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Purpose Human corneal endothelial cells (HCEC) are a potential target of immune attack after corneal transplantation. The aim of this in vitro study was to investigate the role of HCEC during the alloimmune response of T-cells by examining cytokine profiles, function of the immunosuppressive enzyme indoleamine 2,3-dioxigenase (IDO), major histocompatibility complex (MHC-I/-II), T-cell proliferation, and the induction of cell death. Methods Real-time PCR and RP-HPLC were used to determine IDO expression and activity. Multiplex assay was performed for quantification of cytokine levels. T-cell proliferation was assessed by thymidine incorporation, and HCEC cell death was measured by flow cytometry. Results Human corneal endothelial cells induce strong proliferation of allogeneic T-cells and an increase of proinflammatory cytokines such as interleukin-1α (IL-1α), IL-1β, IL-6, interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α). Tumor necrosis factor-alpha (and to a lesser extent IFN-γ) induces apoptosis. Moreover, IFN-γ strongly upregulates MHC-II molecules and IDO activity in HCEC as reflected by high kynurenine (Kyn) concentrations. Interestingly, the T-cell response was not affected by increased IDO activity, since blocking of IDO did not affect the proliferation rate. Indoleamine 2,3-dioxigenase-induced Kyn levels did not exceed concentrations of 175 ± 20 μM. Concentrations of ≥400 μM Kyn were required to suppress T-cell proliferation. Conclusions Our data show that T-cell attack on HCEC leads to increased concentrations of proinflammatory cytokines. Inflammatory cytokines induce apoptosis and upregulate MHC-II molecules and IDO in HCEC. Although increased IDO activity does not influence the T-cell response, it constitutes an inflammatory marker of the alloimmune response toward HCEC.

The purpose of this investigation is to define the location and frequency of tibia pilon fracture lines and impaction injury for the most severe variety (OTA/AO type 43C3).Using axial computed tomography scan images, 38 consecutive OTA/AO type 43C3 fractures treated by a single surgeon were analyzed. For each fracture, a map of the fracture lines and zones of comminution was drawn. Each map was digitized and graphically superimposed to create a compilation of fracture lines and zones of comminution. Based on this compilation, major and minor fracture lines were identified and fracture patterns were defined. Specifically, a basic Y pattern, constant across all patients, was identified where the stem of the Y went into the fibula incisura. All other fracture lines were considered secondary and these defined the comminution.One hundred percent of major fracture lines involved the tibiofibular joint and all exited medially in 2 general zones, anterior and posterior to the medial malleolus best described as a Y-shaped pattern. Therefore, 3 main fragments existed in every single case. Comminution was present in 36 of 38 (95%) cases, and it was predominantly located centrally and in the anterolateral quarter.There is a consistent fracture pattern underlying the majority of OTA/AO type 43C3 pilon fractures that could be defined as 3 main fragments: anterior, medial, and posterior. These result from a major fracture line extending from the fibular incisura and exiting anterior and posterior to the medial malleolus. The comminution commonly distinguishing pilon fractures occurs from secondary fracture lines through the apex of the plafond and in the anterolateral region. Knowledge of this constant pattern should influence surgical approaches and possibly implant design.

It has been demonstrated, that gammadelta T cells play an important role in the development of immune responses to many pathogens. gammadelta T cells play a role in the clearance of viral and microbiological infections, anti-tumor responses, but also in autoimmune diseases. Many different protocols for the isolation and cultivation of gammadelta T cells can be found in the literature. Here we compare three common cultivation protocols for gammadelta T cells derived from peripheral blood with a newly developed protocol depending on SLAM (Signaling Lymphocyte Activation Molecule) stimulation. We demonstrate that the cultivation protocol chosen to raise gammadelta T cells has direct impact on the resulting gammadelta T cell phenotype. We show differences in gammadelta TCR composition, memory phenotype formation, CD8 receptor expression and the expression of NK cell markers depending on the stimulation protocol used. As such, the cultivation protocol chosen for a series of experiments might have significant impact on the outcome of the experiments and should be considered carefully.

