The ratio of cytochrome c oxidase subunit 4 isoform 4I1 and 4I2 mRNA is changed in permanent atrial fibrillation.

AIMS: The conditions of hypoxia are suggested to induce permanent atrial fibrillation (AF). The regulation of COX4I2 and COX4I1 depends on oxygen availability in tissues. A role of COX4I2 in the myocardium of AF patients is supposed for pathogenesis of AF and subsequent alterations in the electron transfer chain (ETC) under hypoxia. METHODS AND RESULTS: In vitro, influence of hypoxia on HeLa 53 cells was studied and elevated parts of COX 4I2 were confirmed. Myocardial biopsies were taken ex vivo from the patients' Right Atria with SR (n = 31) and AF (n = 11), respectively. RT- PCR for mRNA expresson, mitochondrial respiration by polarography and the protein content of cytochrome c oxidase (CytOx) subunit 4I1 and CytOx subunit 4I2 by ELISA were studied. Clinical data were correlated to the findings of gene expressions in parallel. Patients with permanent AF had a change in isoform 4I2/4I1 expression along with a decrease of isoform COX 4I1 expression. The 4I2/4I1 ratio of mRNA expression was increased from 0.630 to 1.058 in comparison. However, the protein content of CytOx subunit 4 was much lower in the AF group, whereas the respiration/units enzyme activity in both groups remained the same. CONCLUSIONS: This study describes a possible molecular correlate for the development of AF. Due to the known functional significance of COX 4I2, mitochondrial dysfunction can be assumed as a part of the pathogenesis of AF.

Long-Term Outcomes Stratified by Age in Patients with a Mechanical versus Biological Mitral Valve Replacement.

Objectives: Balancing anticoagulation and reoperation risks determines prostheses choice (mechanical/biological) for mitral valve replacement. We aimed to re-evaluate the outcomes after biological versus mechanical mitral valve replacement. Methods: We compared long-term benefits and risks of mechanical and biological prostheses in 2056 patients (52% men, 48% women; 65.4 ± 12.1 years) who underwent mitral valve replacements between 1993−2017, in a retrospective single-centre study. Data sources included prospective institutional database, social registry, general practitioner data and follow-up questionnaire. Patients were stratified by age: < = 39 y (n = 82), 40−49 y (n = 164), 50−59 y (n = 335), 60−69 y (n = 593), 70−79 y (n = 743) and > = 80 y (n = 139). Long-term outcomes (mortality, reoperations, bleeding) were analysed. Results: Altogether, 1308 mechanical (53% men, 47% women; 61.5 ± 11.7 years) and 748 biological (50% men, 50% women; 72.3 ± 9.6 years) valves were implanted. The reason for valve replacement was stenosis in 162, insufficiency in 823 and combined in 323 cases for mechanical, while it was 46, 567 and 135 for biological valves, respectively. Overall cumulative survival was higher with mechanical prosthesis (mean: 139 ± 4 vs. 102 ± 5 months, 10 y: 55% vs. 33%, p < 0.0001). Subgroup analysis revealed higher survival among patients receiving mechanical prosthesis up to 60 years (< = 39 y p = 0.047, 40−49 y p < 0.0001, 50−59 y p = 0.001). In patients 60−69 years, overall survival did not differ; however, in survivors beyond 8 years, mechanical prosthesis showed improved survival (p = 0.014). While between 70−79 years survival was nearly identical, for above 80 years, patients had a higher survival with biological prosthesis (p = 0.014). Conclusion: The present data demonstrated a higher survival of mechanical prosthesis in a wide range of patients after mitral valve replacement.

Cytochrome c Oxidase Inhibition by ATP Decreases Mitochondrial ROS Production.

This study addresses the eventual consequence of cytochrome c oxidase (CytOx) inhibition by ATP at high ATP/ADP ratio in isolated rat heart mitochondria. Earlier, it has been demonstrated that the mechanism of allosteric ATP inhibition of CytOx is one of the key regulations of mitochondrial functions. It is relevant that aiming to maintain a high ATP/ADP ratio for the measurement of CytOx activity effectuating the enzymatic inhibition as well as mitochondrial respiration, optimal concentration of mitochondria is critically important. Likewise, only at this concentration, were the differences in ΔΨm and ROS concentrations measured under various conditions significant. Moreover, when CytOx activity was inhibited in the presence of ATP, mitochondrial respiration and ΔΨm both remained static, while the ROS production was markedly decreased. Consubstantial results were found when the electron transport chain was inhibited by antimycin A, letting only CytOx remain functional to support the energy production. This seems to corroborate that the decrease in mitochondrial ROS production is solely the effect of ATP binding to CytOx which results in static respiration as well as membrane potential.

