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The motivation of using ecosystems to innovate services derives from our professional experience in the industry with both traditional product-service systems and with Industry 4.0 enabled product-service systems. In our approach, we integrate elements of service design thinking, visualisation, and some aspects of supply chain management to create the six-step framework. The model helps to find the gaps in the value creation, pointing towards potential disruptive dynamics within the ecosystem and supports the development of new services within complex ecosystems.

The collaboration project VASIB aims at reducing the antibiotic consumption in pig production by integrating information from consulting expertise in clinical inspection, hygiene, epidemiology, microbiology and pharmacology. In this VASIB subproject, we investigated the antimicrobial susceptibility and relatedness of porcine respiratory tract pathogens. Bordetella bronchiseptica (n = 47), Pasteurella multocida (n = 18) and Streptococcus suis (n = 58) were obtained from weaner pigs at two farms. Antimicrobial susceptibility testing was performed by broth microdilution according to CLSI standards. Resistance genes were detected via specific PCR assays. Macrorestriction analysis was conducted to determine the relatedness of the isolates and to identify clones. The B. bronchiseptica isolates showed indistinguishable (farm 1) or two closely related XbaI-patterns (farm 2). Different SmaI-PFGE patterns of P. multocida isolates were obtained at three different time points. In contrast, PFGE analysis of S. suis indicated more than one fragment pattern per pig and time point. Isolates exhibiting indistinguishable PFGE patterns were considered to represent the same clone. This study showed that only two closely related B. bronchiseptica clones were present in both farms, which had low MICs to all antimicrobials, except to β-lactams. Different P. multocida clones were present at the three time points. They showed overall low MIC values, with two clones being resistant and one intermediate to tetracycline. S. suis clones were resistant to tetracycline (n = 19) and/or erythromycin/clindamycin (n = 16). They harboured the tetracycline resistance genes tet(O), tet(M) or tet(L) and/or the macrolide/lincosamide/streptogramin B resistance gene erm(B). Five penicillin-resistant S. suis clones were also detected.

Leptospirosis is a ubiquitous and potentially fatal zoonosis with protean manifestations. Human infection commonly occurs through contact with contaminated water or soil. In developed countries, leisure or household activities are increasingly associated with the disease. Within few months, we encountered five unrelated and autochthonous cases of severe leptospirosis, three of them requiring interim dialysis. In this case series, we present their clinical course. Furthermore, we provide an overview on the spectrum of organ involvement, with an emphasis on kidney injury, and comment on pitfalls in establishing the diagnosis. The considerable variance in presentation-with admissions both to internal and neurological units-emphasises the high index of suspicion required to arrive at the right diagnosis, particularly in countries of perceived low risk such as Austria.

The spatial organisation of the chromosomes in the nucleus is influenced by chromatin regions binding to the nucleic lamina, i.e., the inner part of the nucleic envelope. To investigate the architecture of chromosomes in the interphase nucleus, it is thus of high interest to detect such chromatin segments. This goal can be achieved by considering the fibrous protein Lamin B as a surrogate, since regions of high abundance of Lamin B can indicate chromatin segments attached to the nucleic lamina. We analyse ChIP-Seq (Chromatin-Immunoprecipitation Sequencing) data from an experiment that is designed to record Lamin B abundance. We introduce a Bayesian segmentation procedure in which a Markov Chain Monte Carlo (MCMC) algorithm is used for inference about the desired segmentation. The procedure is based on a Bayesian hierarchical model. Inference allows the distinction between regions of high versus low levels of Lamin B, and therefore, gives an insight into the binding of the chromatin to the nucleic envelope. An implementation of this approach is available in the statistical software environment R. This article is part of a special issue entitled: Computational proteomics in the post-identification era. Guest Editors: Martin Eisenacher and Christian Stephan.

To estimate the risk of dissemination of Clavibacter michiganensis ssp. sepedonicus through potato residues from processing industries, the various processes and the usage of residues from plants from different processing branches were analysed with regard to the effect they can have on the pathogen. The residues were classified into different risk categories, from category 0 (no risk of dissemination) to category 4 (high risk of dissemination). Residues not heated during processing and used in agriculture, e.g., as fertilizer, were pooled in the highest risk category 4. Residues that were sanitised before use in agriculture, e.g., by composting or pasteurisation, were still classified as probably high risk (risk category 3), as no information on these treatments concerning the inactivation of the pathogen was available so far. Therefore the effect of composting and pasteurisation under varying conditions was tested on samples (ready-made compost mould) contaminated with Clavibacter michiganensis ssp. sepedonicus. Viable bacteria could be extracted after all experiments via bioassay on eggplants, and cultivated on semi-selective media from plant sap forming characteristic colonies. The viable pathogen could be extracted after composting for 6 days at maximum temperatures at 70 °C, 13 days at 55 °C and 90 min pasteurisation at 70 °C. It can be concluded that these sanitation treatments are not sufficient to inactivate Clavibacter michiganensis ssp. sepedonicus and the previous classification of treated residues in category 3 (probably high risk) could thus be confirmed.

