A Swelling-Activated Chloride Current in Microglial Cells is Suppressed by Epac and Facilitated by PKA - Impact on Phagocytosis.

BACKGROUND/AIMS: Volume-regulated anion channels (VRACs) are of particular importance in regulating the cell volume (CV) and give rise to the swelling-activated Cl- current (ICl,swell), a main component driving global regulatory volume decrease (RVD) during cell swelling. Because ICl,swell affects numerous CV-regulated processes like migration, we assume that its role is also indispensable for phagocytosis which requires local cell swelling. Noradrenaline (NA) modulates phagocytosis in macrophages and microglial cells, macrophage-related cells in the central nervous system. Therefore we evaluated whether NA modulates ICl,swell and phagocytosis in microglia. METHODS: Experiments were performed in murine microglial BV-2 and primary mouse microglial cells. Patch clamp experiments were performed in BV-2 cells using the amphotericin-perforated method to minimize cytosolic disturbances. Phagocytosis was quantified by scanning electron microscopy. RESULTS: Following activation of ICl,swell by a hypotonic bath solution, noradrenaline, as well as the ß-adrenergic agonist isoproterenol, evoked a transient decrease of ICl,swell. Repeated application of adrenergic agonists caused a decline of this electrical response. Application of the agonist of exchange protein directly activated by cAMP (Epac), 8-pCPT-2-O-Me-cAMP, or the protein kinase A inhibitor H89 caused a persistent suppression of ICl,swell. When isoproterenol was added concomitantly with the hypotonic saline, ICl,swell developed more rapidly compared to control conditions. Uptake of IgG-coated beads was suppressed by NA or H89 when quantified after 15 min of exposure. CONCLUSION: The activation of ß-adrenergic receptors in microglial cells triggers a cAMP-Epac-dependent and a cAMP-PKA-dependent cascade which affects phagocytosis via modulation of the swelling-activated Cl- current ICl,swell.

Long-term remission of a Her2/neu positive primary breast cancer under double monoclonal antibody therapy with trastuzumab and bevacizumab.

BACKGROUND: The attempt to act on several signalling pathways involved in tumour development simultaneously appears to be more attractive than attacking a single target structure alone. Vascular endothelial growth factor (VEGF) over-expression is frequently observed in human epidermal growth factor receptor 2 (Her2/neu) positive patients with breast cancer and over-expression of the proto-oncogene Her2/neu is associated with an up-regulation of VEGF. CASE REPORT: The case of a Her2/neu positive patient with breast cancer who refused cytotoxic chemotherapy with its potential side effects as well as mastectomy is presented. Our patient has been receiving the combined double administration of bevacizumab and trastuzumab for more than 4 years. CONCLUSIONS: This case report shows that (a) the combined double administration of bevacizumab and trastuzumab was be clinically effective. (b) The combination of bevacizumab and trastuzumab is safe and non-toxic. (c) Bevacizumab and trastuzumab can be used as a long-term application.

Validation of a simple assay for the quantification of the capecitabine metabolites 5'-DFCR and 5'-DFUR for drug monitoring in patients receiving outpatient chemotherapy.

A simple and precise analytical method for the determination of 5'deoxy-5-fluorocytidine (DFCR) and 5'deoxy-5-fluorouridine (DFUR), the enzymatically formed metabolites of capecitabine in plasma, was developed using a reversed-phase high performance liquid chromatography gradient method with external standard method. Blood samples were analyzed after separation of DFCR/DFUR by solid-phase extraction from matrix compounds using a C16 amide reversed-phase column operated at a flow rate of 0.8 ml/min in gradient elution mode with a mobile phase composed of water-methanol (10 mM ammonium acetate in water; m/v). Excellent recoveries in plasma ranging from 77.5-99.12% for DFCR and 84.70-99.15% for DFUR, respectively, were obtained. For both compounds the calibration curves were linear over the range from 0.156 to 5.0 µg/ml. The present assay is robust, selective and sensitive, and is being applied in our laboratories to monitor plasma concentrations of DFCR and DFUR in clinical phase I and phase II studies.