Cholate Disrupts Regulatory Functions of Cytochrome c Oxidase.

Cytochrome c oxidase (CytOx), the oxygen-accepting and rate-limiting enzyme of mitochondrial respiration, binds with 10 molecules of ADP, 7 of which are exchanged by ATP at high ATP/ADP-ratios. These bound ATP and ADP can be exchanged by cholate, which is generally used for the purification of CytOx. Many crystal structures of isolated CytOx were performed with the enzyme isolated from mitochondria using sodium cholate as a detergent. Cholate, however, dimerizes the enzyme isolated in non-ionic detergents and induces a structural change as evident from a spectral change. Consequently, it turns off the "allosteric ATP-inhibition of CytOx", which is reversibly switched on under relaxed conditions via cAMP-dependent phosphorylation and keeps the membrane potential and ROS formation in mitochondria at low levels. This cholate effect gives an insight into the structural-functional relationship of the enzyme with respect to ATP inhibition and its role in mitochondrial respiration and energy production.

Gallbladder Cancer Presenting as Mirizzi Syndrome Complicated by Rapidly Evolving 23 rRNA Gene-Linezolid Resistance with Vancomycin-Resistant Enterococcus Infection Resulting in Fatal Cholangial Sepsis.

We describe the case of a 71-year-old woman who presented with obstructive jaundice and subhilar bile duct stenosis. MRI showed extensive cholecystolithiasis with an impacted bile stone in the cystic duct suggesting Mirizzi syndrome. Delayed enhancement of the thickened gallbladder wall suggested inflammation instead of carcinoma. After drainage of the obstructed bile duct via ERCP, the patient developed liver abscesses with a nosocomial vancomycin-resistant enterococcus infection treated by linezolid. After 4 weeks, the VRE infection was complicated by a new-onset 23 rRNA gene-mediated linezolid resistance in the same bacterial strain, which was proven via core genome multilocus sequencing. Meropenem and tigecycline were administered according to a resistogram. Furthermore, percutaneous transhepatic biliary drainage of both sides of the liver was necessary. After demission, the patient had to be admitted again due to septic shock. An emergency operation revealed extended, inoperable gallbladder cancer. The patient died a few days later in the intensive care unit. An earlier diagnosis of bile duct infiltrating gallbladder cancer by cholangioscopy or laparoscopy and treatment of vancomycin-resistant enterococcus infection with daptomycin may have changed the clinical course of the disease.

Fluorescence Microscopy-Based Quantitation of GLUT4 Translocation: High Throughput or High Content?

Due to the global rise of type 2 diabetes mellitus (T2DM) in combination with insulin resistance, novel compounds to efficiently treat this pandemic disease are needed. Screening for compounds that induce the translocation of glucose transporter 4 (GLUT4) from the intracellular compartments to the plasma membrane in insulin-sensitive tissues is an innovative strategy. Here, we compared the applicability of three fluorescence microscopy-based assays optimized for the quantitation of GLUT4 translocation in simple cell systems. An objective-type scanning total internal reflection fluorescence (TIRF) microscopy approach was shown to have high sensitivity but only moderate throughput. Therefore, we implemented a prism-type TIR reader for the simultaneous analysis of large cell populations grown in adapted microtiter plates. This approach was found to be high throughput and have sufficient sensitivity for the characterization of insulin mimetic compounds in live cells. Finally, we applied confocal microscopy to giant plasma membrane vesicles (GPMVs) formed from GLUT4-expressing cells. While this assay has only limited throughput, it offers the advantage of being less sensitive to insulin mimetic compounds with high autofluorescence. In summary, the combined implementation of different fluorescence microscopy-based approaches enables the quantitation of GLUT4 translocation with high throughput and high content.

Reversible dimerization of cytochrome c oxidase regulates mitochondrial respiration.

