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  • DKFZ Publication Database  (1,189)
  • IN-VIVO  (1,189)
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  • DKFZ Publication Database  (1,189)
Keywords
  • 1
    Keywords: GROWTH ; IRRADIATION ; IN-VIVO ; NECK TUMORS ; FRACTIONATED RADIOTHERAPY ; OXYGEN-TENSION ; COMPUTER-SIMULATION ; CONTROL PROBABILITY ; HETEROGENEOUS TUMORS ; VASCULAR DENSITY
    Abstract: Purpose: In radiotherapy, it is important to predict the response of tumors to irradiation prior to the treatment. This is especially important for hypoxic tumors, which are known to be highly radioresistant. Mathematical modeling based on the dose distribution, biological parameters, and medical images may help to improve this prediction and to optimize the treatment plan. Methods: A voxel-based multiscale tumor response model for simulating the radiation response of hypoxic tumors was developed. It considers viable and dead tumor cells, capillary and normal cells, as well as the most relevant biological processes such as (i) proliferation of tumor cells, (ii) hypoxia-induced angiogenesis, (iii) spatial exchange of cells leading to tumor growth, (iv) oxygen-dependent cell survival after irradiation, (v) resorption of dead cells, and (vi) spatial exchange of cells leading to tumor shrinkage. Oxygenation is described on a microscopic scale using a previously published tumor oxygenation model, which calculates the oxygen distribution for each voxel using the vascular fraction as the most important input parameter. To demonstrate the capabilities of the model, the dependence of the oxygen distribution on tumor growth and radiation-induced shrinkage is investigated. In addition, the impact of three different reoxygenation processes is compared and tumor control probability (TCP) curves for a squamous cells carcinoma of the head and neck (HNSSC) are simulated under normoxic and hypoxic conditions. Results: The model describes the spatiotemporal behavior of the tumor on three different scales: (i) on the macroscopic scale, it describes tumor growth and shrinkage during radiation treatment, (ii) on a mesoscopic scale, it provides the cell density and vascular fraction for each voxel, and (iii) on the microscopic scale, the oxygen distribution may be obtained in terms of oxygen histograms. With increasing tumor size, the simulated tumors develop a hypoxic core. Within the model, tumor shrinkage was found to be significantly more important for reoxygenation than angiogenesis or decreased oxygen consumption due to an increased fraction of dead cells. In the studied HNSSC-case, the TCD50 values (dose at 50% TCP) decreased from 71.0 Gy under hypoxic to 53.6 Gy under the oxic condition. Conclusions: The results obtained with the developed multiscale model are in accordance with expectations based on radiobiological principles and clinical experience. As the model is voxel-based, radiological imaging methods may help to provide the required 3D-characterization of the tumor prior to irradiation. For clinical application, the model has to be further validated with experimental and clinical data. If this is achieved, the model may be used to optimize fractionation schedules and dose distributions for the treatment of hypoxic tumors.
    Type of Publication: Journal article published
    PubMed ID: 25563250
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  • 2
    Keywords: IN-VIVO ; MODEL ; VOLUME ; HEPATOCELLULAR-CARCINOMA ; TISSUE ; RADIOFREQUENCY ABLATION ; POWER ; embolization ; PORCINE KIDNEYS ; TRANSARTERIAL CHEMOEMBOLIZATION
    Abstract: PURPOSE: This study was designed to compare technical parameters during ablation as well as CT 3D rendering and histopathology of the ablation zone between sphere-enhanced microwave ablation (sMWA) and bland microwave ablation (bMWA). METHODS: In six sheep-livers, 18 microwave ablations were performed with identical system presets (power output: 80 W, ablation time: 120 s). In three sheep, transarterial embolisation (TAE) was performed immediately before microwave ablation using spheres (diameter: 40 +/- 10 mum) (sMWA). In the other three sheep, microwave ablation was performed without spheres embolisation (bMWA). Contrast-enhanced CT, sacrifice, and liver harvest followed immediately after microwave ablation. Study goals included technical parameters during ablation (resulting power output, ablation time), geometry of the ablation zone applying specific CT 3D rendering with a software prototype (short axis of the ablation zone, volume of the largest aligned ablation sphere within the ablation zone), and histopathology (hematoxylin-eosin, Masson Goldner and TUNEL). RESULTS: Resulting power output/ablation times were 78.7 +/- 1.0 W/120 +/- 0.0 s for bMWA and 78.4 +/- 1.0 W/120 +/- 0.0 s for sMWA (n.s., respectively). Short axis/volume were 23.7 +/- 3.7 mm/7.0 +/- 2.4 cm(3) for bMWA and 29.1 +/- 3.4 mm/11.5 +/- 3.9 cm(3) for sMWA (P 〈 0.01, respectively). Histopathology confirmed the signs of coagulation necrosis as well as early and irreversible cell death for bMWA and sMWA. For sMWA, spheres were detected within, at the rim, and outside of the ablation zone without conspicuous features. CONCLUSIONS: Specific CT 3D rendering identifies a larger ablation zone for sMWA compared with bMWA. The histopathological signs and the detectable amount of cell death are comparable for both groups. When comparing sMWA with bMWA, TAE has no effect on the technical parameters during ablation.