The importance of diet quality in toxicological research with fish was illustrated in several studies. The acute toxicity of chlordane to rainbow trout was dependent upon the type of diet fed before toxicity testing. Rainbow trout were fed four commercial diets or a low (23 percent) or high protein (45 percent) synthetic diet. After 42 days, the trout were tested against chlordane in static toxicity tests. The 96-h median lethal concentration (LC 5 0 ) for the synthetic 45 percent protein group was statistically higher (P = 0.05) than values for all other groups. Also, differences in LC 5 0 s existed among groups fed commercial diets. In flow-through toxicity studies, rainbow trout, bluegill, and channel catfish fed a diet containing casein and gelatin as a protein source had a greater tolerance to polychlorinated biphenyls (PCBs) than those fed a diet with fish meal and soybean meal as the primary protein sources. By increasing the concentration of the amino acid methionine in the diet of rainbow trout, the toxicity of dichlorodiphenyltrichloroethane (DDT) increased while dieldrin toxicity decreased. In studies with channel catfish, the concentration of vitamin C in the diet influenced the chronic effects of toxaphene on growth and skeletal development. These studies illustrate that the type of diet fed before acute toxicity testing and during chronic toxicity testing is important, and that consideration for nutritious standard diets in toxicological research with fish is needed.

Two phosphate ester hydraulic fluids, Pydrauls 50E and 115E, are used principally in industrial systems. A study by the U.S. Environmental Protection Agency indicated that a significant portion of the annual production ofall hydraulic fluid was discharged into the environment as a result of leakage. We studied the mobility, persistence, bioconcentration potential, acute and chronic toxicity, and environmental concentrations of Pydrauls 50E and 115E and their three main components: triphenyl phosphate, nonylphenyl diphenyl phosphate, and cumylphenyl diphenyl phosphate. Physicochemical properties and use-disposal patterns were also considered. Pydrauls 50E and 115E presently do not appear to constitute an unreasonable risk to the environment; however, inadvertent leakage of these materials to the aquatic environment should be avoided. Toxicity tests indicated that the margin of safety between environmental concentrations in water and the expected biological effect concentration is at least 1 order of magnitude for coldwater fish, and about 3 orders of magnitude for warm-water species. Better control of hydraulic fluid leakage would reduce environmental contamination and increase the margin of safety.

The purpose of the present study was to evaluate the potency of the proteasome inhibitor bortezomib ± gemcitabine in vitro and in vivo in pancreatic carcinoma. It could be shown that bortezomib induced apoptosis and inhibited proliferation of pancreatic carcinoma very efficiently in vitro. In contrast, in an orthotopic pancreatic adenocarcinoma mouse model, gemcitabine treatment inhibited tumor growth, whereas bortezomib promoted it. Bortezomib-treated animals showed significantly higher tumor burden compared with gemcitabine-treated and control animals, although bortezomib was locally active and induced a decrease of proteasome activity, which was most pronounced following the simultaneous administration of gemcitabine. Also, tumor progression was not caused by immunosuppression as a result of proteasome inhibition. Interestingly, anti-CD31 staining of tumors showed that angiogenesis was significantly increased in the tumors of bortezomib-treated mice compared with the tumors of control animals. In addition, bortezomib resulted an increase of pericytes, vascular endothelial growth factor, RGS-5, and hypoxia-inducible factor-1α in the tumor. Although this study supports efficacy of bortezomib against pancreatic carcinoma in vitro, it strongly indicates that bortezomib therapy has a significant tumor-promoting effect in vivo by induction of angiogenesis. The data are in accordance with the complete failure of bortezomib in a phase II trial for this indication. Choosing the right schedule of gemcitabine and bortezomib showed some synergistic effects, but the gain might not be big enough to compensate the potentially detrimental effects. [Mol Cancer Ther 2008;7(11):3624–31]

The extent of linkage disequilibrium (LD) is an important factor when designing experiments for mapping disease or trait loci using LD mapping methods. It depends on the population history and hence is a characteristic of each population. Here, we have assessed the extent of LD in a sub-isolate of the general Sardinian population (775 members of one village) using 22 polymorphic markers on chromosome 19. We found high levels of disequilibrium that extended to 8 cM, when based on D', and 11 cM when based on the significance level of the allelic association. The fact that conclusions based on both methods are similar suggests that the estimates are quite robust. We have also shown, through a simple resampling technique, that small sample sizes can overestimate both the mean value of D' and its variance up to a factor of about 2 and 16, respectively, when the number of diplotypes (the pair of haplotypes that compose the genotype) decreased from 186 to 26. We evaluated the effect on D' of the depth of the pedigree available when using phased founders, and compared the estimates with those obtained when using unphased founders, and also the effect of grouping alleles on the value of D' and the significance level. Owing to the high sampling variance of LD, we recommend the use of at least 200 unrelated individuals when characterizing the extent of LD