Beta-adrenergic stimulation suppresses phagocytosis via Epac activation in murine microglial cells.

Endogenous noradrenaline presumably prohibits neuroinflammation by stimulation of ß-adrenergic receptor-dependent suppression of the production of inflammatory mediators. Using the microglial cell line, BV-2, as well as primary murine microglial cells, we show here that the ß-adrenergic agonist, isoproterenol, suppresses uptake of hydrophobic polystyrene microspheres. The number of cells showing a specific number of engulfed microspheres followed a Poisson distribution. Isoproterenol decreased the number of engulfed particles per cell and the number of cells showing at least one incorporated particle. Elevation of intracellular cAMP by activation of adenylyl cyclase activity with forskolin, suppression of phosphodiesterase activity with 3-isobutyl-1-methylxanthine (IBMX), or application of the membrane-permeable cAMP analog, 8-bromo-cAMP, suppressed particle uptake. The protein kinase A inhibitor, H-89, did not prevent isoproterenol-dependent suppression of particle engulfment. However, activation of exchange protein activated by cAMP (Epac), specific guanine nucleotide exchange factors for the Ras GTPase homologues, Rap1 and Rap2, with the Epac1-specific cAMP analog, 8-pCPT-2'-O-Me-cAMP, mimicked the suppressive effect of isoproterenol on particle uptake. Our results suggest that ß-adrenergic receptor stimulation suppresses particle uptake in microglia by cAMP-dependent activation of Epac.

Clinical and economic aspects of KRAS mutational status as predictor for epidermal growth factor receptor inhibitor therapy in metastatic colorectal cancer patients.

Treatment of metastasized colorectal cancer (mCRC) patients with anti-epidermal growth factor receptor (EGFR)-directed monoclonal antibodies is driven by the results of the KRAS mutational status (wild type [WT]/mutated [MUT]). To find out as to what extent the treatment selection based on the KRAS status had impact on overall costs, a retrospective analysis was performed. Of 73 mCRC patients 31.5% were MUT carriers. Costs of EGFR inhibitor treatment for WT patients were significantly higher compared to those for patients with MUT (p = 0.005). Higher treatment costs in WT carriers reflect a significantly higher number of treatment cycles (p = 0.012) in this cohort of patients. Savings of drug costs minus the costs for the determination of KRAS status accounted for EUR 779.42 (SD ±336.28) per patient per cycle. The routine use of KRAS screening is a cost-effective strategy. Costs of unnecessary monoclonal EGFR inhibitor treatment can be saved in MUT patients.

[Organization, administration, and performance of clinical studies]. / Organisation, Administration und Durchfuhrung von klinischen Studien.

This contribution deals with all important organizational and administrative aspects of clinical studies in German speaking countries. All trials are to be executed in accordance with the Good Clinical Practice (GCP) Guidelines. GCP applies to the process of designing, conducting, recording, and reporting of clinical studies. Compliance with GCP facilitates the mutual acceptance of resulting clinical data by the respective regulatory authorities worldwide. Before initiating a clinical study the investigator has to obtain written and dated approval from the responsible ethics committee, the competent authorities, and the hospital administration. The investigator's study file contains all essential study documents. One of the most important tasks of an investigator is to properly inform the prospective subjects and to obtain their informed consent. All relevant treatment-related information has to be recorded in the patient files. These source data are transferred to case report forms. During monitoring visits, audits, and inspections, source data verification will be performed routinely. Any adverse events (AEs) must be documented according to the CTCAE, the Common Terminology Criteria for Adverse Events. All serious adverse events (SAEs) have to be reported to the sponsor immediately. At the end of the study a termination visit is performed, and all authorities are officially informed about the termination of the trial.