Irreversible inflammation is associated with decreased levels of the alpha1-, beta1-, and alpha2-subunits of sGC in human odontoblasts

The nitric oxide (NO) receptor enzyme soluble guanylate cyclase (sGC) contains one prosthetic heme group as an αβ heterodimer, and two heterodimer isoforms (α(1)β(1), α(2)β(1)) were characterized to have enzyme activity. To test the irreversible inflammation-dependent regulation of sGC in odontoblasts, we incubated decalcified frozen sections of healthy and inflamed human third molars with antibodies against β-actin, nitrotyrosine, inducible nitric oxide synthase (iNOS), α(1)-, β(1)-, and α(2)-subunits of sGC and analyzed them at protein levels by quantitative immunohistochemistry. The irreversible inflammation induced an increase in the signal intensities for nitrotyrosine and iNOS and a decrease for the α(1)-, β(1)-, and α(2)-subunits of sGC in odontoblasts. Inflammatory mediators, reactive oxygen, and nitrogen species may impair the expression of the α(1)-, β(1)-, and α(2)-subunits in odontoblasts. The decrease of sGC at the protein level in inflamed odontoblasts is compatible with a critical role for sGC to mediate biological effects of NO in health.     

Mature proteins derived from Epstein–Barr virus fail to feed into the MHC class I antigenic pool

The immediate presentation of peptide epitopes on MHC class I (MHC I) after antigen expression has led to the concept that MHC I ligands are mostly derived from defective ribosomal products (DRiPs), a subset of newly synthesized proteins that are rapidly degraded by the proteasome. Whether and to what extent mature proteins contribute to the antigenic pool, however, has remained elusive. Here, we developed a conditional antigen expression system that allows studying antigen presentation from mature proteins by inducing their rapid proteasomal degradation in the absence of further antigen synthesis. Target cells in which expression of two Epstein–Barr virus (EBV) antigens was induced were rapidly recognized by antigen‐specific CD8⁺ T cells in a time‐ and dosage‐dependent manner, demonstrating that antigen presentation was linked to antigen synthesis. By contrast, T cells failed to recognize target cells containing large amounts of mature protein even after induction of their rapid proteasomal degradation. Thus, the presentation of these antigens proved to be strictly dependent on protein synthesis whereas mature proteins failed to furnish the antigenic pool. These results have implications for the design of immunotherapeutic strategies that aim at targeting proteins with increased half‐lives and are hence overexpressed in tumors.     

eLearning in education and advanced training in neuroradiology: introduction of a web-based teaching and learning application

Introduction New information technologies offer the possibility of major improvements in the professional education and advanced training of physicians. The web-based, multimedia teaching and learning application Schoolbook has been created and utilized for neuroradiology.Methods Schoolbook is technically based as a content management system and is realized in a LAMP environment. The content is generated with the help of the developed system and stored in a database. The layout is defined by a PHP application, and the webpages are generated from the system.Results Schoolbook is realized as an authoring tool so that it can be integrated into daily practice. This enables the teacher to autonomously process the content into the web-based application which is used for lectures, seminars and self-study. A multimedia case library is the central building block of Schoolbook for neuroradiology, whereby the learner is provided with original diagnostic and therapeutic data from numerous individual cases. The user can put individual emphasis on key learning points as there are various ways to work with the case histories. Besides the case-based way of teaching and learning, a systematically structured way of dealing with the content is available.Conclusion eLearning offers various opportunities for teaching and learning in academic and scientific as well as in economic contexts. Web-based applications such as Schoolbook may be beneficial not only for basic university education but also for the realization of international educational programmes such as the European Master of Medical Science with a major in neuroradiology.     

