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Background: The neutrophil-lymphocyte ratio (NLR) is a recognized marker of inflammation associated with poor outcomes in various clinical situations. We analyzed the prognostic significance of preoperative elevated NLR in patients undergoing cardiac surgery., Methods: We performed a retrospective review of 3,027 consecutive patients undergoing cardiac surgery. Receiver-operating-characteristic was used to determine the cutoff value for elevated NLR. Multivariate regression was used to determine the predictive value of preoperative NLR on clinical outcomes. Cox proportional hazards functions were used to determine predictors of late events. Late survival data to 16 years was obtained from the Ministry of Interior., Results: The cutoff value for elevated NLR was 2.6. Patients with elevated NLR were older (p < 0.0001), had a higher incidence of cardiac comorbidity (p < 0.0001), and higher European System for Cardiac Operative Risk Evaluation score (p < 0.0001). An elevated NLR emerged as an independent predictor of operative mortality (hazard ratio [HR] 2.15, 95% confidence interval [CI]: 1.51 to 3.08, p < 0.0001); pleural effusion (HR 1.42, 95% CI: 1.13 to 1.80, p = 0.003); low output syndrome (HR 1.54, 95% CI: 1.23 to 1.93, p = 0.0002); prolonged ventilation (HR 1.49, 95% CI: 1.23 to 1.82, p = 0.0001); or composite outcomes (HR 1.61, 95% CI: 1.36 to 1.91, p < 0.0001). The NLR emerged as an independent predictor of late mortality (HR 1.19, 95% CI: 1.11 to 1.28; p < 0.0001)., Conclusions: Elevated NLR is associated with a higher incidence of adverse outcomes after cardiac surgery. It is a predictor of operative as well as late mortality. Further studies are warranted to determine whether prophylactic treatment with antiinflammatory agents can prevent such outcomes. It may be warranted to include the baseline NLR as another variable in risk stratification of patients about to undergo cardiac surgery., (Copyright © 2018 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Epithelial ovarian cancer (EOC) is worldwide the sixth most lethal form of cancer occurring in women. More than one third of ovarian patients have ascites at the time of diagnosis and almost all of them have it when recurrence occurs. Although its effect on tumor cell microenvironment remains poorly understood, its presence is correlated with bad diagnosis. In previous studies, we proposed a novel glycan-based biomarker for the diagnosis of EOC, which showed an improved sensitivity and specificity at any stage of the disease and an improved discrimination between malignant and benign ovarian tumors. In this work, we report for the first time the N-glycome profiles of ascitic fluid from primary serous EOC patients and compare them with the serum N-glycomes of the same patients as well as of healthy controls. N-Glycans were digested from equivalent amount of ascites and serum from 18 EOC patients and from serum of 20 age-matched controls and measured by MALDI-TOF-MS. Ascites N-glycome showed increased antennarity, branching, sialylation and Lewis X motives compared to healthy serum. In addition, a correlation was established between ascites volume and degree of sialylation., Significance: Malignant ascitic fluid is the build-up of large volumes of fluid in the peritoneal cavity secondary to cancer. At least one-third of ovarian cancer patients develop ascites, a generally voluminous fluid containing cells of tumor origin, in the course of cancer and almost all when recurrence occurs. The proteome of ascites is known to be as complex as that of serum and contains high amount of proteins shed from inflammatory cells as well as from tumor cells. Although many attempts have been made to provide molecular insight into the proteomic and peptidomic content of malignant ascites, no data about the N-glycome of the ascitic fluid fraction from cancer patients has been reported to date. In this study, the N-glycosylation profile of ascites from 20 patients suffering from epithelial ovarian cancer (EOC) was analyzed by MALDI-TOF-mass spectrometry and compared to the pathologically modified N-glycan pattern obtained from serum of the same patients as well as to the pattern of serum from healthy individuals. Significant quantitative differences were observed in the ascites of EOC patients when compared to the serum of healthy subjects. The glycome of ascites shows typical features of inflammatory conditions, what was also found in the serum of patients suffering from EOC when compared to healthy serum. In addition, a correlation was established between ascites volume and degree of sialylation, showing that the high-volume ascites contains a higher amount of sialylated structures than the low-volume ascites., (Copyright © 2017 Elsevier B.V. All rights reserved.)