Multidrug resistance is a major obstacle in the treatment of pancreatic cancer. Immunochemotherapy including interferon-alpha increases response rates and survival.Pancreatic cancer was induced in an orthotopic mouse model. Animals received standard chemotherapy or combinative treatment with interferon-alpha. Expression and function of drug-resistance proteins were analyzed. Immunological phenotyping, cytotoxic activity assays and analysis of T-cell activation status were performed.Addition of interferon-alpha to chemotherapeutic regimes significantly reduced chemotherapy-induced expression of multidrug resistance proteins and drug efflux activity of cancer cells. Tumor size and metastatic seeding decreased significantly upon combination therapy and survival was prolonged. A significantly higher proportion of activated and cytotoxic active CD8+ tumor infiltrating lymphocytes was detectable after induction of drug resistance.Restitution of chemosensitivity by the addition of interferon alpha to chemotherapy was demonstrated in experimental pancreatic cancer for the first time. Since drug-resistance proteins may function as tumor antigens, our data support immunochemotherapy as an encouraging new approach.

Proteomic technologies, such as yeast two-hybrid, mass spectrometry (MS), protein/peptide arrays and fluorescence microscopy, yield multi-dimensional data sets, which are often quite large and either not published or published as supplementary information that is not easily searchable. Without a system in place for standardizing and sharing data, it is not fruitful for the biomedical community to contribute these types of data to centralized repositories.

Signaling lymphocyte activation molecule (SLAM, CD150) is a co-stimulatory receptor involved in T cell activation. The activity of CD150 is dependent on the intracellular signaling molecule SAP. Here, we investigated anti-CD3 activated human lymphocytes, transfected either with CD150-plasmid or with CD150- or SAP-siRNA in cytotoxicity assays against human colon cancer cells in vitro and in a xenograft model (CB/Scid/CrL mice) in vivo . Up-regulation or silencing of CD150 was accompanied by increased or decreased cytotoxic activity, respectively. Similar effects could also be shown in an IFN-γ ELISpot assay. Furthermore, CD150 co-localized after activation with lipid rafts in specific membrane compartments on CD8 T cells. Treatment of xenografted mice with CD150 over-expressing lymphocytes decelerated tumor growth significantly. Lymphocytes were detectable in spleen 18 days after injection and expressed mainly CD8, CD45RO and CD150 above average. In conclusion, over-expression of CD150 in lymphocytes is accompanied with enhanced cytotoxic activity and IFN-γ secretion in vitro and anti-tumor activity in vivo , whereas silencing of CD150 down-regulates effector functions. Adoptive cell transfer of CD150 over-expressing lymphocytes results in an accumulation of CD8, CD45RO and CD150 cells in tumor and spleen indicating together with the observed CD150 co-localization with lipid rafts that CD150 mediates a Th1 response.

Food process engineering, 031, ISBN:978-3-905609-36-3

Stent fracture is uncommon but may have consequences including restenosis. To date, stent fractures reported have been related to aggressive post dilation. We describe a case that involves fracture of a stent deployed to nominal pressure. Unlike most stent fractures reported that involve stent struts only our case demonstrated circumferential disruption with complete separation of the stent segments. © 2006 Wiley-Liss, Inc.

Background The identification of patterns in biological sequences is a key challenge in genome analysis and in proteomics. Frequently such patterns are complex and highly variable, especially in protein sequences. They are frequently described using terms of regular expressions (RegEx) because of the user-friendly terminology. Limitations arise for queries with the increasing complexity of patterns and are accompanied by requirements for enhanced capabilities. This is especially true for patterns containing ambiguous characters and positions and/or length ambiguities. Results We have implemented the 3of5 web application in order to enable complex pattern matching in protein sequences. 3of5 is named after a special use of its main feature, the novel n-of-m pattern type. This feature allows for an extensive specification of variable patterns where the individual elements may vary in their position, order, and content within a defined stretch of sequence. The number of distinct elements can be constrained by operators, and individual characters may be excluded. The n-of-m pattern type can be combined with common regular expression terms and thus also allows for a comprehensive description of complex patterns. 3of5 increases the fidelity of pattern matching and finds ALL possible solutions in protein sequences in cases of length-ambiguous patterns instead of simply reporting the longest or shortest hits. Grouping and combined search for patterns provides a hierarchical arrangement of larger patterns sets. The algorithm is implemented as internet application and freely accessible. The application is available at http://dkfz.de/mga2/3of5/3of5.html. Conclusion The 3of5 application offers an extended vocabulary for the definition of search patterns and thus allows the user to comprehensively specify and identify peptide patterns with variable elements. The n-of-m pattern type offers an improved accuracy for pattern matching in combination with the ability to find all solutions, without compromising the user friendliness of regular expression terms.