Almost all energy consumed by higher organisms, either in the form of ATP or heat, is produced in mitochondria by respiration and oxidative phosphorylation through five protein complexes in the inner membrane. High-resolution x-ray analysis of crystallized cytochrome c oxidase (CytOx), the final oxygen-accepting complex of the respiratory chain, isolated by using cholate as detergent, revealed a dimeric structure with 13 subunits in each monomer. In contrast, CytOx isolated with non-ionic detergents appeared to be monomeric. Our data indicate in vivo a continuous transition between CytOx monomers and dimers via reversible phosphorylation. Increased intracellular calcium, as a consequence of stress, dephosphorylates and monomerises CytOx, whereas cAMP rephosphorylates and dimerises it. Only dimeric CytOx exhibits an "allosteric ATP-inhibition" which inhibits respiration at high cellular ATP/ADP-ratios and could prevent oxygen radical formation and the generation of diseases.

Protamine Sulfate Induces Mitochondrial Hyperpolarization and a Subsequent Increase in Reactive Oxygen Species Production.

Protamine sulfate (PS) is widely used in heart surgery as an antidote for heparin, albeit its pharmacological effects are not fully understood and applications are often accompanied by unwanted side effects. Here we show the effect of PS on mitochondrial bioenergetics profile resulting in mitochondrial reactive oxygen species (ROS) production. Polarographic measurements were performed in parallel to membrane potential and ROS measurements by FACS analyzer using tetramethylrhodamine ethyl ester and MitoSOX fluorescent dyes, respectively. PS inhibited intact rat heart mitochondrial respiration (stimulated by ADP) to 76% (P < 0.001) from the baseline of 51.6 ± 6.9 to 12.4 ± 2.3 nmol O2⋅min-1⋅ml-1 The same effect was found when respiration was inhibited by antimycin A (101.0 ± 8.9 vs. 38.0 ± 9.9 nmol O2 ⋅min-1⋅ml-1, P < 0.001) and later stimulated by substrates of cytochrome oxidase (CytOx) i.e., ascorbate and tetramethyl phenylene diamine, suggesting that PS exerted its effect through inhibition of CytOx activity. Furthermore, the inhibition of mitochondrial respiration by PS was concentration dependent and accompanied by hyperpolarization of the mitochondrial membrane potential (Δψ m), i.e., 18% increase at 50 µg/ml and an additional 3.3% increase at 250 µg/ml PS compared with control. This effect was associated with a strong consequent increase in the production of ROS, i.e., 85% and 88.6% compared with control respectively. We propose that this excessive increase in ROS concentrations results in mitochondrial dysfunction and thus might relate to the "protamine reaction," contributing to the development of various cardiovascular adverse effects.

Mitochondrial active and relaxed state respiration after heat shock mRNA response in the heart.

Induction of Heat Shock Proteins results in cytoprotection. Beneficial effect results from transcription and translational cellular components' involvement that defends metabolism and thus induce ischemic protection of the tissue. Mitochondrial respiration is also involved in stress- induced conditions. It is not a uniform process. Cytochrome c Oxidase (CytOx) representing complex IV of the Electron Transfer Chain (ETC) has a regulatory role for mitochondrial respiratory activity, which is tested in our study after hsp induction. Moreover, protein translation for mitochondrial components was probed by the detection of MT-CO1 for Subunit 1 of CytOx neosynthesis. Wistar rats were subjected to whole-body hyperthermia at 42.0-42.5 °C for 15 min followed by a normothermic recovery period. Heat shock response was monitored time dependent from LV biopsies of all control and heat treated animals with PCR-analysis for hsp 32, 60, 70.1, 70.2, 90 and MT-CO1 expression at 15, 30, 45, 60, 120 and 360 min recovery (n = 5 in each group), respectively. Enzymatic activity of CytOx were evaluated polarographically. High energy phosphates were detected by chromatographic analysis. The mRNA expression of MT-CO1 peaked at 60 min and was accompanied by hsp 32 (r = 0.457; p = 0.037) and hsp 70.2 (r = 0.615; p = 0.003) upregulation. With hsp induction, mitochondrial respiration was increased initially. Enzymatic activity reconciled from active into relaxed status wherein CytOx activity was completely inhibited by ATP. Myocardial ATP content increased from stress induced point i.e. < 1 µmol g-1 protein w/w to finally 1.5 ±â€¯0.53 µmol g-1 protein w/w at 120 min recovery interval. Hyperthermic, myocardial hsp- induction goes along with increased CytOx activity representing an increased "active" mitochondrial respiration. In parallel, de -novo holoenzyme assembly of CytOx begins as shown by MT-CO1 upregulation at 60 min recovery time crossing with a final return to the physiological "relaxed" state and ATP -inhibited respiration.

