased HIF-1 alpha stabilization and subsequently to a reduced expression of its downstream targets and hypoxic fraction in vivo. In contrast, hypoxia-inducible factor 2-alpha (HIF-2 alpha) expression levels in these xenografts were enhanced. Inhibition of mitochondrial function is therefore an interesting approach to increase therapeutic efficacy in hypoxic tumors.

Extracorporeal membrane oxygenation (ECMO) is associated with frequent hemorrhagic and thromboembolic complications. The multiple effects of ECMO include inflammatory response on contact with the circuit, hemolysis acquired von Willebrand syndrome likely affects the function of red blood cells (RBC) and platelets. The aim of this prospective observational study was to analyze RBC aggregation and elongation (deformability) and platelet aggregation in the first week of ECMO. Sixteen patients were included. Blood samples were taken prior to initiation of ECMO and on days 1, 2, 3, 5, and 7. RBC aggregation and elongation were analyzed using the laser-assisted optical rotational red cell analyzer (Lorrca). Upstroke, top, and amplitude as indices of aggregation showed significant time effects. RBC elongation was not affected at low shear stress. At high shear stress there was an increase in the elongation index at day 2 (p = 0.004), followed by a decrease. Platelet function was analyzed using multiple electrode aggregometry (Multiplate&reg, ). In pairwise comparison in the days 1&ndash, 7 to the value prior to ECMO there was no significant difference in platelet aggregation by any of the three agonists (ADP p = 0.61, TRAP p = 0.77, Ristocetin p = 0.25). This implies that the rheology of RBCs seemed to be more affected by ECMO than platelets. Especially the red blood cell deformability continues to decline at higher shear stress.

COPD is characterized by chronic lung inflammation and irreversible lung tissue damage. Inhaled noxious gases, including cigarette smoke, are the major risk factor for COPD. Inhaled smoke first encounters the epithelial lining of the lungs, causing oxidative stress and mitochondrial dysfunction. We investigated whether a mitochondrial defect may contribute to increased lung epithelial pro-inflammatory responses, impaired epithelial repair and reduced corticosteroid sensitivity as observed in COPD. We used wild-type alveolar epithelial cells A549 and mitochondrial DNA-depleted A549 cells (A549 Rho-0) and studied pro-inflammatory responses using (multiplex) ELISA as well as epithelial barrier function and repair (real-time impedance measurements), in the presence and absence of the inhaled corticosteroid budesonide. We observed that A549 Rho-0 cells secrete higher levels of pro-inflammatory cytokines than wild-type A549 cells and display impaired repair upon wounding. Budesonide strongly suppressed the production of neutrophil attractant CXCL8, and promoted epithelial integrity in A549 wild-type cells, while A549 Rho-0 cells displayed reduced corticosteroid sensitivity compared to wild-type cells. The reduced corticosteroid responsiveness may be mediated by glycolytic reprogramming, specifically glycolysis-associated PI3K signaling, as PI3K inhibitor LY294002 restored the sensitivity of CXCL8 secretion to corticosteroids in A549 Rho-0 cells. In conclusion, mitochondrial defects may lead to increased lung epithelial pro-inflammatory responses, reduced epithelial repair and reduced corticosteroid responsiveness in lung epithelium, thus potentially contributing to the pathogenesis of COPD.

Several clinically used drugs are mitotoxic causing mitochondrial DNA (mtDNA) variations, and thereby influence cancer treatment response. We hypothesized that radiation responsiveness will be enhanced in cellular models with decreased mtDNA content, attributed to altered reactive oxygen species (ROS) production and antioxidant capacity. For this purpose BEAS-2B, A549, and 143B cell lines were depleted from their mtDNA (ρ0). Overall survival after irradiation was increased (p