L-selectin mediates extravasation of leukocytes from blood into the surrounding tissue during inflammation and is therefore a therapeutical target in certain overwhelming immune reactions. In this study, we characterized an L-selectin specific blocking DNA aptamer with respect to nucleotide composition and target binding. Introduction of deletions and nucleotide exchanges resulted in an optimized DNA sequence but preservation of the IC50 in the low nanomolar range. The inhibitory potential was significantly increased when the aptamer was displayed as a di- and trimer connected via appropriate linker length. Similar to monoclonal antibodies, trimer yielded picomolar IC50 values in a competitive binding assay. In comparison to the monovalent aptamer, the trivalent assembly reduced PBMC interactions to L-selectin ligands 90-fold under shear and exerted superior inhibition of PBMC rolling in vivo. In conclusion, our work demonstrates the feasibility of optimizing aptamer sequences and shows that multivalent ligand presentation enables superior adhesion receptor targeting., From the Clinical Editor: During inflammation, leukocytes extravasate from blood vessels under chemotaxic signals. The presence of L-selectin on endothelium acts as a mediator for the extravasation process. In this study, the authors investigated an L-selectin specific blocking DNA aptamer in various forms, as inhibitors to leukocyte binding and extravasation. This new approach confirmed the potential use of aptamers in clinical setting., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Conflicting results have been published regarding the induction of genotoxic effects by exposure to radiofrequency electromagnetic fields (RF-EMF). Various results indicating a genotoxic potential of RF-EMF were reported by the collaborative EU-funded REFLEX (Risk Evaluation of Potential Environmental Hazards From Low Energy Electromagnetic Field Exposure Using Sensitive in vitro Methods) project. There has been a long-lasting scientific debate about the reliability of the reported results and an attempt to reproduce parts of the results obtained with human fibroblasts failed. Another part of the REFLEX study was performed in Berlin with the human lymphoblastoid cell line HL-60; genotoxic effects of RF-EMF were measured by means of the comet assay and the micronucleus test. The plausibility and reliability of these results were also questioned. In order to contribute to a clarification of the biological significance of the reported findings, a repeat study was performed, involving scientists of the original study. Comet-assay experiments and micronucleus tests were performed under the same experimental conditions that had led to genotoxic effects in the REFLEX study. Here we report that the attempts to reproduce the induction of genotoxic effects by RF-EMF in HL-60 cells failed. No genotoxic effects of RF-EMF were measured in the repeat experiments. We could not find an explanation for the conflicting results. However, the negative repeat experiments suggest that the biological significance of genotoxic effects of RF-EMF reported by the REFLEX study should be re-assessed., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Background: Prolonged intensive care unit (ICU) stay is a surrogate for advanced morbidity or perioperative complications, and resource utilization may become an issue. It is our policy to continue full life support in the ICU, even for patients with a seemingly grim outlook. We examined the effect of duration of ICU stay on early outcomes and late survival., Methods: Between 1993 and 2011, 6,385 patients were admitted to the ICU after cardiac surgery. Patients were grouped according to length of stay in the ICU: group 1, 2 days or less (n = 4,631; 73%); group 2, 3 to 14 days (n = 1,423; 22%); group 3, more than 14 days (n = 331; 5%). Length of stay in ICU for group 3 patients was 38 ± 24 days (range, 15 to 160; median 31). Clinical profile and outcomes were compared between groups., Results: Patients requiring prolonged ICU stay were older, underwent more complex surgery, had greater comorbidity, and a higher predicted operative mortality (p < 0.0001). They had a higher incidence of adverse events and increased mortality (p < 0.0001). Of the 331 group 3 patients, 60% were discharged: survival of these patients at 1, 3, and 5 years was 78%, 65%, and 52%, respectively. Operative mortality as well as late survival of discharged patients was proportional to duration of ICU stay., Conclusions: Current technology enables keeping sick patients alive for extended periods of time. Nearly two thirds of patients requiring prolonged ICU leave hospital, and of these, 50% attain 5-year survival. These data support offering full and continued support even for patients requiring very prolonged ICU stay., (Copyright © 2013 The Society of Thoracic Surgeons. Published by Elsevier Inc. All rights reserved.)