The emergence of new laser-based mid-infrared (MIR) sources, such as quantum cascade lasers (QCL), led to substantial developments in the field of MIR spectroscopy in the last decade. Recently, also MIR supercontinuum (SC) sources became available. They combine broadband spectral emission known from thermal sources emission with coherent properties known from laser sources like QCLs. Nevertheless, while the latter already find practical application in the field of optical sensing, SC sources have yet to prove their applicability. In this contribution we present the development, characterization and application of a measurement concept consisting of a fiber-coupled broadband MIR SC source (1.75 μm-4.2 μm, 75 mW optical power) and a fully-integrated MOEMS-based Fabry-Perot microspectrometer (FPMS) for MIR spectroscopy. The main hindrance for the use of SC sources in spectroscopy so far, are the significant pulse-to-pulse fluctuations arising from the non-linear nature of the SC generation process. We show to what extent spectral averaging makes sense and evaluate the noise performance. By combining a SC source and a FPMS it was possible to significantly reduce noise in spectral, time and polarization domain, resulting in a set-up suitable for MIR spectroscopy. The performance of the set-up was characterized both in transmission and reflection geometry. Low-noise absorption spectra of oils, polymers and aqueous solutions of acetic acid were acquired . Furthermore, time-resolved measurements of the curing process of ethyl-2-cyanoacrylate and results of the chemical mapping of a painted metal surface are reported. The obtained results prove the concept of SC-FPMS promising for MIR spectroscopy, characterized by its simplicity and versatility.

The interaction of mesenchymal stem cells (MSCs) with endothelium in vivo is significant for regenerative processes in organisms. To design concepts for tissue engineering for bone regeneration based on this interaction, the osteogenic differentiation of human bone marrow-derived MSCs in a co-culture with human dermal microvascular endothelial cells (HDMECs) was studied. The experiments were focussed on the regulation of MSCs in a co-culture with HDMECs on different calcium phosphate scaffolds. Alkaline phosphatase (ALP) activity and mRNA expression of various osteogenic markers increased significantly when cells were co-cultured on materials with calcium phosphate scaffolds compared to tissue culture polystyrene or when MSCs were cultured alone. In addition, it was observed that the expression of osteopontin and osteocalcin was highly sensitive to the substrate for cell adhesion. Whereas these late osteogenic markers were down-regulated in co-cultures on polystyrene, they were up-regulated on calcium phosphate and moreover, were differentially expressed on the three calcium phosphate scaffolds tested. To enhance the osteogenic differentiation of MSCs in a co-culture, direct cell-cell interactions were required. Concerning molecular mechanisms in the interactions between both cell types, it was found that connexin 43 was expressed in contact sites and more apparently, endothelial cells grew over the MSCs, which facilitated direct cellular interactions mediated by various adhesion receptors. This study revealed significant findings for the design of implant materials suitable for regeneration of bone by stimulating the functional interaction of MSCs with endothelial cells. Copyright © 2012 John Wiley & Sons, Ltd.

The two quarantine pests Synchytrium endobioticum, the causal agent of potato wart disease and Globodera rostochiensis, the yellow potato cyst nematode are currently present in Germany. Winter sporangia of Synchytrium endobioticum and cysts of Globodera rostochiensis can be spread with waste from potato processing industries, if infected tubers are processed. The German Biowaste Ordinance prescribes sanitation of organic waste before it can be used on arable land as fertilizer or filling material. Sanitation parameters prescribed by the German Biowaste Ordinance include composting for 7 days at 65°C or 14 days at 55°C or pasteurisation for 60 min at 70°C. The effect of composting and pasteurisation processes on winter sporangia of Synchytrium endobioticum and cysts of Globodera rostochiensis was tested with varying time-temperature relations. Cysts of Globodera rostochiensis were killed by composting for 7 days at 50–55°C and by pasteurisation for 30 min at 70°C. In contrast, viable winter sporangia of Synchytrium endobioticum could be extracted from sample material after composting for 70 days at 30–45°C, composting for 21 days at 50–55°C and after composting for 12 days at 60–65°C. Likewise viable winter sporangia could be extracted after pasteurisation for 90 min at 70°C and heating in a water bath at 80°C and in a dry oven at 90°C for 8 h. The parameters prescribed in the German Biowaste Ordinance are sufficient to kill cysts of Globodera rostochiensis but not sufficient to kill winter sporangia of Synchytrium endobioticum in organic waste.

The control of mesenchymal stem cells (MSC) by physical cues is of great interest in regenerative medicine. Because integrin receptors function as mechanotransducers, we applied drag forces to β1 integrins on the apical surface of adherent human MSC. In addition to mechanical forces, the technique we used involved also the exposure of the cells to an inhomogeneous magnetic field. In order to assess the influence of the substrate on cell adhesion, cells were cultured on plain tissue culture polystyrene (TCP) or on coated well plates, which allowed only adhesion to embedded fibronectin or RGD peptides. We found that the expression of collagen I, which is involved in osteogenesis, and VEGF, a factor which stimulates angiogenesis, increased as a result of short-term mechanical integrin stress. Whereas, collagen I expression was stimulated by mechanical forces when the cells were cultured on fibronectin and RGD peptides but not on TCP, VEGF expression was enhanced by physical stimulation on TCP. The study further revealed that magnetic forces enhanced Sox 9 expression, a marker of chondrogenesis, and reduced the expression of ALP. Concerning the intracellular mechanisms involved, we found that the expression of VEGF induced by physical forces depended on Akt activation. Together, the results implicate that biological functions of MSC can be stimulated by integrin-mediated mechanical forces and a magnetic field. However, the responses of cells depend strongly on the substrate to which they adhere and on the cross-talk between integrin-mediated signals and soluble factors.