Background We hypothesised that increased oxidative stress, as present in the airways of asthma and chronic obstructive pulmonary disease (COPD) patients, induces epithelial damage and reduces epithelial responsiveness to suppressive effects of corticosteroids on proinflammatory cytokine production and barrier function.Methods We induced oxidative stress by H2O2 and/or cigarette smoke extract (CSE) in human bronchial epithelial 16HBE cells and primary bronchial epithelial cells (PBEC) derived by brushings from asthma patients, COPD patients, and smoking and non-smoking control individuals. We investigated effects of budesonide on barrier function (electrical resistance) and TNF-alpha-induced proinflammatory cytokine production (IL-8/CXCL8, granulocyte macrophage-colony stimulating factor (GM-CSF)).Results We observed that H2O2 and CSE reduce epithelial resistance. Budesonide significantly counteracted this effect, likely by protection against epidermal growth factor receptor-dependent cell-cell contact disruption. Furthermore, budesonide suppressed proinflammatory cytokine production. H2O2 pretreatment reduced this effect of budesonide on cytokine production in both 16HBE cells and PBECs. Importantly, PBECs from asthma and COPD patients were less sensitive to budesonide with respect to cytokine production and barrier function than PBECs from control subjects.Conclusions Together, our data indicate that budesonide suppresses epithelial proinflammatory responses and barrier dysfunction and that oxidative stress reduces these effects in airway epithelium from asthma and COPD patients. Therefore, restoration of corticosteroid responsiveness in asthma and COPD may act to improve the airway epithelial barrier.

Betaine uptake in Listeria monocytogenes is mediated by three independent transport systems, the simplest of which in genetic terms is the secondary transporter BetL. Using a random mutagenesis approach, based on the E. coli XL1 Red mutator strain, we identified a single point mutation in a putative promoter region upstream of the BetL coding region which leads to a significant increase in betL transcript levels under osmo- and chill-stress conditions and a concomitant increase in stress tolerance. Furthermore, the mutation appears to counter the heretofore unreported “twisted” cell morphology observed for L. monocytogenes grown at elevated osmolarities in tryptone soy broth.

Betaine uptake in Listeria monocytogenes is mediated by three independent transport systems, the simplest of which in genetic terms is the secondary transporter BetL. Using a random mutagenesis approach, based on the E. coli XL1 Red mutator strain, we identified a single point mutation in a putative promoter region upstream of the BetL coding region which leads to a significant increase in betL transcript levels under osmo- and chill-stress conditions and a concomitant increase in stress tolerance. Furthermore, the mutation appears to counter the heretofore unreported "twisted" cell morphology observed for L. monocytogenes grown at elevated osmolarities in tryptone soy broth.

A subset of asthma patients suffer from glucocorticoid (GC) insensitivity. T-helper cell type 17 cells have an emerging role in GC insensitivity, although the mechanisms are still poorly understood.We investigated whether interleukin (IL)-17A induces GC insensitivity in airway epithelium by studying its effects on responsiveness of tumour necrosis factor (TNF)-alpha-induced IL-8 production to budesonide in human bronchial epithelial 16HBE cells. We unravelled the underlying mechanism by the use of specific pathway inhibitors, reporter and overexpression constructs and a histone deacetylase (HDAC) activity assay.We demonstrated that IL-17A-induced IL-8 production is normally sensitive to GCs, while IL-17A pre-treatment significantly reduced the sensitivity of TNF-alpha-induced IL-8 production to budesonide. IL-17A activated the p38, extracellular signal-related kinase (ERK) and phosphoinositide-3-kinase (PI3K) pathways, and the latter appeared to be involved in IL-17A-induced GC insensitivity. Furthermore, IL-17A reduced HDAC activity, and overexpression of HDAC2 reversed IL-17A-induced GC insensitivity. In contrast, IL-17A did not affect budesonide-induced transcriptional activity of the GC receptor, suggesting that IL-17A does not impair the actions of the ligated GC receptor.In conclusion, we have shown for the first time that IL-17A induces GC insensitivity in airway epithelium, which is probably mediated by PI3K activation and subsequent reduction of HDAC2 activity. Thus, blockade of IL-17A or downstream signalling molecule PI3K may offer new strategies for therapeutic intervention in GC-insensitive asthma.

