Large-aperture coatings for fusion-class laser systems.

Optical coatings for fusion-class laser systems pose unique challenges, given the large substrate sizes, the high intensities incident on the coatings, and the system-focusing requirements, necessitating a well-controlled optical wavefront. Significant advancements have taken place in the past 30 years to achieve the coating capabilities necessary to build laser systems such as the National Ignition Facility, Laser Mégajoule, OMEGA EP, and OMEGA. This work summarizes the coating efforts and advancements to support such system construction and maintenance.

[Point-of-care ultrasonography of the abdomen in emergency and intensive care medicine]. / Point-of-care-Sonographie des Abdomens in der Notfall- und Intensivmedizin.

Point-of-care ultrasound is a fundamental part of diagnostic and therapeutic management in emergency and intensive care medicine. The availability of high-resolution mobile ultrasound systems allows high-quality imaging at the bedside of the patient. Point-of-care ultrasound is not a comprehensive differential diagnostic abdominal ultrasound examination. Rather, the aim of the method is to integrate easily detectable sonographic findings into the clinical context. From this, the necessary diagnostic or therapeutic procedures are derived. This article shows opportunities and limitations of this method. The structure of the article is given by the leading clinical symptoms. The focus is on the ultrasound examination and the characteristic sonographic findings with illustrative ultrasound images. This is followed by a short differential diagnostic interpretation. Further diagnostic or therapeutic management is also briefly addressed.

Development of a hard X-ray split-and-delay line and performance simulations for two-color pump-probe experiments at the European XFEL.

A hard X-ray Split-and-Delay Line (SDL) under construction for the Materials Imaging and Dynamics station at the European X-Ray Free-Electron Laser (XFEL) is presented. This device aims at providing pairs of X-ray pulses with a variable time delay ranging from -10 ps to 800 ps in a photon energy range from 5 to 10 keV for photon correlation and X-ray pump-probe experiments. A custom designed mechanical motion system including active feedback control ensures that the high demands for stability and accuracy can be met and the design goals achieved. Using special radiation configurations of the European XFEL's SASE-2 undulator (SASE: Self-Amplified Spontaneous Emission), two-color hard x-ray pump-probe schemes with varying photon energy separations have been proposed. Simulations indicate that more than 109 photons on the sample per pulse-pair and up to about 10% photon energy separation can be achieved in the hard X-ray region using the SDL.

Label-free protein quantification of sodium butyrate treated CHO cells by ESI-UHR-TOF-MS.

Effects of butyrate on CHO producer cells are contradictory, promoting productivity and at the same time repressing proliferation. Though in previous omics studies the background of butyrate impact on producer cells has been investigated, the knowledge about the mechanism is still very limited. As previous proteomic results on this field are mainly based on 2DE-gels, we conducted a label-free MS quantification, based on fast high resolution ESI-MS and a straight forward software solution, to gain insight in shifted cellular processes of CHO cells 25h after butyrate treatment. 118 proteins or subunits with significantly altered abundances were identified suggesting changes in carbohydrate, protein metabolic and cell cycle processes. Effects of butyrate on the nucleosome assembly as a known direct epigenetic influence on HDAC activity turned out to be unexpectedly fast and persistent, as confirmed by Western blots of histone-H4 acetylation. Contradictory to increased cell specific productivity, most elements of protein metabolism exhibited decreased levels after butyrate treatment. In comparison to published results some overlap of our label free MS data could be observed but also apparently diverging findings, showing the need for complementary omics techniques for a holistic view on cellular processes such as response to butyrate.

Extracellular ATP attenuates ischemia-induced caspase-3 cleavage in human endothelial cells.