Background: Inguinal lymph node dissection (LND) is often associated with wound complications. The aim of this study was to evaluate epidermal vacuum therapy for the prevention of wound complications following inguinal LND., Methods: From January 2009 to March 2012, a total of 24 patients with penile cancer or cancer of the urethra received uni- or bilateral inguinal lymphadenectomy in our institution. Postoperative wound care consisted of conventional wound care (CWC) in 16 patients or epidermal vacuum dressings (VAC) in eight patients. Maximum drained fluid per day, duration of drainage, duration of hospitalisation and inguinal complications (formation of lymphocele, persistent lymphorrhoea or lymphoedema of the lower extremity) as well as rate of reinterventions were evaluated retrospectively. Mann-Whitney U-tests were performed to compare treatment groups for maximal drained fluid per day, duration of drainage and duration of hospitalisation. Binary data were compared with Fisher's exact test. Statistical calculations were performed on a patient level., Results: Patients treated with CWC showed a slight tendency to higher values of maximum drained fluid per day (p=0.632), duration of drainage (p=0.496) and a significantly longer time of hospitalisation (p=0.049). Epidermal VAC treatment resulted in significantly fewer complications such as formation of lymphoceles (62% vs. 20%), persistent lymphorrhoea (45% vs. 7%) or lymphoedema of the lower extremity (46% vs. 0%) (p=0.032). Reinterventions had to be performed in 23% of inguinal wounds (four patients) treated with CWC and for 7% of inguinal wounds (one patient) treated with epidermal VAC (p=0.631)., Conclusions: Epidermal VAC following inguinal LND might be advantageous for the prevention of postoperative wound complications. Prospective, controlled studies are warranted to further evaluate efficacy and cost-effectiveness., (Copyright © 2012 British Association of Plastic, Reconstructive and Aesthetic Surgeons. Published by Elsevier Ltd. All rights reserved.)

Sample preparation is the rate-limiting step in glycan analysis workflows. Among all of the steps, enzymatic digestions, which are usually performed overnight, are the most time-consuming. In the current study, we report an economical and fast preparation of N-glycans from serum, including microwave-assisted enzymatic digestion in the absence of denaturing chemicals and solvents during the release. To this end, we used a household microwave oven to accelerate both pronase and endo-β-N-acetylglucosaminidase H (Endo H) digestions. Purification was then performed using self-made SP20SS and carbon tips. We were able to prepare samples in 55 min instead of 21 h. Finally, the method was applied in the context of oncological biomarker discovery exemplarily to ovarian and colon cancer. We observed a significant downregulation of sialylated hybrid structures in ovarian cancer samples using capillary electrophoresis-laser-induced fluorescence (CE-LIF). Furthermore, sepsis, a systemic inflammatory response syndrome, was also included in the study to understand whether the changes observed in ovarian cancer patients were due to the cancer itself or to the inflammation that usually accompanies its development. Because sialylated hybrid structures were upregulated in sepsis samples, the downregulation of these structures in ovarian cancer is specific to the cancer itself and, therefore, could be used as a biomarker., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The leukocytic cell adhesion receptor L-selectin mediates the initial step of the adhesion cascade, the capture and rolling of leukocytes on endothelial cells. This event enables leukocytes to migrate out of the vasculature into surrounding tissues during inflammation and immune surveillance. Distinct domains of L-selectin contribute to proper leukocyte migration. In this review, we discuss the contributions of these domains with respect to L-selectin function: the regulation by serine phosphorylation of the cytoplasmic tail, the role of the transmembrane domain in receptor positioning on the cell surface as well as the N-glycosylation of the extracellular part and the identification of novel binding partners., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

Selectins are attractive targets for specific anti-inflammatory therapies. Using human lymphocytes as well as an L-selectin-transfected pre-B-cell line in dynamic flow chamber experiments, we could demonstrate that the small-molecule compound efomycine M blocks L-selectin-mediated lymphocyte rolling on sialylated Lewis(X), an action that was confirmed by plasmon resonance spectroscopy. Recruitment of naive lymphocytes to peripheral lymph nodes depends on L-selectin-mediated adhesion to high endothelial venules. We performed intravital microscopy studying lymphocyte rolling in peripheral lymph nodes and showed a 53% reduction (P=0.0006) of lymphocyte rolling in mice treated with efomycine M or a function-blocking antibody against L-selectin. In addition, the number of lymph node-homing T cells was reduced by >60% using either efomycine M or L-selectin-blocking antibodies. As recruitment of naive lymphocytes is a prerequisite for sensitization in T-cell-mediated immune reactions and allergic responses, mice were treated with efomycine M or an L-selectin-specific antibody during contact sensitization with DNFB. After adoptive transfer of corresponding T cells into non-sensitized recipient mice, the capacity of these cells to induce contact hypersensitivity was significantly reduced (P=0.0002 and P=0.0001, respectively). Our data demonstrate that it is possible, in principle, to diminish T-cell-mediated allergic reactions through interference with L-selectin functions during the early sensitization phase.