Several mutations in nuclear genes encoding for mitochondrial components have been associated with an increased cancer risk or are even causative, e.g. succinate dehydrogenase (SDHB, SDHC and SDHD genes) and iso-citrate dehydrogenase (IDH1 and IDH2 genes). Recently, studies have suggested an eminent role for mitochondrial DNA (mtDNA) mutations in the development of a wide variety of cancers. Various studies associated mtDNA abnormalities, including mutations, deletions, inversions and copy number alterations, with mitochondrial dysfunction. This might, explain the hampered cellular bioenergetics in many cancer cell types. Germline (e.g. m.10398A>G; m.6253T>C) and somatic mtDNA mutations as well as differences in mtDNA copy number seem to be associated with cancer risk. It seems that mtDNA can contribute as driver or as complementary gene mutation according to the multiple-hit model. This can enhance the mutagenic/clonogenic potential of the cell as observed for m.8993T>G or influences the metastatic potential in later stages of cancer progression. Alternatively, other mtDNA variations will be innocent passenger mutations in a tumor and therefore do not contribute to the tumorigenic or metastatic potential. In this review, we discuss how reported mtDNA variations interfere with cancer treatment and what implications this has on current successful pharmaceutical interventions. Mutations in MT-ND4 and mtDNA depletion have been reported to be involved in cisplatin resistance. Pharmaceutical impairment of OXPHOS by metformin can increase the efficiency of radiotherapy. To study mitochondrial dysfunction in cancer, different cellular models (like p(o) cells or cybrids), in vivo murine models (xenografts and specific mtDNA mouse models in combination with a spontaneous cancer mouse model) and small animal models (e.g. Danio rerio) could be potentially interesting to use. For future research, we foresee that unraveling mtDNA variations can contribute to personalized therapy for specific cancer types and improve the outcome of the disease. (C) 2015 The Authors. Published by Elsevier B.V.

Rationale: Cigarette smoke is the major risk factor in the development of chronic obstructive pulmonary disease (COPD). Lipidomics is a novel and emerging research field that may provide new insights in the origins of chronic inflammatory diseases, such as COPD.Objectives: To investigate whether expression of the sputum lipidome is affected by COPD or cigarette smoking.Methods: Lipid expression was investigated with liquid chromatography and high-resolution quadrupole time-of-flight mass spectrometry in induced sputum comparing smokers with and without COPD, and never-smokers. Changes in lipid expression after 2-month smoking cessation were investigated in smokers with and without COPD.Measurements and Main Results: More than 1,500 lipid compounds were identified in sputum. The class of sphingolipids was significantly higher expressed in smokers with COPD than in smokers without COPD. At single compound level, 168 sphingolipids, 36 phosphatidylethanolamine lipids, and 5 tobacco-related compounds were significantly higher expressed in smokers with COPD compared with smokers without COPD. The 13 lipids with a high fold change between smokers with and without COPD showed high correlations with lower lung function and inflammation in sputum. Twenty (glyco)sphingolipids and six tobacco-related compounds were higher expressed in smokers without COPD compared with never-smokers. Two-month smoking-cessation reduced expression of 26 sphingolipids in smokers with and without COPD.Conclusions: Expression of lipids from the sphingolipid pathway is higher in smokers with COPD compared with smokers without COPD. Considering their potential biologic properties, they may play a role in the pathogenesis of COPD.

This paper describes a novel strategy to confine transplanted β-cells in a natural immune suppressive matrix barrier which could provide new avenues for insights into β-cell transplantation without the use of harmful medication. Type I diabetes is characterized by autoimmune destruction of the endocrine pancreas islets. In our hypothetical strategy we include various aspects that could be beneficial for successful β-cell transplantation, dependent on the pathology-specific expression of immune response dampening molecules. Here we propose a novel strategy involving local inflammation-specific induction of immunosuppressive proteins by a Symbiotic Defence Cell (SDC). SDC is a helper cell line that could form a surrounding matrix barrier disallowing autoimmune cells to reach and destroy the pancreatic β-cells. This immunosuppressive barrier consists of usable secretion of immunosuppressive proteins like Tumor Necrosis Factor inhibitor (TNFi), Soluble Cytotoxic T Lymphocyte Antigen-4 ligand (CTLA-4 ligand) and Insulin-like Growth Factor-I (IGF-I).