BACKGROUND: Apoptotic death of endothelial cells (EC) plays a crucial role for the development of ischemic injury. In the present study we investigated the impact of extracellular Adenosine-5'-triphosphate (ATP), either released from cells or exogenously added, on ischemia-induced apoptosis of human EC. METHODS AND RESULTS: To simulate ischemic conditions, cultured human umbilical vein endothelial cells (HUVEC) were exposed to 2 h of hypoxia (Po(2)<4mm Hg) in serum-free medium. Ischemia led to a 1.7-fold (+/-0.4; P<0.05) increase in EC apoptosis compared to normoxic controls as assessed by immunoblotting and immunocytochemistry of cleaved caspase-3. Ischemia-induced apoptosis was accompanied by a 2.3-fold (+/-0.5; P<0.05) increase of extracellular ATP detected by using a luciferin/luciferase assay. Addition of the soluble ecto-ATPase apyrase, enhancing ATP degradation, increased ischemia-induced caspase-3 cleavage. Correspondingly, inhibition of ATP breakdown by addition of the selective ecto-ATPase inhibitor ARL67156 significantly reduced ischemia-induced apoptosis. Extracellular ATP acts on membrane-bound P2Y- and P2X-receptors to induce intracellular signaling. Both, ATP and the P2Y-receptor agonist UTP significantly reduced ischemia-induced apoptosis in an equipotent manner, whereas the P2X-receptor agonist αß-me-ATP did not alter caspase-3 cleavage. The anti-apoptotic effects of ARL67156 and UTP were abrogated when P2-receptors were blocked by Suramin or PPADS. Furthermore, extracellular ATP led to an activation of MEK/ERK- and PI3K/Akt-signaling pathways. Accordingly, inhibition of MEK/ERK-signaling by UO126 or inhibition of PI3K/Akt-signaling by LY294002 abolished the anti-apoptotic effects of ATP. CONCLUSION: The data of the present study indicate that extracellular ATP counteracts ischemia-induced apoptosis of human EC by activating a P2Y-receptor-mediated signaling reducing caspase-3 cleavage.

Effects of high passage cultivation on CHO cells: a global analysis.

Cell lines for industrial pharmaceutical protein production processes need to be robust, fast-growing, and high-producing. In order to find such cells, we performed a high passage cultivation of monoclonal antibody producing Chinese hamster ovary (CHO) cells in shaking flasks for more than 420 days. Examinations of cell growth, productivity, intracellular protein, and metabolite characteristics as well as product transcript and genomic integrate levels revealed substantial differences between subpopulations that were cryopreserved from long-term cultivation at different time points. Detected growth performance as well as intracellular adenylate energy charge increased during high passage cultivation. In addition, proteome analysis indicated an augmented utilization of glycolysis with higher passage number and an enhanced robustness based on anti-stress proteins. Interestingly, the product formation increased at first but decreased dramatically during the later subcultivations, although selection pressure was applied. Utilizing flow cytometry and quantitative real-time polymerase chain reaction, we further examined the translational, transcriptional, and genomic basis for the observed phenotypes. The detected reduction of antibody expression, in particular of the heavy chain, was ascribed to a decrease of antibody transcript, caused by loss of gene copy number and assumably a malfunctioning splicing mechanism of the dicistronic mRNA. To our knowledge, this is the first systematic approach using process analytics and targeted omic techniques to elucidate the effects of long-term cultivation of CHO cells expressing a therapeutic protein.

Intermedin (adrenomedullin2) stabilizes the endothelial barrier and antagonizes thrombin-induced barrier failure in endothelial cell monolayers.