Although studies in vivo revealed promising results in bone regeneration after implantation of scaffolds together with osteogenic progenitor cells, basic questions remain how material surfaces control the biology of mesenchymal stem cells (MSC). We used human MSC derived from bone marrow and studied the osteogenic differentiation on calcium phosphate surfaces. In osteogenic differentiation medium MSC differentiated to osteoblasts on hydroxyapatite and BONITmatrix®, a degradable xerogel composite, within 14 days. Cells revealed a higher alkaline phosphatase (ALP) activity and increased RNA expression of collagen I and osteocalcin using real-time RTPCR compared with cells on tissue culture plastic. To test whether material surface characteristics alone are able to stimulate osteogenic differentiation, MSC were cultured on the materials in expansion medium without soluble additives for osteogenic differentiation. Indeed, cells on calcium phosphate without osteogenic differentiation additives developed to osteoblasts as shown by increased ALP activity and expression of osteogenic genes, which was not the case on tissue culture plastic. Because we reasoned that the stimulating effect on osteogenesis by calcium phosphate surfaces depends on an altered cell–extracellular matrix interaction we studied the dynamic behaviour of focal adhesions using cells transfected with GFP labelled vinculin. On BONITmatrix®, an increased mobility of focal adhesions was observed compared with cells on tissue culture plastic. In conclusion, calcium phosphate surfaces are able to drive MSC to osteoblasts in the absence of osteogenic differentiation supplements in the medium. An altered dynamic behaviour of focal adhesions on calcium phosphate surfaces might be involved in the molecular mechanisms which promote osteogenic differentiation.

We read with interest the case report by Witczak et al .1 The authors describe a young woman who presented with headache, neck stiffness and confusion and was eventually found to have primary adrenal insufficiency. The authors speculate that the patient’s neurological manifestation was a consequence of the underlying adrenal failure, although they acknowledge that there is no reported association of the patient's initial presentation with adrenal crisis. We wonder whether a better recognized entity was considered by the authors, which could easily account for the patient's …

Intracellular replication of Salmonella enterica requires the formation of a unique organelle termed Salmonella-containing vacuole (SCV). The type III secretion system (T3SS) encoded by Salmonella Pathogenicity Island 2 (SPI2-T3SS) has a crucial role in the formation and maintenance of the SCV. The SPI2-T3SS translocates a large number of effector proteins that interfere with host cell functions such as microtubule-dependent transport. We investigated the function of the effector SseF and observed that this protein is required to maintain the SCV in a juxtanuclear position in infected epithelial cells. The formation of juxtanuclear clusters of replicating Salmonella required the recruitment of dynein to the SCV but SseF-deficient strains were highly reduced in dynein recruitment to the SCV. We performed a functional dissection of SseF and defined domains that were important for translocation and the specific effector functions of this protein. Of particular importance was a hydrophobic domain in the C-terminal half that contains three putative transmembrane (TM) helices. Deletion of one of these TM helices ablated the effector functions of SseF. We observed that this domain was essential for the proper intracellular positioning of the SCV to a juxtanuclear, Golgi-associated localization. These data show that SseF, in concert with the effector proteins SifA and SseG mediate the precise positioning of the SCV by differentially modulating the recruitment of microtubule motor proteins to the SCV.

Crops and weeds were tested for their ability to host Clavibacter michiganensis ssp. sepedonicus (Cms), the causal agent of bacterial ring rot in potato. Ten crops grown in rotation with potato in Europe, namely maize, wheat, barley, oat, bush bean, broad bean, rape, pea and onion and five cultivars of sugar beet were tested by stem and root inoculation. About 6-8 weeks after inoculation, Cms could be detected in most crops except onion and sugar beet, in larger numbers in stems (105-106 cells/g of tissue) than in roots (?103 cells/g of tissue) in immunofluorescence cell-staining (IF). Cms was successfully re-isolated only from IF-positive stem samples of maize, bush bean, broad bean, rape and pea, but not from roots. Twelve solanaceous weeds and 13 other weeds, most commonly found in potato fields in Europe, were tested in IF as hosts of Cms by stem and root inoculations. Only in Solanum rostratum, a weed present in northern America, Cms persisted in high numbers (108 cells/g tissue) in stems and leaves, where it caused symptoms. In the other solanaceous weeds, Cms persisted at low numbers (approximately 105 cells/g of tissue) in stems but less so in roots. The bacteria could be frequently re-isolated from stem but not from root tissues. In 2 consecutive years, plants from 14 different weed species were collected from Cms-contaminated potato field plots and tested for the presence of Cms by dilution plating or immunofluorescence colony-staining (IFC), and by AmpliDet RNA, a nucleic acid-based amplification method. Cms was detected in roots but not in stems of Elymus repens plants growing through rotten potato tubers, and in some Viola arvensis and Stellaria media plants, where they were detected both in stems and roots, but more frequently by AmpliDet RNA than by IFC.