Combining TIR and FRET in Molecular Test Systems.

Pharmaceutical agents or drugs often have a pronounced impact on protein-protein interactions in cells, and in particular, cell membranes. Changes of molecular conformations as well as of intermolecular interactions may affect dipole-dipole interaction between chromophoric groups, which can be proven by measuring the Förster resonance energy transfer (FRET). If these chromophores are located within or in close proximity to the plasma membrane, they are excited preferentially by an evanescent electromagnetic wave upon total internal reflection (TIR) of an incident laser beam. For the TIR-FRET screening of larger cell collectives, we performed three separate steps: (1) setting up of a membrane associated test system for probing the interaction between the epidermal growth factor receptor (EGFR) and the growth factor receptor-bound protein 2; (2) use of the Epac-SH188 sensor for quantitative evaluation under the microscope; and (3) application of a TIR fluorescence reader to probe the interaction of GFP with Nile Red. In the first two steps, we measured FRET from cyan (CFP) to yellow fluorescent protein (YFP) by spectral analysis and fluorescence lifetime imaging (FLIM) upon illumination of whole cells (epi-illumination) as well as selective illumination of their plasma membranes by TIR. In particular, TIR excitation permitted FRET measurements with high sensitivity and low background. The Epac sensor showed a more rapid response to pharmaceutical agents, e.g., Forskolin or the A2B adenosine receptor agonist NECA, in close proximity to the plasma membrane compared to the cytosol. Finally, FRET from a membrane associated GFP to Nile Red was used to test a multi-well TIR fluorescence reader with simultaneous detection of a larger number of samples.

Echocardiographic criteria to detect unicuspid aortic valve morphology.

Aims: Unicuspid aortic valve (UAV) is a rare congenital malformation associated with severe aortic stenosis or regurgitation. This study aimed to systematically determine echocardiographic criteria to identify UAV. Methods and results: All patients underwent a preoperative baseline examination, including echocardiography. A total of 69 patients with intraoperatively confirmed UAV underwent an aortic valve repair procedure between August 2001 and May 2011. To compare the findings of UAV cases with those of other valve morphologies, we examined 99 consecutive patients with a bicuspid aortic valve (BAV) and 103 consecutive patients with a tricuspid aortic valve (TAV) undergoing isolated aortic valve surgery before May 2016. The mean age of the 271 patients was 44.2 ± 12.8 years; 85% were male, with a mean body mass index of 26.2 ± 4.0 kg/m2. Patients with UAV were younger and had fewer co-morbidities than patients with BAV or TAV, respectively. The major criteria for the echocardiographic diagnosis of UAV were defined based on our preoperative examination as follows: (i) single commissural attachment zone, (ii) rounded, leaflet-free edge on the opposite side of the commissural attachment zone, (iii) eccentric valvular orifice during systole, and (iv) patient age <20 years and mean transvalvular gradient >15 mmHg. The minor criteria were defined as an associated thoracic aortopathy and age <40 years. Three out of the four major criteria or two out of the four major criteria and one minor criterion were met in all patients with UAV and in none of the patients with BAV or TAV. Associated 95% confidence intervals were calculated for each estimate of sensitivity (94.7-100%) and specificity (98.1-100%), indicating that an adequate number of patients were included in each of the three groups. Conclusion: The proposed echocardiographic score appears to be a specific and sensitive method to distinguish UAV from BAV and TAV.

Myocardial insufficiency is related to reduced subunit 4 content of cytochrome c oxidase.