BACKGROUND AND PURPOSE: Intermedin is a member of the calcitonin gene-related-peptide (CGRP) family expressed in endothelial cells and acts via calcitonin receptor-like receptors (CLRs). Here we have analysed the receptors for intermedin and its effect on the endothelial barrier in monolayers of human umbilical vein endothelial cells (HUVECs). EXPERIMENTAL APPROACH: We analysed the effect of intermedin on albumin permeability, contractile machinery, actin cytoskeleton and VE-cadherin in cultured HUVECs. KEY RESULTS: Intermedin concentration-dependently reduced basal endothelial permeability to albumin and antagonized thrombin-induced hyperpermeability. Intermedin was less potent (EC(50) 1.29 ± 0.12 nM) than adrenomedullin (EC(50) 0.24 ± 0.07 nM) in reducing endothelial permeability. These intermedin effects were inhibited by AM(22-52) and higher concentrations of αCGRP(8-37), with pA(2) values of αCGRP(8-37) of 6.4 for both intermedin and adrenomedullin. PCR data showed that HUVEC expressed only the CLR/RAMP2 receptor complex. Intermedin activated cAMP/PKA and cAMP/Epac signalling pathways. Intermedin's effect on permeability was blocked by inhibition of PKA but not of eNOS. Intermedin antagonized thrombin-induced contractile activation, RhoA activation and stress fibre formation. It also induced Rac1 activation, enhanced cell-cell adhesion and antagonized thrombin-induced loss of cell-cell adhesion. Treatment with a specific inhibitor of Rac1 prevented intermedin-mediated barrier stabilization. CONCLUSION AND IMPLICATIONS: Intermedin stabilized endothelial barriers in HUVEC monolayers via CLR/RAMP2 receptors. These effects were mediated via cAMP-mediated inactivation of contractility and strengthening of cell-cell adhesion. These findings identify intermedin as a barrier stabilizing agent and suggest intermedin as a potential treatment for vascular leakage in inflammatory conditions.

CellViCAM--Cell viability classification for animal cell cultures using dark field micrographs.

Online monitoring of cell density and cell viability is a challenging but essential task to control and optimize biotechnical processes and is of particular interest for the growing field of animal cell cultures. For this purpose, we introduce an optical approach for automated cell detection and viability classification of suspended mammalian cells. Our proposed system CellViCAM is capable of evaluating dark field micrographs by means of several image processing and supervised machine learning techniques without the use of any dyes or fluorescent labeling. Using a human cell line as the reference culture, an efficient cell detection procedure has been established also enabling a cell density estimation. Furthermore, a comprehensive but reagent-free viability analysis, based on a semi-automatic training data generation, has been developed. By means of an extensive validation dataset we can show that the CellViCAM approach can be considered as an equivalent to staining-based methods and moreover, how it provides a technical platform for a more differentiated cell state classification into living, necrotic, early and late apoptosis.

Transient hypoxia induces ERK-dependent anti-apoptotic cell survival in endothelial cells.

Ischemia-induced apoptosis of endothelial cells may contribute to tissue injury, organ failure, and transplantation rejection. However, little is known about survival mechanisms capable to counteract endothelial apoptosis. This study investigated the potential role of an endogenous anti-apoptotic response elicited by transient hypoxia, capable to avert ongoing apoptosis in endothelial cells. Experiments were carried out in three different types of cultured endothelial cells (human umbilical vein, pig aorta, and from rat coronary microvasculature). As a pro-apoptotic challenge endothelial cells were cultured in serum-free medium and subjected to hypoxia for 2 h. We found that transient hypoxia reduced caspase 3 activation within 1 h of hypoxia. Accordingly, the number of apoptotic cells was reduced after 24 h of reoxygenation. This was true for all three cell types analyzed. Analysis of Akt and mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathways revealed that hypoxia induced a transient activation of ERK 2 but not of Akt. ERK 2 phosphorylation preceded the phosphorylation of pro-apoptotic molecule Bad at Ser112, an inhibitory phosphorylation site specific for ERK. The protective effects of hypoxia regarding Bad phosphorylation, caspase 3 activation, and apoptosis were abolished by MEK 1/2 inhibitors, PD98059 or UO126, as well as by antisense oligonucleotides directed against ERK 1/2. Furthermore, inhibition of this pathway inhibited hypoxia-induced increase in mitochondrial membrane potential. The present study demonstrates that transient hypoxia induces a novel survival mechanism that protects endothelial cells against apoptosis. This endogenous process involves MEK/ERK-mediated inhibition of the pro-apoptotic molecule Bad and caspase 3.