Am Institut fur Pflanzenschutz (IPS) der Bayerischen Landesanstalt fur Landwirtschaft (LfL) in Freising wurde in Feldversuchen in den Jahren 2001 und 2002 die Ubertragbarkeit des Erregers der Bakteriellen Ringfaule der Kartoffel, Clavibacter michiganensis ssp. sepedonicus (Cms), von kontaminierten Holzlagerkisten auf darin eingelagertes Pflanzgut untersucht. In drei Versuchen wurden gesunde Knollen wahrend der Lagerung in Kontakt mit Cms gebracht: (i) 4-wochige Lagerung in frisch kontaminierten Kisten bei 8°C, (ii) 8-monatige Lagerung bei unregulierter Kellertemperatur (zwischen 1 und 15°C) in Kisten, die bereits 5 Monate kontaminiert und seitdem unbefullt waren und (iii) 5-monatige Lagerung in frisch kontaminierten Kisten bei 4°C. Anschliesend wurden die Knollen ausgepflanzt und der Befall an Kraut und Knollen der Stauden mit der Polymerasekettenreaktion (PCR) und dem Immunfluoreszenz(IF)-Test ermittelt. Die Lagerung des Pflanzguts in kontaminierten Kisten fuhrte in allen Versuchen zu infizierten Pflanzen: 2 (ii) bis 9% (i) der Stauden wiesen positive PCR-Ergebnisse der Tochterknollen auf.

In den Jahren 2001 und 2002 wurden am Pflanzenschutzamt der Landwirtschaftskammer (LWK) Weser-Ems und am Institut fur Pflanzenschutz der Bayerischen Landesanstalt fur Landwirtschaft (LfL) in Freising jeweils zwei Feldversuche zur Ubertragbarkeit der Bakteriellen Ringfaule der Kartoffel (Clavibacter michiganensis ssp. sepedonicus) mit dem Krautschlager durchgefuhrt. An der LWK Weser-Ems wurde fur die Untersuchung der Fragestellung ein praxisnahes Modellsystem eingesetzt, in dem das Krautschlagen durch die Verwendung eines Rasentrimmers simuliert wurde und die Schnittflachen der Pflanzenstangel anschliesend mit Bakteriensuspension bespruht wurden. Es wurden dabei zwei verschiedene Sorten (fruh und spat) verwendet als auch die Wirkung einer zusatzlichen chemischen Behandlung zur Reiferegulation getestet. An der LfL in Freising wurden Parzellen mit infizierten Pflanzen fur die Kontamination des Krautschlagers angelegt. Die Pflanzen auf diesen Parzellen wurden jeweils direkt vor dem Befahren der Parzellen mit gesunden Stauden geschlagen. Der Befall an Kraut (Wiederaustrieb) und Knollen der Stauden wurde mit der Polymerasekettenreaktion (PCR) und dem Immunfluoreszenz(IF)-Test ermittelt.

In Mikrokosmosuntersuchungen wurde das Uberleben von Ralstonia solanacearum Biovar 2 (Rasse 3) in Boden bei einem Wassergehalt am permanenten Welkpunkt (WP) und bei Feldkapazitat (FC) sowie auf verschiedenen Materialien gepruft. Die Boden wurden bei konstanten –5°C, 4°C, 15°C und 20°C sowie wochentlich wechselnden –10/0/+10°C aufgestellt und das Material bei 5°C, 15°C, 20°C, wobei die relative Luftfeuchte (rLF) entweder ungeregelt oder bei konstant 10 bzw. 90% blieb.

In den Jahren 1999 bis 2003 wurde in Freiland-, Klimakammer- und Lagerungsversuchen uberpruft, ob ein Risiko fur die Ubertragung des Erregers der Bakteriellen Ringfaule der Kartoffel (Clavibacter michiganensis ssp. sepedonicus) besteht, wenn (a) gesunde Kartoffelknollen in Kontakt mit Maschinen und Geraten kommen, die mit dem Erreger kontaminiert sind (indirekter Kontakt) und (b) gesunde Kartoffelknollen direkt in Kontakt mit infizierten Knollen kommen (direkter Kontakt). Nach indirektem Kontakt konnte nur beim nachfolgenden Anbau der kontaminierten Knollen in der Klimakammer Befall in Kraut und Knollen festgestellt werden. Im Freiland konnte der Erreger, auch bei wiederholtem Nachbau der geernteten Knollen, nicht nachgewiesen werden. Nach direktem Kontakt und nachfolgendem Anbau der kontaminierten Knollen in der Klimakammer und im Freiland, wurde der Erreger in allen Fallen in den geerntete Knollen nachgewiesen. Befall im Kraut wurde nur in dem Klimakammerversuch und in einem Freilandversuch ermittelt. Wurden durch direkten Kontakt kontaminierte Knollen eingelagert, konnte der Erreger in allen untersuchten Knollen festgestellt werden. Insgesamt besteht ein hohes Risiko, dass gesunde Knollen infiziert werden, wenn oberflachliche Kontaminationen mit dem Erreger erfolgen. Die Wahrscheinlichkeit von Infektionen steigt mit zunehmender Kontaminationsstarke.