The toxic oil syndrome (TOS) caused by ingestion of rapeseed oil adulterated with aniline is characterized by symptoms of an allergic and/or autoimmune illness associated with vessel wall lesions similar to those of atherosclerosis. Fatty acid esters of 3-(N-phenylamino)-1,2-propanediol (PAP) have been incriminated as the etiologic agents of TOS. However, the pathogenesis of TOS is yet unknown. Here, we addressed whether PAP fatty acid esters are incorporated into lipoproteins, which after transport to vascular endothelial cells are taken up to initiate TOS vasculopathy. After loading (14)C-dioleyl-ester of PAP into LDL labeled with 1,1'-dioctadecyl-3,3,3',3'-tetramethylindolcarbocyanine (DiI) we assessed receptor mediated endocytosis and intracellular localization of these lipopoproteins in vascular endothelial cells. Our data suggest that these lipoprotein-derivatives are internalized into endothelial cells by LDL receptor mediated endocytosis. Confocal microscopy revealed that DiI-LDL loaded with dioleyl-ester of PAP and incubated for 60 min with endothelial cells colocalizes with the lysosomotropic compound LysoTracker Green, indicating that internalized PAP-loaded LDL are targetted to the endolysosomal compartment for further processing. Subcellular fractionation of endothelial-like ECV-304 cells after incubation with LDL loaded with the (14)C-dioleyl-ester of PAP for 6h showed that the radioactive label accumulated in fractions containing endosomes, the Golgi apparatus and the endoplasmic reticulum.

Ca(2+) influx across the plasma membrane after stimulation of G protein-coupled receptors is important for many physiological functions. Here we studied the regulation of an inwardly rectifying whole cell current and its putative role in Ca(2+) entry in Xenopus oocytes. Expression of P2Y(1) or M1 receptors in Xenopus oocytes elicited a characteristic inwardly rectifying current without receptor stimulation. This current displayed distinct activation and inactivation kinetics and was highly Ca(2+)-dependent. After stimulation of endogenous G(q)-coupled receptors in water-injected cells similar currents were observed. We therefore speculated that the current could be activated via Ca(2+) store depletion induced by constitutive stimulation of the IP(3) cascade in cells overexpressing G(q)-coupled receptors. Receptor-independent Ca(2+) store depletion also induced the current. In conclusion, this current is activated after store depletion suggesting a role in Ca(2+) entry after stimulation of G(q)-coupled receptors. Finally, our data do not support the proposed ionotropic properties of the P2Y(1) receptor.

Adenine quantitation is required for a variety of applications. To date, the prevalent method for quantifying free adenine, in a variety of applications, is the detection of fluorescent-derivatized adenine by HPLC. For the present study, we developed a high-throughput, nonradioactive, enzyme-based colorimetric adenine quantitation assay that is performed in one multireaction incubation step. The assay does not require adenine derivatization and is designed for microplates. The key step is the conversion of adenine to adenosine monophosphate by adenine phosphoribosyl transferase. Subsequent reactions finally produce three inorganic phosphate ions per adenine molecule. Phosphate is quantitated by the color-generating phosphorylysis of a particular purine derivate. Ribosome-inactivating proteins that release adenine from polynucleotides are often used to investigate intracellular protein trafficking and are important for the design of immunotoxins. We therefore used ricin, dianthin, saporin, and a variety of saporin fusion proteins to show that this method is suitable for quantifying adenine release using different substrates. The measured rate of adenine release and substrate specificity are comparable to those determined by HPLC and radioactive detection techniques.

The transferrin receptor (TfR) is a N- and O-glycosylated transmembrane protein mediating the cellular iron uptake by binding and internalization of diferric transferrin. In this study, rate constants and dissociation constants of 125I-ferri-transferrin binding to the human TfR were examined dependent on receptor glycan composition, pH, bivalent cations, and temperature. To do so, purified human placental TfR was noncovalently immobilized to polystyrene surfaces and subjected to alterations in various parameters. We found that transferrin binding was clearly dependent on a receptor pretreatment with buffers of various pH in that most of the TfR molecules irreversibly lost transferrin binding activity below pH 6.5. However, the dissociation constant of the remaining active binding sites was not affected. Similarly, we were able to define the thermal stability of the receptor as a function of transferrin binding ability. Binding of transferrin was completely lost provided that the receptor was pretreated at temperatures of at least 65 degrees C. Treatment with EDTA also caused an irreversible loss of transferrin binding activity, indicating that the functionally active conformation of the mature TfR depends on bivalent cations. In order to examine the role of the receptor glycans, we enzymatically removed the sialic acid residues, the hybrid and oligomannosidic N-glycans, or all types of N-glycans. In contrast to the parameters described above, all desialylated and N-deglycosylated TfR variants had exactly the same transferrin binding properties as the native TfR. To assess changes in the secondary structure of the receptor, circular dichroic spectra were recorded from TfR at pH 5.0, from heat pretreated receptor and from deglycosylated TfR. Since the receptor did not exhibit detectable changes in the CD spectrum of the deglycosylated receptor, it can be concluded that the N-linked carbohydrates of the mature, fully processed TfR are not essential for transferrin binding and conformational stability.