BACKGROUND: Treatment of heart failure remains one of the most challenging task for intensive care medicine, cardiology and cardiac surgery. New options and better indicators are always required. Understanding the basic mechanisms underlying heart failure promote the development of adjusted therapy e.g. assist devices and monitoring of recovery. If cardiac failure is related to compromised cellular respiration of the heart, remains unclear. Myocardial respiration depends on Cytochrome c- Oxidase (CytOx) activity representing the rate limiting step for the mitochondrial respiratory chain. The enzymatic activity as well as mRNA expression of enzyme's mitochondrial encoded catalytic subunit 2, nuclear encoded regulatory subunit 4 and protein contents were studied in biopsies of cardiac patients suffering from myocardial insufficiency and dilated cardiomyopathy (DCM). METHODS: Fifty-four patients were enrolled in the study and underwent coronary angiography. Thirty male patients (mean age: 45 +/- 15 yrs.) had a reduced ejection fraction (EF) 35 ± 12% below 45% and a left ventricular end diastolic diameter (LVEDD) of 71 ± 10 mm bigger than 56 mm. They were diagnosed as having idiopathic dilated cardiomyopathy (DCM) without coronary heart disease and NYHA-class 3 and 4. Additionally, 24 male patients (mean age: 52 +/- 11 yrs.) after exclusion of secondary cardiomyopathies, coronary artery or valve disease, served as control (EF: 68 ± 7, LVEDD: 51 ± 7 mm). Total RNA was extracted from two biopsies of each person. Real-time PCR analysis was performed with specific primers followed by a melt curve analysis. Corresponding protein expression in the tissue was studied with immune-histochemistry while enzymatic activity was evaluated by spectroscopy. RESULTS: Gene and protein expression analysis of patients showed a significant decrease of subunit 4 (1.1 vs. 0.6, p < 0.001; 7.7 ± 3.1% vs. 2.8 ± 1.4%, p < 0.0001) but no differences in subunit 2. Correlations were found between reduced subunit 2 expression, low EF (r = 0.766, p < 0.00045) and increased LVEDD (r = 0.492, p < 0.0068). In case of DCM less subunit 4 expression and reduced shortening fraction (r = 0.524, p < 0.017) was found, but enzymatic activity was higher (0.08 ± 0.06 vs. 0.26 ± 0.08 U/mg, p < 0.001) although myocardial oxygen consumption continued to the same extent. CONCLUSION: In case of myocardial insufficiency and DCM, decreased expression of COX 4 results in an impaired CytOx activity. Higher enzymatic activity but equal oxygen consumption contribute to the pathophysiology of the myocardial insufficiency and appears as an indicator of oxidative stress. This kind of dysregulation should be in the focus for the development of diagnostic and therapy procedures.

Axial tomography in live cell laser microscopy.

Single cell microscopy in a three-dimensional (3-D) environment is reported. Cells are grown in an agarose culture gel, located within microcapillaries and observed from different sides after adaptation of an innovative device for sample rotation. Thus, z -stacks can be recorded by confocal microscopy in different directions and used for illustration in 3-D. This gives additional information, since cells or organelles that appear superimposed in one direction, may be well resolved in another one. The method is tested and validated with single cells expressing a membrane or a mitochondrially associated green fluorescent protein, or cells accumulating fluorescent quantum dots. In addition, axial tomography supports measurements of cellular uptake and distribution of the anticancer drug doxorubicin in the nucleus (2 to 6 h after incubation) or the cytoplasm (24 h). This paper discusses that upon cell rotation an enhanced optical resolution in lateral direction compared to axial direction can be utilized to obtain an improved effective 3-D resolution, which represents an important step toward super-resolution microscopy of living cells.

Mitochondrial cytochrome c oxidase is inhibited by ATP only at very high ATP/ADP ratios.

In the past, divergent results have been reported based on different methods and conditions used for enzymatic activity measurements of cytochrome c oxidase (CytOx). Here, we analyze in detail and show comparable and reproducible polarographic activity measurements of ATP-dependent inhibition of CytOx kinetics in intact and non-intact rat heart mitochondria and mitoplasts. We found that this mechanism is always present in isolated rat heart mitochondria and mitoplasts; however, it is measurable only at high ATP/ADP ratios using optimal protein concentrations. In the kinetics assay, measurement of this mechanism is independent of presence or absence of Tween-20 and the composition of measuring buffer. Furthermore, the effect of atractyloside on intact rat heart mitochondria confirms that (i) ATP inhibition occurs under uncoupled conditions [in the presence of carbonly cyanide m-chlorophenyl hydrazone (CCCP)] when the classical respiratory control is absent and (ii) high ATP/ADP ratios in the matrix as well as in the cytosolic space are required for full ATP inhibition of CytOx. Additionally, ATP inhibition measured in intact mitochondria extends in the presence of oligomycin, thus indicating further that the problem to measure the inhibitory effect of ATP on CytOx is apparently due to the lack of very high ATP/ADP ratios in isolated mitochondria.