Primary cells as feeder cells for coculture expansion of human hematopoietic stem cells from umbilical cord blood--a comparative study.

Although umbilical cord blood (UCB) has been widely accepted as an alternative source of hematopoietic stem cells (HSC) for transplantation, its use in adults is restricted because of low absolute HSC numbers. To overcome this obstacle, expansion of HSC in coculture with feeder cells is a promising possibility. In this study, we compared the potential of three human primary cell types, namely, mesenchymal stem cells (MSC), human umbilical cord vein endothelial cells (HUVEC), and Wharton's jelly cells (WJC), for use as feeder cells in a potentially clinically applicable coculture system. In first experiments, we evaluated procedures needed to obtain feeder cells, the possibility to separate them from cells derived from CD34(+) cells after coculture, their ability to activate allogeneic T cells, and their survival in CD34(+)-adapted medium. Finally, we compared their support for UCB-derived CD34(+) expansion. MSC and WJC were superior to HUVEC in terms of ease and reliability of isolation procedures needed. None of the potential feeder cells expressed CD34 or CD45, thus providing markers for cell sorting after coculture. Other markers (CD31, CD90, CD105, CD166) were expressed differently on feeder cell types. While MSC in higher concentrations did not activate allogeneic T cells, those were stimulated by lower concentrations of MSC as shown by CD25, CD69, and CD71 expression. In contrast, HUVEC and WJC were proven to activate T cells at all ratios tested. Feeder cells survived a 7-day culture in CD34(+)-adapted medium. In cocultures of UCB CD34(+)cells with primary feeder cells, mononuclear cell expansion was 30- to 60-fold, colony-forming cell expansion 20- to 40-fold, and cobblestone area-forming cell expansion 10- to 50-fold. We conclude that after a careful further evaluation especially of their immunological properties, all three primary cell types might possibly be suitable for use in a potentially clinically applicable system for expansion from UCB CD34(+)cells, with WJC being best choice and MSC still superior to HUVEC.

Spatio-temporal coherence of free electron laser pulses in the soft x-ray regime.

The temporal coherence properties of soft x-ray free electron laser pulses at FLASH are measured at 23.9 nm by interfering two time-delayed partial beams directly on a CCD camera. The partial beams are obtained by wave front beam splitting in an autocorrelator operating at photon energies from h nu = 30 to 200 eV. At zero delay a visibility of (0.63+/- 0.04) is measured. The delay of one partial beam reveals a coherence time of 6 fs at 23.9 nm. The visibility further displays a non-monotonic decay, which can be rationalized by the presence of multiple pulse structure.

Immunisation with 'naïve' syngeneic dendritic cells protects mice from tumour challenge.

Dendritic cells (DCs) 'pulsed' with an appropriate antigen may elicit an antitumour immune response in mouse models. However, while attempting to develop a DC immunotherapy protocol for the treatment of breast cancer based on the tumour-associated MUC1 glycoforms, we found that unpulsed DCs can affect tumour growth. Protection from RMA-MUC1 tumour challenge was achieved in C57Bl/6 MUC1 transgenic mice by immunising with syngeneic DCs pulsed with a MUC1 peptide. However, unpulsed DCs gave a similar level of protection, making it impossible to evaluate the effect of immunisation of mice with DCs pulsed with the specific peptide. Balb/C mice could also be protected from tumour challenge by immunisation with unpulsed DCs prior to challenge with murine mammary tumour cells (410.4) or these cells transfected with MUC1 (E3). Protection was achieved with as few as three injections of 50,000 naïve DCs per mouse per week, was not dependent on injection route, and was not specific to cell lines expressing human MUC1. However, the use of Rag2-knockout mice demonstrated that the adaptive immune response was required for tumour rejection. Injection of unpulsed DCs into mice bearing the E3 tumour slowed tumour growth. In vitro, production of IFN-gamma and IL-4 was increased in splenic cells isolated from mice immunised with DCs. Depleting CD4 T cells in vitro partially decreased cytokine production by splenocytes, but CD8 depletion had no effect. This paper shows that naïve syngeneic DCs may induce an antitumour immune response and has implications for DC immunotherapy preclinical and clinical trials.