We present a novel method for the rapid determination of association constants. The method is based on the direct calibration of an enzyme-linked immunosorbent assay (dcELISA) and does not require any external calibration. It combines kinetic and equilibrium binding experiments and can be performed on a single microtiter plate. The absorbance data are evaluated by several linearized plots without the need for sophisticated computations. The dcELISA has been used to analyze the binding of a monoclonal antibody, OKT9, to its cognate antigen, the human transferrin receptor, and yielded an association constant of Ka = 2.2 x 10(9) l/mol and a complex formation rate constant of kc = 2.7 x 10(-4) s-1. A 26% larger association constant was obtained with a radioimmunoassay (RIA)-based Scatchard analysis using 125I-labeled OKT9. By quantifying the binding of the same iodinated antibody with the dcELISA we were able to verify that the iodination modifies the binding properties of the antibody. The dcELISA thus appears to be superior to all methods requiring covalent modifications. In principle, the direct calibration method can also be combined with all other solid phase assays. It should thus expand their scope in quantifying the binding properties of biologically important molecules.

A method for the modification of the oligosaccharide moiety of even small amounts of purified glycoproteins by enzymatic glycosylation and deglycosylation is described. The method includes noncovalent immobilization of the glycoproteins onto the polystyrene surface of the wells of microtiter plates used as reaction tubes, deglycosylation or glycosylation by incubation either with exoglycosidases or endoglycosidases or with glycosyltransferases, and the characterization of the modified glycan structures by probing them with lectins. Placental transferrin receptor employed as a model glycoprotein was modified in amounts of as little as 100 ng removing sialic acid residues, hybrid-type glycans or all types of N-glycans with neuraminidase, endo-beta-N-acetylglucosaminidase H or peptide-N4-(acetyl-beta-glucosaminyl) asparagine amidase. Asialotransferrin receptor was alpha-2,6-sialylated with alpha-2,6-sialyltransferase from rat liver, but could not be alpha-2,3-sialylated with alpha-2,3-sialyltransferase from porcine liver. Changes in the structure and in the relative amount of the oligosaccharides could be monitored semiquantitatively with high sensitivity by the binding of digoxigenin-labeled lectins and anti-digoxigenin Fab fragments. The method is easy to use, does not require immobilization of the enzymes employed, offers simple separation of the enzymes and the product, and leaves the protein intact for further studies.

A method was developed to isolate cell surface proteins by a simple two-step procedure. Hepatocyte cell surface proteins were labeled by a cleavable biotin derivative in a covalent pulse reaction. Under the described conditions, NHS-SS-biotin proved to be an impermeant, cell surface-specific label which does not affect the impermeant, cell surface-specific label which does not affect the viability of rat hepatocytes. Biotinylated cell surface proteins could be selectively separated under non-denaturing conditions from non-biotinylated proteins and biotin-containing carboxylases by avidin affinity chromatography and sulfhydryl-mediated elution. Subsequent to alkylation of the eluted protein, individual cell surface proteins could be isolated by immunoprecipitation as shown for a selected Mr 120,000 glycoprotein gp120 of the hepatocyte plasma membrane. Using this technique, a transit time of gp120 from the endoplasmic reticulum to the cell surface of 2 h was determined. The results show that the combination of labeling with a cleavable biotin derivative, non-denaturing avidin affinity chromatography and immunoprecipitation is a useful method to isolate and study individual cell surface proteins.

To evaluate the role of lysosomes in the breakdown of the carbohydrate and the polypeptide moiety of plasma membrane glycoproteins, degradation of the plasma membrane glycoprotein gp120 was studied in the liver of rats treated with the lysosomotropic amine chloroquine. Half-lives of degradation of the terminal sugar L-fucose and of L-methionine of gp120 were measured in isolated plasma membranes after pulse-chase experiments in vivo. Chloroquine extended the plasma membrane half-life of the polypeptide moiety of gp120 from 51 h to 143 h. By contrast, L-fucose of gp120 in the plasma membrane was not affected by chloroquine, but decayed with the same short half-lives of 22 h and 23 h in both controls and chloroquine-treated rats. The data suggest that the protein portion of gp120 is degraded within the lysosomes. Conversely, the terminal sugar L-fucose is removed from the glycoprotein independent from proteolysis before segregation of the glycoprotein into the lysosomal compartment.