Revisiting Kadenbach: Electron flux rate through cytochrome c-oxidase determines the ATP-inhibitory effect and subsequent production of ROS.

Mitochondrial respiration is the predominant source of ATP. Excessive rates of electron transport cause a higher production of harmful reactive oxygen species (ROS). There are two regulatory mechanisms known. The first, according to Mitchel, is dependent on the mitochondrial membrane potential that drives ATP synthase for ATP production, and the second, the Kadenbach mechanism, is focussed on the binding of ATP to Cytochrome c Oxidase (CytOx) at high ATP/ADP ratios, which results in an allosteric conformational change to CytOx, causing inhibition. In times of stress, ATP-dependent inhibition is switched off and the activity of CytOx is exclusively determined by the membrane potential, leading to an increase in ROS production. The second mechanism for respiratory control depends on the quantity of electron transfer to the Heme aa3 of CytOx. When ATP is bound to CytOx the enzyme is inhibited, and ROS formation is decreased, although the mitochondrial membrane potential is increased.

Modifications of carbon black nanoparticle surfaces modulate type II pneumocyte homoeostasis.

Inhalation uptake of carbon black nanoparticles (CBNP) bears the risk of morphological and functional lung impairment attributed to the highly reactive particle surface area. Chemical particle surface modifications might affect particle-cell interactions; however, thus far these alterations have not been determined. This is the first in vivo study comparing particle-induced acute lung injury using Printex(®)90 (Pr90, 7 µg), Printex®90 covered by benzo[a]pyrene or 9-nitroanthracene (BaP-Pr90, NA-Pr90, 7 µg, 15% BaP or NA by weight), and acetylene carbon black (CB) with polycyclic aromatic hydrocarbons (PAH-AB, 7 µg, 20% PAH by weight). All particles were suspended in distilled water with bovine serum albumin (BSA). In addition, the influence of suspension media was tested using Printex®90 suspended without BSA (Pr90(-BSA), 7 µg). Quartz (DQ12, 7 µg), 70 µl saline (NaCl), and distilled water with or without BSA (H2O(+/-BSA)) were used as reference and controls. It was postulated that CBNP surface modifications trigger pulmonary responses. After oropharyngeal particle aspiration, lung functions were measured 2 d postexposure, followed by lung preparation for histological or bronchoalveolar lavage fluid (BALF) examinations and type II pneumocyte isolation on d 3. Head-out body plethysmography revealed reduced flow rates induced by PAH-AB. Examinations of BALF demonstrated reduced influx of macrophages after exposure to Pr90(-BSA) and decreased lymphocyte levels after Pr90(+BSA) or BaP-Pr90 treatment. Further, CBNP induced changes in mRNA expressions (surfactant proteins) in type II pneumocytes. These findings indicate that CBNP surface area and media modulate interactions between NP and lung cells in short-term experiments.

Monitoring of apoptosis in 3D cell cultures by FRET and light sheet fluorescence microscopy.

Non-radiative cell membrane associated Förster Resonance Energy Transfer (FRET) from an enhanced cyan fluorescent protein (ECFP) to an enhanced yellow fluorescent protein (EYFP) is used for detection of apoptosis in 3-dimensional cell cultures. FRET is visualized in multi-cellular tumor spheroids by light sheet based fluorescence microscopy in combination with microspectral analysis and fluorescence lifetime imaging (FLIM). Upon application of staurosporine and to some extent after treatment with phorbol-12-myristate-13-acetate (PMA), a specific activator of protein kinase c, the caspase-3 sensitive peptide linker DEVD is cleaved. This results in a reduction of acceptor (EYFP) fluorescence as well as a prolongation of the fluorescence lifetime of the donor (ECFP). Fluorescence spectra and lifetimes may, therefore, be used for monitoring of apoptosis in a realistic 3-dimensional system, while light sheet based microscopy appears appropriate for 3D imaging at low light exposure.