In den Jahren 1999 bis 2001 wurde in Freilandversuchen uberpruft, ob wahrend der Vegetationsperiode ein Risiko besteht, den Erreger der Bakteriellen Ringfaule der Kartoffel (Clavibacter michiganensis ssp. sepedonicus) durch kontaminierte Maschinen und Gerate in das Kraut von gesunden Kartoffelpflanzen zu ubertragen und wenn diese Ubertragung moglich ist, ob eine Ableitung der Bakterien in die neu gebildeten Knollen erfolgt. Es wurden zwei verschiedene Methoden gewahlt: einerseits wurde eine scharfkantige Metallschiene, die mit einer Bakteriensuspension kontaminiert war, bis zu 6-mal in der Vegetationsperiode durch das Kraut von Kartoffelpflanzen gezogen. In dem anderen Fall wurde eine Suspension des Erregers mit einer Spritze direkt in die Stangel der Kartoffelpflanzen injiziert. In allen Versuchen wurden die geernteten Knollen untersucht, in den Versuchen mit der kontaminierten Metallschiene wurden auch Untersuchungen des Krauts durchgefuhrt.

The determination of the fractal dimension is an interesting way for the characterization of rough surfaces. Microstructured titanium surfaces produced by different technologies (polishing, glass and corundum blasting, vacuum plasma spraying) were investigated by a number of physical and electrochemical methods, i.e. surface profiling, scanning electron microscopy, voltammetry and electrochemical impedance spectroscopy. The data sets obtained by these very different methods were used for the estimation of the fractal dimension of all structures. The aim of this work is to examine the suitability of this additional parameter for a better characterization of structured surfaces and to compare the various applied methods regarding their feasibility. Accompanying cell biological examinations were used to verify whether there is a relation between the parameter fractal dimension and cell behaviour.

BACKGROUND The etiology of tumors arising in the biliary tract remains unclear. Several previous studies have detected Helicobacter pylori organisms in bile from patients with gallstones or cholecystitis. The objective of this study was to determine whether there is an association between H. pylori in bile and biliary tract carcinoma. METHODS The authors used polymerase chain reaction (PCR) assays to detect the presence of H. pylori in the stomach and bile from 89 patients: Sixty-three disease free patients had biliary calculi, 15 patients had carcinoma of the biliary tract, and 11 patients had neither gallstones nor carcinoma. Bile was considered to contain H. pylori only if the results of PCR determinations were positive in two or more samples assayed independently in two separate laboratories. RESULTS There was a strong association between the presence of H. pylori in the stomach and in the bile (P ≤ 0.01). Biliary H. pylori was associated with age but not with gender, and it was associated strongly with the clinical diagnosis. Patients with gallstones were 3.5 times as likely to have H. pylori in the bile compared with patients in a control group (95% confidence interval [95%CI], 0.8–15.8; P = 0.100), and H. pylori was 9.9 times more frequent in patients with biliary tract carcinoma compared with patients in the control group (95%CI, 1.4–70.5; P = 0.022). CONCLUSIONS There is a strong association between biliary tract carcinoma and H. pylori in bile. If these results are confirmed by prospective studies, H. pylori may be responsible for a significant proportion of malignant biliary tract disease. Cancer 2002;95:1946–53. © 2002 American Cancer Society. DOI 10.1002/cncr.10893

Coping with illness and the process of dying can be interpreted as an antinomy for a physician's work from ancient times. Successful coping is targeted by definition--at least implicitly--on preserving human life at the best quality possible. On the contrary, the process of dying leading to an individual's death remains out of scope. The aim of our study is to describe present concepts concerning the process of dying and successful coping with illness (i.e., with cancer) in order to look for the specific place of competent medical attendance and terminal care within this context.

Following the idea that integrin receptors function as mechanotransducers, we applied defined physical forces to integrins in osteoblastic cells using a magnetic drag force device to show how cells sense different modes of physical forces. Application of mechanical stress to the beta1-integrin subunit revealed that cyclic forces of 1 Hz were more effective to stimulate the cellular calcium response than continuous load. Cyclic forces also induced an enhanced cytoskeletal anchorage of tyrosine-phosphorylated proteins and increased activation of the focal adhesion kinase (FAK) and mitogen activated protein (MAP) kinase. These events were dependent on an intact cytoskeleton and the presence of intracellular calcium. Analyses of the intracellular spatial organization of the calcium responses revealed that calcium signals originate in a restricted region in the vicinity of the stressed receptors, which indicates that cells are able to sense locally applied stress on the cell surface via integrins. The calcium signals can spread throughout the cell including the nucleus, which shows that calcium also is a candidate to transmit mechanically induced information into different cellular compartments.

Designing of implant surfaces using a suitable ligand for cell adhesion to stimulate specific biological responses of stem cells will boost the application of regenerative implants. For example, materials that facilitate rapid and guided migration of stem cells would promote tissue regeneration. When seeded on fibronectin (FN) that was homogeneously immmobilized to NCO-sP(EO-stat-PO), which otherwise prevents protein binding and cell adhesion, human mesenchymal stem cells (MSC) revealed a faster migration, increased spreading and a more rapid organization of different cellular components for cell adhesion on fibronectin than on a glass surface. To further explore, how a structural organization of FN controls the behavior of MSC, adhesive lines of FN with varying width between 10 mu m and 80 mu m and spacings between 5 mu m and 20 mu m that did not allow cell adhesion were generated. In dependance on both line width and gaps, cells formed adjacent cell contacts, were individually organized in lines, or bridged the lines. With decreasing sizes of FN lines, speed and directionality of cell migration increased, which correlated with organization of the actin cytoskeleton, size and shape of the nuclei as well as of focal adhesions. Together, defined FN lines and gaps enabled a fine tuning of the structural organization of cellular components and migration. Microstructured adhesive substrates can mimic the extracellular matrix in vivo and stimulate cellular mechanisms which play a role in tissue regeneration.