Background Well known for its gene density and the large number of mapped diseases, the human sub-chromosomal region Xq28 has long been a focus of genome research. Over 40 of approximately 300 X-linked diseases map to this region, and systematic mapping, transcript identification, and mutation analysis has led to the identification of causative genes for 26 of these diseases, leaving another 17 diseases mapped to Xq28, where the causative gene is still unknown. To expedite disease gene identification, we have initiated the functional characterisation of all known Xq28 genes. Results By using a systematic approach, we describe the Xq28 genes by RNA in situ hybridisation and Northern blotting of the mouse orthologs, as well as subcellular localisation and data mining of the human genes. We have developed a relational web-accessible database with comprehensive query options integrating all experimental data. Using this database, we matched gene expression patterns with affected tissues for 16 of the 17 remaining Xq28 linked diseases, where the causative gene is unknown. Conclusion By using this systematic approach, we have prioritised genes in linkage regions of Xq28-mapped diseases to an amenable number for mutational screens. Our database can be queried by any researcher performing highly specified searches including diseases not listed in OMIM or diseases that might be linked to Xq28 in the future.

Wir konnten zeigen, dass beim experimentellen Pankreaskarzinom die vermehrte Expression von MDR-assoziierten Proteinen durch die Behandlung mit 5-FU, Cisplatin und Gemcitabine induziert wird. Der Peptidchemosensitizer R121 kann die therapieinduzierte MRP1 und MRP3 Expression senken, scheint jedoch den klinischen Verlauf nicht zu beeinflussen.

The graphical representation of three-dimensional data obtained from tomographic imaging has been the centralproblem since this technology is available. Neither the representation as a set of two-dimensional slices nor the2D projection of three-dimensional models yields satisfactory results.In this paper a way is outlined which permits the investigation of volumetric clinical data obtained fromstandard CT, MR, PET, SPECT or experimental very high resolution CT-scanners in a three dimensionalenvironment within a few worksteps. Volumetric datasets are converted into surface data (segmentation process)using the 3D-Slicer software tool and saved as .vtk “les and exported as a collection of primitives in any common“le format (.iv, .pfb). Subsequently this “les can be displayed and manipulated in the CAVE virtual realitycenter.The CAVE is a multiuser walkable virtual room consisting of several walls on which stereoscopic images areprojected by rear panel beamers. Adequate tracking of the head position and separate image calculation foreach eye yields a vivid impression for one or several users. With the use of a seperately tracked 6D joystickmanipulations such as rotation, translation, zooming, decomposition or highlighting can be done intuitively.The usage of the CAVE technology opens new possibilities especially in surgical training (Žhands-on-eectŽ)and as an educational tool (availability of pathological data). Unlike concurring technologies the CAVE permitsa walk-through into the virtual scene but preserves enough physical perception to allow interaction between mul-tiple users. e.g. gestures and movements. By training in a virtual environment on one hand the learning processof students in complex anatomic “ndings may be improved considerably and on the other hand unaccustomedviews such as the one through a microscope or endoscope can be trained in advance. The availability of low-costPC based CAVE-like systems and the rapidly decreasing price of high-performance video beamers makes theCAVE an aordable alternative to conventional surgical training techniques and without limitations in handlingcadavers.Keywords: CAVE, Virtual Reality, Medical Imaging, Surgical Planning, Stereoscopic Visualization, Tomo-graphic Data

Signalling lymphocyte activation molecule (SLAM)-associated protein (SAP) is a small protein that is mutant in humans with X-linked lymphoproliferative (XLP) disease. Patients with XLP disease are affected by fatal EBV infection and malignant B-cell lymphomas. The increased risk for B-cell lymphomas is suggested to result from impaired immunosurveillance of B-cell proliferation by T cells. In this study, we investigated the role of SLAM and SAP in activation of effector cells with cytotoxic activity, cytokine-induced killer (CIK) cells, which are generated by non-specific stimulation of the TCR and addition of exogenous IL-2. Agonistic TCR activation 1 day after preparation (day +1) resulted in cell activation, with a peak of SLAM on day +6 visible at both the protein and mRNA level as well as membrane detectable SLAM. This increase in SLAM expression correlated significantly with SAP expression at the mRNA level as well as at the protein level. Cytotoxic activity peaked 1 day after the observed SAP and SLAM peaks. At that point in time, IL-10 secretion, which was high during the early days of culture, decreased. In conclusion, activation of peripheral blood cells with agonistic anti-CD3 antibody and exogenous IL-2, as used for generation of CIK cells, results in significant SLAM and SAP activation 5 days after TCR stimulation. This peak correlates with cytotoxic activity against tumour cells. Expression of SLAM and SAP seems to be important in the activation of cytotoxic effector cells.