INTRODUCTION: Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease with pulmonary and extra-pulmonary manifestations. Although COPD is a complex disease, diagnosis and staging are still based on simple spirometry measurements. Different COPD phenotypes exist based on clinical, physiological, immunological and radiological observations. Cigarette smoking is the most important risk factor for COPD, but only 15-20% of smokers develop the disease, suggesting a genetic predisposition. Unfortunately, little is known about the pathogenesis of COPD, and even less on the very first steps that are associated with an aberrant response to smoke exposure. This study aims to investigate the underlying local and systemic inflammation of different clinical COPD phenotypes, and acute effects of cigarette smoke exposure in individuals susceptible and non-susceptible for the development of COPD. Furthermore, we will investigate mechanisms associated with corticosteroid insensitivity. Our study will provide valuable information regarding the pathogenetic mechanisms underlying the natural course of COPD.METHODS AND ANALYSIS: This cross-sectional study will include young and old individuals susceptible or non-susceptible to develop COPD. At a young age (18-40 years) 60 'party smokers' will be included who are called susceptible or non-susceptible based on COPD prevalence in smoking family members. In addition, 30 healthy smokers (age 40-75 years) and 110 COPD patients will be included. Measurements will include questionnaires, pulmonary function, low-dose CT scanning of the lung, body composition, 6 min walking distance and biomarkers in peripheral blood, sputum, urine, exhaled breath condensate, epithelial lining fluid, bronchial brushes and biopsies. Non-biased approaches such as proteomics will be performed in blood and epithelial lining fluid.ETHICS AND DISSEMINATION: This multicentre study was approved by the medical ethical committees of UMC Groningen and Utrecht, the Netherlands. The study findings will be presented at conferences and will be reported in peer-reviewed journals.TRIAL REGISTRATION: ClinicalTrials.gov, NCT00807469 (study 1) and NCT00850863 (study 2).

Background Cigarette smoking is the major risk factor for COPD, leading to chronic airway inflammation. We hypothesized that cigarette smoke induces structural and functional changes of airway epithelial mitochondria, with important implications for lung inflammation and COPD pathogenesis. Methods We studied changes in mitochondrial morphology and in expression of markers for mitochondrial capacity, damage/biogenesis and fission/fusion in the human bronchial epithelial cell line BEAS-2B upon 6-months from ex-smoking COPD GOLD stage IV patients to age-matched smoking and never-smoking controls. Results We observed that long-term CSE exposure induces robust changes in mitochondrial structure, including fragmentation, branching and quantity of cristae. The majority of these changes were persistent upon CSE depletion. Furthermore, long-term CSE exposure significantly increased the expression of specific fission/fusion markers (Fis1, Mfn1, Mfn2, Drp1 and Opa1), oxidative phosphorylation (OXPHOS) proteins (Complex II, III and V), and oxidative stress (Mn-SOD) markers. These changes were accompanied by increased levels of the pro-inflammatory mediators IL-6, IL-8, and IL-1β. Importantly, COPD primary bronchial epithelial cells (PBECs) displayed similar changes in mitochondrial morphology as observed in long-term CSE-exposure BEAS-2B cells. Moreover, expression of specific OXPHOS proteins was higher in PBECs from COPD patients than control smokers, as was the expression of mitochondrial stress marker PINK1. Conclusion The observed mitochondrial changes in COPD epithelium are potentially the consequence of long-term exposure to cigarette smoke, leading to impaired mitochondrial function and may play a role in the pathogenesis of COPD. © 2013 Hoffmann et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.