Programmed cell death via the Fas receptor/Fas Ligand and DR4, DR5/TRAIL plays a major role in tumor escape and elimination mechanisms. It also promises to be an effective therapy alternative for aggressive tumors, as has been recently shown for colon, breast, and lung cancer cells. We attempted to clarify the role of these molecules in aggressivity of pancreatic carcinomas and to identify possible pathways as targets for therapy.Five pancreatic cell lines were investigated for the expression of FasL/Fas, DcR3, DR4, DR5/TRAIL, DcR1, DcR2, and other death pathways related molecules such as Bax, bcl-xL, bcl-2, FADD, and caspase-3 by flow cytometry, immunoblotting, and RT/PCR, both semiquantitative and real time (TaqMan). The susceptibility of these cell lines to apoptosis mediated by recombinant TRAIL was investigated. The effect of therapeutic agents (gemcitabine) on their susceptibility to TRAIL induced apoptosis was studied as well.Pancreatic adenocarcinomas expressed high levels of apoptosis-inducing receptors and ligands. They showed differential susceptibility to cell death induced by TRAIL, despite expressing intact receptors and signaling machineries. Treatment with commonly used therapeutic agents did not augment their susceptibility to apoptosis. This could be explained by the fact that they expressed differentially high levels of decoy receptors, as well as molecules known as inhibitors of apoptosis.The data suggest that pancreatic carcinoma cells have developed different mechanisms to evade the immune system. One is the expression of nonfunctional receptors, decoy receptors, and molecules that block cell death, such as bcl2 and bcl-xL. The second is the expression of apoptosis-inducing ligands, such as TRAIL, that could induce cell death of immune cells. The success in treating malignant tumors by recombinant TRAIL might apply to some but not all pancreatic tumors because of their differential resistance to TRAIL-induced cell death.

Background The diagnosis of biliary disease, namely malignant disorders, is frequently hampered by the inconclusive cytology. We investigated prospectively the frequency of molecular changes in p53 and ras compared with cytology in patients with primary or secondary hepato-biliary disease. Material and methods We investigated 118 consecutive patients, aged 24-89 with the following clincal diagnoses: choledocho/cholecystolithiasis (28), cholangiocellular carcinoma (21), gall bladder tumor (8), liver metastasis (3), autoimmune disease (8), chronic pancreatitis (16), pancreatic carcinoma (11), papillary disease (4), hepatic cirrhosis (6), cholangitis (2), anomalies (2), and normal (9). Bile was aspirated during routine endoscopic retrograde cholangio pancreatography (ERCP) or percutaneous transhepatic cholangiography (PTC). DNA was prepared freshly from a native aliquot, p53 mutations were detected by polymerase chain reaction (PCR) for exons 5 through 8 followed by TGGE. PCR for ras mutations was performed as RFLP-PCR with sequencing. Results In four cases, mutations in p53 could be found in exons 6 and 7. Twenty-two samples showed ras mutations; ras mutations were found in choledocholithiasis (4/28), bile duct (5/21), gall bladder (3/8) and pancreatic (1/11) carcinoma, liver metastasis (3/3), ulcerative colitis (2/3), PSC (1/2), and chronic pancreatitis (1/16). Cytology was clearly positive in seven cases, suspicious in three other, inconclusive in six, and negative in the rest. The molecular analysis resulted in a sensitivity of 33% and specificity of 87%, respectively, for the diagnosis of a malignant condition. Conclusion PCR for p53 and ras mutations may aid the diagnosis of primary and secondary (metastatic) hepatobiliary disease if a malignant condition of the bile ducts and the liver is suspected and cytology is inconclusive or negative. However, the incidence of p53 and ras mutations in bile seems less frequent than in other malignant conditions of the gastrointestinal tract and the pancreas and lower than in tissue, leaving a poor sensitiviy and specificity. Nevertheless, the presence of a p53 and/or ras mutation per se supports a clinical suspicion of malignancy, even when the conventional cytology is negative or inconclusive.

The prognosis of pancreatic cancer is poor, and current medical treatment is mostly ineffective. The aim of this study was to design a new treatment modality in an animal model system. We describe here a novel treatment strategy employing a mouse model system for pancreatic carcinoma. Embryonal kidney epithelial cells were genetically modified to express the cytochrome P450 subenzyme 2B1 under the control of a cytomegalovirus (CMV) immediate early promoter. This CYP2B1 gene converts ifosfamide to its active cytotoxic compounds, phosphoramide mustard, which alkylates DNA, and acrolein, which alkylates proteins. The cells were then encapsulated in a cellulose sulphate formulation and implanted into preestablished tumors derived from a human pancreatic tumor cell line. Intraperitoneal administration of low-dose ifosfamide to tumor bearing mice that received the encapsulated cells results in partial or even complete tumor ablation. Such an in situ chemotherapy strategy utilizing genetically modified cells in an immunoprotected environment may prove useful for solid tumor therapy in man.