Extracellular ATP induces assembly and activation of the myosin light chain phosphatase complex in endothelial cells.

OBJECTIVES: Extracellular ATP stabilizes the endothelial barrier and inactivates the contractile machinery of endothelial cells. This inactivation relies on dephosphorylation of the regulatory myosin light chain (MLC) due to an activation of the MLC phosphatase (MLCP). To date, activation and function of MLCP in endothelial cells are only partially understood. METHODS: Here, the mechanism of extracellular ATP-mediated activation of MLCP was analyzed in human endothelial cells from umbilical veins. Cells were transfected with the endogenous protein phosphatase 1 (PP1)-specific inhibitor-2 (I-2). RESULTS: Overexpression of I-2 led to inhibition of PP1 activity and abrogation of the ATP-induced dephosphorylation of MLC. This indicates that the PP1 catalytic subunit is the principal phosphatase catalyzing the MLC dephosphorylation induced by extracellular ATP. As demonstrated by immunoprecipitation analysis, extracellular ATP recruits the PP1delta catalytic subunit and the myosin phosphatase targeting subunit (MYPT1) to form a complex. ATP stimulated dephosphorylation of MYPT1 at the inhibitory phosphorylation sites threonine 850 and 696. However, extracellular ATP failed to stimulate MYPT1 dephosphorylation in I-2-overexpressing cells. CONCLUSIONS: The present study shows for the first time that, in endothelial cells, extracellular ATP causes activation of MLCP through recruitment of PP1delta and MYPT1 into a MLCP holoenzyme complex and PP1-mediated reduction of the inhibitory phosphorylation of MYPT1.

[The predictive quality of the Psychopathy Checklist-Revised (PCL-R) for violent and sex offenders in Switzerland. A validation study]. / Die prädiktive Qualität der Psychopathy Checklist-Revised (PCL-R) bei Gewalt- und Sexualstraftätern in der Schweiz. Eine Validierungsstudie.

In Switzerland, the Hare Psychopathy Checklist-Revised (PCL-R) is administered rather restrictively for risk assessment of recidivism among violent and sexual offenders. The aim of the present study was a first-time evaluation of the predictive validity of the PCL-R for violent and sexual recidivism in Switzerland. The PCL-R scores of 96 violent and sex offenders were evaluated by collecting the data in their psychiatric expert opinions. The scores were then compared to the rates of recidivism as shown in the criminal records. Consistent with previous studies in North America and Europe, the determined predictive accuracy was satisfying. This degree of precision supports the use of the PCL-R for risk assessment of sexual and violent recidivism in Switzerland, as long as the instrument does not constitute the sole criterion to determine future recidivism, but is applied only in combination with a thorough clinical evaluation. The use of precise cut-off scores did not prove to be a valid criterion for the prognosis of recidivism and can therefore not be recommended for Swiss offenders.

[Mechanisms influencing cellular physiology as a concept of treatment for wound healing disturbances]. / Mechanismen zur Beeinflussung der Zellphysiologie als Wundheilungskonzept.

AIM: Recent knowledge about repair mechanisms in different types of tissue is the basic of actual therapeutic efforts. Center of several experimental and clinical approaches is the influencing of angiogenesis with an also distinct meaning concerning wound healing. Therefore, application of growth factors, gene transfer, and employment of genetically manipulated cells often aim at angiogenesis. Nevertheless, manipulation of angiogenesis also leads to secondary problems such as hyperpermeability followed by impairment of local wound milieu. Our study was done to identify mechanisms to protect from disturbances of endothelial barrier function. METHOD: In a first experimental investigation on cultured endothelial cells, the influence of plasma-transglutaminase (Factor XIII) to endothelial barrier function was studied. In a second step, the influence of Factor XIII on wound healing properties was investigated in patients with a chronic venous ulceration. RESULTS: Activated Factor XIII (FXIIIA*) led to a dose-dependent reduction of endothelial cell permeability of 30 % compared to control with a maximum effect using 1 to 5 U/mL. Clinical investigation revealed a nearly complete reduction of wound secretion. CONCLUSION: Experimental studies revealed that activated Factor XIII stabilizes endothelial barrier under basic conditions as well as under conditions of induced hyperpermeability. Clinical study revealed that Factor XIII also distinctly reduces wound secretion. Therefore, plasma-transglutaminase may offer a new therapeutic option to treat the local or generalized leakage-syndrome.