Nitration of protein without allergenic potential triggers modulation of antioxidant response in type II pneumocytes.

Inhalation of nitrogen and reactive oxygen species (ROS) is known to induce lung inflammation, which is prevented by enzymatic and nonenzymatic antioxidant systems. These agents form nitrated allergens that were shown to enhance allergenicity. The aim of this study was to examine the influence of nitrated proteins on inflammation and antioxidant status of the lung. Ovalbumin (OVA) in nitrated form (nOVA) was intraperitoneally (ip) injected in mice for sensitization and in nitrated or unmodified form for challenge to induce allergic bronchial inflammation. To study the allergen potential of unrelated protein and verify cross-reactivity, nitrated and unmodified keyhole limpet hemocyanin (nKLH, KLH) was used for challenge. Challenge with OVA or nOVA reduced lung function and increased eosinophilia and protein content in bronchoalveolar lavage fluid (BALF). Challenge with nitrated or native OVA or KLH elevated glutathione (GSH) ratio in type II pneumocytes. Reduced mRNA expression of glutathione peroxidase (GPX) 3, glutathione reductase (GR), superoxide dismutase (SOD) 2, and catalase (CAT) was most prominent after challenge with nitrated OVA and nitrated KLH, respectively. Challenge with nOVA enhanced SOD1 mRNA reduction. Immunostaining of GPX 3 and SOD2 increased after challenge with OVA or nOVA, while reactivity of GR and reactivity of SOD2 were reduced after challenge with KLH or nKLH. SOD1 immunostaining was diminished after challenge with nonnitrated OVA or KLH. CAT immunoreaction was similar in all groups. Nitrated proteins without allergenic potential triggered mRNA reduction of antioxidants in type II cells after sensitization with a nitrated allergen but did not induce bronchial inflammation.

C1-esterase-inhibitor for primary graft dysfunction in lung transplantation.

BACKGROUND: Primary graft dysfunction (PGD) is the most important cause of early morbidity and mortality in lung transplantation (LTX) with an incidence of 8% to 20%. We hypothesized that application of C1-esterase-inhibitor (C1-INH) in LTX-recipients showing early signs of severe PGD would attenuate the condition. METHODS: Starting as of May 2010, all recipients showing a PaO2/FiO2 ratio of less than 100 as early sign of PGD at first measurement in the OR were immediately treated with C1-INH. Postoperative courses of C1-INH-treated recipients were compared with a subgroup of recipients that developed severe PGD (PGD3-group) within 72 hours after LTX but did not receive C1-INH. Additionally, a third group consisting of all remaining recipients was assembled. RESULTS: A total of 275 LTX were performed between May 2010 and September 2012 at our center. Among these, 24 patients (8.7%) revealed a first PaO2/FiO2 ratio less than 100 and were treated with C1-INH (C1-INH-group). The PGD3-group consisted of 14 patients; the control cohort consisted of 237 patients. PGD scores were significantly higher in the C1-INH-group and PGD3-group as compared with the control group at all times postoperatively. ICU stay was longest in the PGD3 cohort and prolonged in C1-INH patients compared with the control group (29 [2-70] vs. 9 [2-83] vs. 3 [1-166] days, P=0.002). One-year survival in the PGD3-cohort was 71.4%, the C1-INH-treated-group had a one-year-survival of 82.5%, the control group had the best outcome (95%) (P=0.001). CONCLUSION: Treatment of PGD with C1-INH led to acceptable outcome. Although survival in the C1-INH treated patients was lower than in the remaining collective, it was as good or better, compared with the PGD3 group and as what is internationally regarded as reasonable after LTX.

Nanosecond ratio imaging of redox states in tumor cell spheroids using light sheet-based fluorescence microscopy.

A new concept of three-dimensional imaging of tumor cell spheroids by light sheet-based fluorescence microscopy and nanosecond ratio imaging is described. Due to its low light dose and alternative excitation by two laser wavelengths (391 and 470 nm), this method maintains cell viability and permits recording of real-time kinetics. A genetically encoded sensor permits measurement of the redox state of glutathione and visualization of the impact of oxygen radicals. The pharmaceutically relevant system is tested upon addition of an oxidizing agent (H2O2), as well as upon addition of the apoptosis-inducing agent staurosporine.