NO-cGMP signaling molecules in cells of the rat molar dentin-pulp complex

By the formation of cyclic guanosine 3',5'-monophosphate (cGMP), nitric oxide (NO)-sensitive enzyme-soluble guanylate cyclase (sGC) plays a receptor role for NO within the NO-cGMP signaling cascade, which is involved in vasodilatation and neurotransmission. The hypothesis that NO-cGMP signaling molecules modulate cells of the dentin-pulp complex was investigated in rat molars by histochemical, immunohistochemical, immuno-ultrastructural, and organ bath techniques. NO synthase (NOS) I-III, the sGC alpha(2)-subunit/beta(1)-subunit, and cGMP were detected in odontoblasts and blood vessels. NOS I, sGC alpha(2), and cGMP were identified in nerve fibers. Treatment of rat molars with the NO donor NONOate (10(-5) M) increased cGMP staining intensities in blood vessels and odontoblasts, while NO synthase inhibitor L-NAME (10(-4) M) attenuated intensity of the reaction products for cGMP, suggesting an effect of endogenous NO on sGC. These correlations of patterns and alterations of cGMP staining intensities after treatment with the NO donor or NO inhibitor might represent an NO-sGC-cGMP signaling-dependent modulation of odontoblasts, blood vessels, and nerve fibers in the dentin-pulp complex.     

Contribution of viral recombinants to the study of the immune response against the Epstein-Barr virus

Over the past two decades, Epstein-Barr virus (EBV) mutants have become valuable tools for the analysis of viral functions. Several experimental strategies are currently used to generate recombinant mutant genomes that carry alterations in one or several viral genes. The probably most versatile approach utilizes bacterial artificial chromosomes (BAC) carrying parts or the whole EBV genome, which permits extensive genetic manipulations in Escherichia coli cells. The 'mini-EBVs', for example, which contain roughly half of the wild type viral information, efficiently transform primary B cells and have been used as gene vectors for foreign antigens. After expression in lymphoblastoid cell lines (LCLs), these antigens are efficiently presented on MHC molecules and recognized by antigen-specific T cells. These vectors, however, cannot undergo lytic replication and require a helper cell line for efficient replication and DNA packaging. Further experimental systems include the complete viral genome cloned onto a BAC. These mutants can typically be complemented by expression plasmids, some of which are expressed on EBV-derived vectors and can be propagated without requirement of a helper cell line. Over the last years, these viral recombinants have been utilized increasingly to analyse different aspects of the immune response against EBV. Immunological applications are manifold and steadily growing and include crude screening of T cell clones for their specificity towards latent versus lytic antigens, or more detailed analyses in which the exact specificity of T cells is determined using EBV mutants that lack a single viral antigen. Other applications include detailed analysis of protein domains important for immune recognition, e.g. Gly-Ala repeats in the EBV nuclear antigen 1 (EBNA1) protein, expansion of T cell clones directed against virion structures using virus-like particles and phenotypic analysis of virus mutants defective in infection. Future developments might include the genetic identification and characterization of viral proteins involved in the modulation of the immune response and, in particular, immune evasion. Recombinant viral strains are already being used experimentally for the expansion of T cells in vitro prior to in vivo cellular therapy and have been proposed as potential prophylactic vaccines.     

Glyceryl trinitrate treatment up-regulates soluble guanylyl cyclase in rat dura mater.

Nitric oxide (NO) is a key molecule in vascular headaches and the dura mater has been implicated as a tissue where vascular headache develops. Here we demonstrate expression, enzyme activity and cellular distribution of the intracellular receptor for NO, soluble guanylyl cyclase (sGC), in rat dura mater. Subcutaneous treatment of rats with the NO-donor glyceryl trinitrate (GTN) induced an increase of sGC expression and activity in dural blood vessels after 20-30 min. It has previously been shown that GTN induces headache in normal subjects after 20-30 min. Our findings suggest that an up-regulation of the NO target enzyme contributes to the pathogenesis of GTN-induced headache explaining the subacute rather than acute onset of symptoms.     

Editorial Commentary: Neuraxial Anesthesia Improves Pain After Hip Arthroscopy but Risks Ambulatory Discharge Delay

Hip arthroscopy continues to be one of the fastest-growing orthopaedic procedures nationally, and pain control following these procedures can be challenging. As regional anesthesia techniques for this population have shown to have limited benefits, pain management for hip arthroscopy focused on multimodal analgesia and preventive analgesia, interventions that reduce postoperative hyperalgesia. The use of neuraxial anesthesia such as spinal and epidural anesthesia, established preventive analgesic anesthetic techniques, has demonstrated to improve postoperative pain in orthopaedic surgery when compared with general anesthesia. This promising finding highlights that despite potential disadvantages of neuraxial anesthesia, such as a small risk for complications or delayed resolution of the neuraxial block that could delay discharge, neuraxial anesthesia could be a suitable anesthetic technique for ambulatory orthopaedic surgery.     