Plasma transglutaminase factor XIII induces microvessel ingrowth into biodegradable hydroxyapatite implants in rats.

Coagulation factor XIII is a member of the transglutaminase-family. Transgluaminases cross-link either fibrin monomers in blood coagulation or extracellular proteins in extracellular matrix formation. In early stages of bone healing migration and proliferation of endothelial cells lead to formation of new vessels. The aim of this study was to investigate the angiogenetic activity of plasma factor XIII in bone defects filled with nanoparticulate hydroxyapatite paste. A critical size defect was created in the tibial head of rats which was not filled in group I. In group II the defect was filled with hydroxyapatite paste, and in group III with hydroxyapatite paste enriched with factor XIII. Ten days after surgery angiogenesis in the defects was assessed using immunohistochemistry and confocal laser scanning microscopy. Ac16 antibody was used to detect activation of factor XIII into factor XIIIA. In defects without biomaterial (group I) vessel-rich connective tissue and diffuse distribution of capillaries was observed. In defects filled with pure hydroxyapatite (group II) formation of capillaries was limited to the host bone-hydroxyapatite interface. In contrast, addition of plasma factor XIII to hydroxyapatite (group III) stimulated formation of vessels within the biomaterial. The current study reveals that factor XIII can improve angiogenesis in hydroxyapatite.

Opposite effect of cAMP signaling in endothelial barriers of different origin.

cAMP-mediated signaling mechanisms may destabilize or stabilize the endothelial barrier, depending on the origin of endothelial cells. Here, microvascular coronary [coronary endothelial cells (CEC)] and macrovascular aortic endothelial cell (AEC) monolayers with opposite responses to cAMP were analyzed. Macromolecule permeability, isometric force, activation state of contractile machinery [indicated by phosphorylation of regulatory myosin light chains (MLC), activity of MLC kinase, and MLC phosphatase], and dynamic changes of adhesion complex proteins (translocation of VE-cadherin and paxillin) were determined. cAMP signaling was stimulated by the adenosine receptor agonist 5'-N-(ethylcarboxamido)-adenosine (NECA), the beta-adrenoceptor agonist isoproterenol (Iso), or by the adenylyl cyclase activator forskolin (FSK). Permeability was increased in CEC and decreased in AEC on stimulation with NECA, Iso, or FSK. The effects could be inhibited by the PKA inhibitor Rp-8-CPT-cAMPS and imitated by the PKA activator Sp-cAMPS. Under cAMP/PKA-dependent stimulation, isometric force and MLC phosphorylation were reduced in monolayers of either cell type, due to an activation of MLC phosphatase. In CEC but not in AEC, FSK induced delocalization of VE-cadherin and paxillin from cellular adhesion complexes as indicated by cell fractionation and immunofluorescence microscopy. In conclusion, decline in contractile activation and isometric force contribute to cAMP/PKA-mediated stabilization of barrier function in AEC. In CEC, this stabilizing effect is overruled by cAMP-induced disintegration of cell adhesion structures.

Bioprocess development for the production of a recombinant MUC1 fusion protein expressed by CHO-K1 cells in protein-free medium.