Microencapsulation, as a tool for immunoisolation for allogenic or xenogenic implants, is a rapidly growing field. However most of the approaches are based on alginate/polylysine capsules, despite this system's obvious disadvantages such as its pyrogenicity. Here we report a different encapsulation system based on sodium cellulose sulfate and polydiallyldimethyl ammonium chloride for the encapsulation of mammalian cells. We have characterized this system regarding capsule formation, strength and size of the capsules as well as viability of the cells after encapsulation. In addition, we demonstrate the efficacy of these capsules as a "microfactory" in vitro and in vivo. Using encapsulated hybridoma cells we were able to demonstrate long-term release of antibodies up to four months in vivo. In another application we could show the therapeutic relevance of encapsulated genetically modified cells as an in vivo activation center for cytostatic drugs during tumor therapy.

Since 2004 discussions on the need for greater international co-operation and coordination between diagnostic laboratories have taken place at several meetings of the EU Heads of National Plant Protection Organisations (NPPOs) and of the European and Mediterranean Plant Protection Organization (EPPO). Technical discussions on criteria for, and tasks and responsibilities of, National Reference Laboratories (NRLs) took place in a Commission Expert Working Group in September 2007. The meeting was attended by representatives from 13 Member States. The experts discussed the issues broadly and very actively and it was possible to elaborate an agreed Guidance Paper containing the main elements for the establishment of NRLs, including a priority list of individual pests for which such laboratories are needed. At their meeting in December 2007, the EU Heads of the NPPOs supported the establishment of NRLs as far as possible and where necessary. It was agreed that the Guidance Paper is a suitable common basis for the establishment of NRLs in the Member States and that it should be reviewed on the basis of the experience gained at national level.

The prognosis of pancreatic adenocarcinoma is poor and current treatment ineffective. A novel treatment strategy is described here using a mouse model system for pancreatic cancer. Cells that have been genetically modified to express the cytochrome P450 2B1 enzyme are encapsulated in cellulose sulphate and implanted into pre-established tumours derived from human pancreatic cells. Cytochrome P450 2B1 converts the chemotherapeutic agent ifosfamide to toxic metabolites. Administration of ifosfamide to tumour-bearing mice that were recipients of implanted encapsulated cells results in partial or even complete tumour ablation. These results suggest that in situ chemotherapy with genetically modified cells in an immunoprotected environment may prove useful for application in man.

CD2 is a co-stimulatory receptor involved in T cell activation. Here we report on immunosuppressive effects of three mouse CD2 monoclonal antibodies (OX34, OX54, OX55) directed against non-overlapping epitopes of the rat CD2 receptor on various modes of T cell activation in vitro and in vivo. Although non-ligand-blocking OX54 and OX55, in concert, activated T cells through CD2 in vitro, they individually suppressed the mixed lymphocyte reaction (MLR) and significantly prolonged allograft survival after rat heart transplantation in vivo. Phenotype analysis revealed that OX55 significantly down-modulated CD2 in vivo, whereas OX54 depleted T cells. Graft rejection coincided with re-expression of CD2 and clearance of OX55 from serum, whereas T cell depletion by OX54 outlasted the period of graft survival. The most suppressive antibody, OX34, down-modulated CD2 and inhibited T cell activation through the TCR or CD2 and the MLR and prolonged median allograft survival time from 7 days in controls to 45 days in the absence of any additional treatment. Graft survival was clearly dose dependent and correlated with the duration of CD2 down-modulation and the presence of circulating CD2 antibody in serum. Importantly, the specific antibody production to a T cell-dependent antigen as demonstrated by immunization with keyhole limpet hemocyanin in vivo remained unaffected after treatment with OX34. These results demonstrate the pivotal role of CD2 signaling in mediating allogeneic immune reactions after vascularized organ transplantation while allowing specific humoral immune responses in vivo.

Epidemiological data show an association between Helicobacter pylori gastritis and gastric carcinoma. However, most people infected with H. pylori do not develop gastric cancer. We have therefore evaluated histological criteria indicating an increased risk for gastric cancer. H. pylori gastritis was investigated in 117 patients with small (∅

CD48, the murine homolog of human CD58, binds to CD2 in rats and mice. Whereas inhibition of CD2 signaling leads to profound immunosuppression, no information is available on CD48-targeted therapy in the rat. We could show that anti-CD2 treatment (OX34) efficiently inhibited TCR-driven as well as CD2-mediated proliferation, whereas blocking of ligand binding (OX45) remained completely uneffective. Inhibition of allogeneic MLR by OX45 turned out to be due to induction of unspecific suppressive mechanisms. In vivo, OX45 failed to prolong rat heart allograft survival in contrast to that seen with OX34. Grafts were rejected despite persistent and complete downmodulation of CD48 on lymphocytes without any cell depleting effect, rendering receptor/ligand interactions physically impossible. Combined application of CD2 and CD48 mAb did not enhance immunosuppression induced by CD2 mAb alone. Provided that there is no alternative CD2 ligand in the rat, we conclude that CD2-directed immunotherapy is mediated by suppressive events induced by modulation of the CD2 receptor (“negative signaling”) rather than by mere disruption of the CD2–CD48 interaction.