As several model genomes have been sequenced, the elucidation of protein function is the next challenge toward the understanding of biological processes in health and disease. We have generated a human ORFeome resource and established a functional genomics and proteomics analysis pipeline to address the major topics in the post-genome-sequencing era: the identification of human genes and splice forms, and the determination of protein localization, activity, and interaction. Combined with the understanding of when and where gene products are expressed in normal and diseased conditions, we create information that is essential for understanding the interplay of genes and proteins in the complex biological network. We have implemented bioinformatics tools and databases that are suitable to store, analyze, and integrate the different types of data from high-throughput experiments and to include further annotation that is based on external information. All information is presented in a Web database (http://www.dkfz.de/LIFEdb). It is exploited for the identification of disease-relevant genes and proteins for diagnosis and therapy.

Problem: Three-dimensional models are quite common now, but are still restricted to a 2-dimensional view by a screen or beamer. We use CAVE technology for education of complex anatomical findings and for surgical simulation. Methods: We create models by segmentation of datasets using the “3D-Slicer.” For the small structures in the temporal bone we used an experimental cone-beam Volume CT prototype that offers 160 micron isotropic resolutions. The CAVE is a Virtual Reality Center (VRC) which allows real-time visualization of interactive, complex graphical data in a walkable room. The graphical visualization is achieved by rear-panel beamers on 3 walls and a projection surface on the floor. Multiple users can interact in this virtual surrounding with different types of graphical data by rotating, zooming, or performing any programmable function. Results: Teaching pathologic findings or surgical planning in challenging cases are the most common fields of application. We use this technology for students’ and residents’ learning difficult anatomic findings. By simulating different pathways we can experience the changes of the anatomy or pathology, which is presented in its variations to the surgeons’ view. Conclusion: Using CAVE technology for medical imaging is the next step of 3D visualization. Most advantages are in the field of education because of the possibility for several dozen individuals to see 3D models simultaneously and to interact with real patients’ data (in other words, allowing a “hands-on effect”). Significance: By using virtual reality we presume to optimize the learning curve of students and especially residents, who are in the first training stages of acquiring the skill of surgery. Another possibility of training surgeons is to simulate the surgeons’ view through a microscope, which is harder because of the loss of landmarks. Walking around the model helps to train the “mental eye” and improves the surgical skills of young surgeons. Support: None reported.

To functionally characterize numerous novel proteins encoded by cDNAs sequenced by the German Consortium, 800 were tagged with green fluorescent protein. The subcellular localizations of the fusion proteins were examined in living cells, enabling their classification in subcellular groups. Their activity in cell growth, cell death, and protein transport was screened in high throughput using robotic liquid handling and reading stations. The resulting information is integrated with functional genomics and proteomics data for further understanding of protein functions in the cellular context. To cite this article: S. Wiemann et al., C. R. Biologies 326 (2003).

The process of pickling is an important intermediate step in the production line of steel processing. The strip surface is cleaned from grease and scales before further processing by immersion into an acid bath. Problems arising at higher process speeds with increasing inclination of the free surface are reduced strip immersion length and increased mass outflow. In this paper a differential equation is derived describing the influence of the bath depth on the local surface inclination for a simplified two-dimensional case. Since it can only be solved analytically for trivial boundary conditions a numerical solution has been computed giving an estimation for the order of growth of the bath inclination and mass outflow with the strip velocity. Further, a series of CFD simulations of the complete three-dimensional geometry at different strip velocities have been carried out calibrating the formulas of mass outflow. In the course of the CFD simulations the deformation of the free surface was calculated by a VOF model with explicit reconstruction of the interface. A standard κ–e turbulence model was applied and special considerations have been made regarding the boundary conditions. Finally the resulting formula has been verified making use of data from a small scale model. It was found that the overflow at the far end of the tank is the dominant mass transfer mechanism at process velocities of the current generation of pickling tanks. Still, due to the superior order of growth, mass drag-out via the upper side of the strip becomes important for process velocities of 8 to 10 m/s. The good accordance of the analytical solution, CFD simulation and experiment indicate that the formula derived in the first part of the paper is a good estimation for the mass outflow from the pickling tank, hence making time and resource consuming CFD simulations obsolete for the design layout. Further the validity of geometrically non similar small scale models could be showed.Copyright © 2004 by ASME