The mucin MUC1 is a candidate for use in specific immunotherapy against breast cancer, but this requires the large-scale production of a MUC1 antigen. In this study, a bioprocess for the expression of a recombinant MUC1 fusion protein with a cancer associated glycosylation in CHO-K1 cells has been developed. Cells permanently expressing parts of the extracellular portion of MUC1 fused to IgG Fc were directly transferred from adherent growth in serum-containing medium to suspension culture in the protein-free ProCHO4-CDM culture medium. Using the Cellferm-pro system, optimal culture parameter as pH and pO(2) were determined in parallel spinner flask batch cultures. A pH of 6.8-7.0 and a pO(2) of 40% of air saturation was found to give best cell growth and productivity of secreted recombinant protein. Specific productivity strongly depended the pO(2) and correlated with the online monitored oxygen uptake rate (OUR) of the cells, which indicates a positive influence of the rate of oxidative phosphorylation on productivity. The optimised conditions were applied to continuous perfusion culture which gave very high cell densities and space time yields of the recombinant MUC1 fusion protein, allowing production at gram scale. The product degradation was much lower in supernatants from continuous perfusion culture compared to batch mode. Antibodies reacting with cancer associated MUC1 glycoforms strongly bound to the fusion protein, indicating that the desired glycoforms were obtained and suggesting that the recombinant MUC1 protein could be tested for use in immunotherapy.

MEK/MAPK as a signaling element in ATP control of endothelial myosin light chain.

Phosphorylation of endothelial myosin light chains (MLC) is a key mechanism in control of endothelial contractile machinery. Extracellular ATP influences endothelial MLC phosphorylation by either activation of Ca(2+)-dependent MLC kinase or Ca(2+)-independent MLC phosphatase. Here, the role of the MEK/MAPK pathway in this signaling was investigated in porcine aortic endothelial cells. Phosphorylation of ERK2 and phosphorylation of MLC were analyzed in cultured aortic endothelial cells. ATP (10 microM) increased ERK2 phosphorylation from basal 17 +/- 3 to 53 +/- 4%, an effect suppressed in the presence of the MEK inhibitors PD-98059 (20 microM) or U0126 (10 microM). Phosphorylation of ERK2 was not dependent on the ATP-induced cytosolic Ca(2+) rise, because it was unaltered when this was suppressed by the Ca(2+) chelator BAPTA (10 microM) or xestospongin C (3 microM), an inhibitor of the inositol 1,4,5-trisphosphate-sensitive Ca(2+) release mechanism of the endoplasmic reticulum. Phosphorylation of ERK2 was neither induced by the adenosine analog 5'-(N-ethylcarboxamido)adenosine (1 microM) nor inhibited in the presence of the adenosine receptor antagonist 8-phenyltheophylline (10 microM). ATP increased MLC kinase activity, and this was blocked in presence of PD-98059. ATP also increased MLC phosphatase activity, which was not inhibited by PD-98059. The MEK/MAPK pathway is a Ca(2+)-independent part of ATP signaling toward MLC kinase but not of ATP signaling toward MLC phosphatase.

Development of a standardized protocol for reproducible generation of matured monocyte-derived dendritic cells suitable for clinical application.

There is increasing interest in the generation of dendritic cells (DC) for cancer immunotherapy. In order to utilize DC in clinical trials it is necessary to have standardized, reproducible and easy to use protocols. We describe here the process development for the generation of DC as the result of investigation of culture conditions as well as consumption rates of medium and cytokines. Our studies demonstrate that highly viable DC (93 +/- 2%) can be produced from CD14(+) enriched monocytes via immunomagnetic beads in a high yield (31 +/- 6%) with X-VIVO 15, 400 U ml(-1) GM-CSF and 2000 U ml(-1) IL-4 without serum and feeding. For the maturation of DC different cocktails (TNF-alpha, IL-1beta, IL-6, PGE(2) and TNF-alpha, PGE(2)) were compared. In both cases cells expressed typical surface molecules of mature DC and induced high proliferative responses in mixed lymphocyte reactions which led to IFN-gamma producing T-lymphocytes. The data suggest that the use of this optimized, easy to use protocol results in highly mature DC.