For the treatment of acute ischemic stroke (AIS), IV recominant tissue plasminogen activator (rtPA) has proven efficacy n eligible patients, but carries an increased risk of intracereral hemorrhage [1]. Preexisting anticoagulation, including the irect thrombin inhibitor dabigatran, usually contraindicates the se of rtPA. This notwithstanding, several single cases of IV tPA treatment in patients with AIS under dabigatran have een reported, with mixed results [2]. Endovascular therapy has merged as a potential, yet still experimental, alternative treatment ption for a number of clinical scenarios with contraindications o IV rtPA [3]. We report successful rescue thrombectomy in two patients who uffered AIS under treatment with dabigatran for atrial fibrillation AF).

During intracellular life, the bacterial pathogen Salmonella enterica translocates a complex cocktail of effector proteins by means of the SPI2-encoded type III secretions system. The effectors jointly modify the endosomal system and vesicular transport in host cells. SseF and SseG are two effectors encoded by genes within Salmonella Pathogenicity Island 2 and both effector associate with endosomal membranes and microtubules and are involved in the formation of Salmonella-induced filaments. Our previous deletional analyses identified protein domains of SseF required for the effector function. Here we present a detailed mutational analysis that identifies a short hydrophobic motif as functionally essential. We demonstrate that SseF and SseG are still functional if translocated as a single fusion protein, but also mediate effector function if translocated in cells co-infected with sseF and sseG strains. SseF has characteristics of an integral membrane protein after translocation into host cells.

BackgroundAdolescents with type 1 diabetes mellitus (T1DM) often show low adherence to complex insulin regimens, leading to poor glycemic control. The benefit of pharmaceutical care in adults with diabetes mellitus type 2 (T2DM) has been widely explored; however, evidence in adolescents with T1DM remains scarce.ObjectiveTo evaluate the impact of pharmaceutical care in adolescents with T1DM provided by a multidisciplinary team on multiple important clinical outcomes.SettingAt the outpatient Helios Paediatric Clinic and at the 12 regular community pharmacies of the study patients with 14 pharmacists in the Krefeld area, Germany, and at the University Pediatric Clinic with one pharmacist on-site in Sarajevo, Bosnia-Herzegovina.MethodsA randomized, controlled, prospective, multicenter study in 68 adolescents with T1DM. The intervention group received monthly structured pharmaceutical care delivered by pharmacists plus supplementary phone calls for 6 months. The control group received usual diabetic care. Data were collected at baseline and after 3 and 6 months. In addition, HbA1c was measured after 12 months.Main outcome measures The between-group difference in the change from baseline in glycosylated hemoglobin (HbA1c), the number of severe hypoglycemic events in both groups, and patient well-being in the intervention group.ResultsThe improvement from baseline in HbA1c was significantly greater in the intervention group than in the control group after 6 months (change from baseline −0.54 vs. +0.32%, p=0.0075), even after adjustment for country-specific variables (p=0.0078). However, the effect was more pronounced after only 3 months (−1.09 vs. +0.23%, p=0.00002). There was no significant between-group difference in the number of severe hypoglycemia events. After 6 months, the well-being according to the WHO-5 index in the intervention group increased significantly from 52.8% to 63.3%. After 12 months the mean total HbA1c remained significantly reduced in the intervention compared to the control group (8.6% vs. 9.5%, p=0.0184).ConclusionThe improved outcomes seen in this study provide new evidence that pharmaceutical care adds value to the management of T1DM in adolescents. However, the optimal methods of achieving sustained long-term improvements in this specific patient population require further study.

The interaction of mesenchymal stem cells (MSCs) with endothelium in vivo is significant for regenerative processes in organisms. To design concepts for tissue engineering for bone regeneration based on this interaction, the osteogenic differentiation of human bone marrow-derived MSCs in a co-culture with human dermal microvascular endothelial cells (HDMECs) was studied. The experiments were focussed on the regulation of MSCs in a co-culture with HDMECs on different calcium phosphate scaffolds. Alkaline phosphatase (ALP) activity and mRNA expression of various osteogenic markers increased significantly when cells were co-cultured on materials with calcium phosphate scaffolds compared to tissue culture polystyrene or when MSCs were cultured alone. In addition, it was observed that the expression of osteopontin and osteocalcin was highly sensitive to the substrate for cell adhesion. Whereas these late osteogenic markers were down-regulated in co-cultures on polystyrene, they were up-regulated on calcium phosphate and moreover, were differentially expressed on the three calcium phosphate scaffolds tested. To enhance the osteogenic differentiation of MSCs in a co-culture, direct cell-cell interactions were required. Concerning molecular mechanisms in the interactions between both cell types, it was found that connexin 43 was expressed in contact sites and more apparently, endothelial cells grew over the MSCs, which facilitated direct cellular interactions mediated by various adhesion receptors. This study revealed significant findings for the design of implant materials suitable for regeneration of bone by stimulating the functional interaction of MSCs with endothelial cells.