Analyse der Personalkosten nach Reorganisation der Intensivmedizin mithilfe kalkulierter DRG-Vergleichsdaten

BACKGROUND: In an extensive project intensive care units (ICUs) of the Charité University Hospital were reorganized. The aim of this investigation was to determine if staff costs after this reorganization are financed by modular profits of diagnosis-related groups (DRGs).METHODS: Staff costs of all non-pediatric intensive care units, including ICUs, intermediate care units and post-anaesthesia care units (PACUs) in the Charité University Hospital were compared with the modular profits of all DRGs of patients older than 14 years in 2005. These DRGs were converted into the German refined DRG (GDRG) system 4.0 from 2006 with calculations based on actual income for medical doctors and nurses in 2006. Due to changed wage agreements for the incomes of physicians in 2006 there was an increase of costs. For the other professional groups an increase in income is expected, which cannot be estimated at present.RESULTS: The calculation revealed that staff costs of the ICUs at the Charité University Hospital based on a current German mean base rate of 2,836 EUR were 4.2% above the modular profits of the DRGs. As a result of a structural reorganization of the ICUs, the costs of staff could be adapted to the modular profits. Under the conditions of the actual reduced base rate of Berlin of 2,955 EUR the costs and profits were nearly equal. As the financial impact of the reorganization of the ICUs will take full effect in the coming years, it can be anticipated that with an expected base rate of 2,949 EUR in 2010 the intensive care medicine of a University hospital in Germany can become profitable.DISCUSSION: The spectrum of intensive care medicine at the Charité University Hospital covers the maximum range of operative and non-operative medicine. After an extensive reorganization of the ICUs under the aspect of staff costs, intensive care medicine can become profitable under the 4.0 G-DRG system. With consequent reorganization the cost efficiency of staff can be optimized, particularly in the setting of high-end intensive care medicine.     

Blockade of interleukin-13-mediated cell activation by a novel inhibitory antibody to human IL-13 receptor alpha1

Interleukin-13 (IL-13) is a cytokine with a crucial role in the development of allergic asthma. The IL-13 receptor shares the IL-4Ralpha subunit with the IL-4R system, but contains as a specific component the IL-13Ralpha1 chain. Blocking signal release by IL-13 without affecting IL-4 function is a potentially interesting therapeutical option for the treatment of asthma. Employing genetic immunization, we generated a set of novel monoclonal antibodies to the IL-13Ralpha1 receptor that proved very specific and efficient inhibitors of human IL-13 activity. Receptor binding antibodies were identified by their specific reactivity with both human monocytes and a murine pro-B cell line overexpressing human IL-13Ralpha1 by flow cytometry and cell ELISA. A luciferase reporter cell system based on STAT6-mediated promoter activation in murine Ba/F3 cells was employed to screen the antibodies for IL-13 antagonistic properties. Inhibitory antibody effects were quantified by interference with IL-13-dependent proliferation of TF-1 cells. The capability of blocking IL-13-driven responses of primary, inflammation-relevant cells was tested by Western blot analysis of STAT6 tyrosine phosphorylation and expression of 15-lipoxygenase in monocytes from fresh blood. The most potent inhibitory antibody identified, GM1E7, inhibited IL-13-driven gene activation and cell proliferation in immune cell lines with IC(50) values in the low nanomolar range. Both short-term (STAT6 activation) and long-term (15-LO induction) responses of primary human blood cells to IL-13 were almost entirely blocked, whereas IL-4 effects remained virtually unaffected. GM1E7 is superior to available agents interfering with IL-13 activity in terms of specificity and efficiency and offers potential novel therapeutic perspectives for the treatment of allergic asthma.