    Type of Publication: Journal article published
    PubMed ID: 25167958
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  • 3
    Keywords: GROWTH-FACTOR ; IN-VIVO ; PROTEIN-KINASE ; cell lines ; DRUG DISCOVERY ; SELECTIVITY ; STRATEGY ; RHO-KINASE ; KINOME ; PEPTIDE IDENTIFICATION
    Abstract: Solid supported probes have proven to be an efficient tool for chemical proteomics. The kinobeads technology features kinase inhibitors covalently attached to Sepharose for affinity enrichment of kinomes from cell or tissue lysates. This technology, combined with quantitative mass spectrometry, is of particular interest for the profiling of kinase inhibitors. It often leads to the identification of new targets for medicinal chemistry campaigns where it allows a two-in-one binding and selectivity assay. The assay can also uncover resistance mechanisms and molecular sources of toxicity. Here we report on the optimization of the kinobead assay resulting in the combination of five chemical probes and four cell lines to cover half the human kinome in a single assay ( approximately 260 kinases). We show the utility and large-scale applicability of the new version of kinobeads by reprofiling the small molecule kinase inhibitors Alvocidib, Crizotinib, Dasatinib, Fasudil, Hydroxyfasudil, Nilotinib, Ibrutinib, Imatinib, and Sunitinib.
    Type of Publication: Journal article published
    PubMed ID: 25660469
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  • 4
    Keywords: CELLS ; EXPRESSION ; IN-VIVO ; resistance ; SINGLE NUCLEOTIDE POLYMORPHISMS ; CIRCULATING LEVELS ; GENETIC-VARIATION ; QUANTITATIVE PCR ; IGFBP-3 POLYMORPHISMS ; PATHWAY GENES
    Abstract: Intrinsic and acquired resistance to the monoclonal antibody drug trastuzumab is a major problem in the treatment of HER2-positive breast cancer. A deeper understanding of the underlying mechanisms could help to develop new agents. Our intention was to detect genes and single nucleotide polymorphisms (SNPs) affecting trastuzumab efficiency in cell culture. Three HER2-positive breast cancer cell lines with different resistance phenotypes were analyzed. We chose BT474 as model of trastuzumab sensitivity, HCC1954 as model of intrinsic resistance, and BTR50, derived from BT474, as model of acquired resistance. Based on RNA-Seq data, we performed differential expression analyses on these cell lines with and without trastuzumab treatment. Differentially expressed genes between the resistant cell lines and BT474 are expected to contribute to resistance. Differentially expressed genes between untreated and trastuzumab treated BT474 are expected to contribute to drug efficacy. To exclude false positives from the candidate gene set, we removed genes that were also differentially expressed between untreated and trastuzumab treated BTR50. We further searched for SNPs in the untreated cell lines which could contribute to trastuzumab resistance. The analysis resulted in 54 differentially expressed candidate genes that might be connected to trastuzumab efficiency. 90% of 40 selected candidates were validated by RT-qPCR. ALPP, CALCOCO1, CAV1, CYP1A2 and IGFBP3 were significantly higher expressed in the trastuzumab treated than in the untreated BT474 cell line. GDF15, IL8, LCN2, PTGS2 and 20 other genes were significantly higher expressed in HCC1954 than in BT474, while NCAM2, COLEC12, AFF3, TFF3, NRCAM, GREB1 and TFF1 were significantly lower expressed. Additionally, we inferred SNPs in HCC1954 for CAV1, PTGS2, IL8 and IGFBP3. The latter also had a variation in BTR50. 20% of the validated subset have already been mentioned in literature. For half of them we called and analyzed SNPs. These results contribute to a better understanding of trastuzumab action and resistance mechanisms.
    Type of Publication: Journal article published
    PubMed ID: 25710561
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  • 5
    Keywords: carcinoma ; ENDOTHELIAL GROWTH-FACTOR ; IN-VIVO ; MICE ; adenocarcinoma ; ANTITUMOR IMMUNITY ; inflammation ; CLINICAL-SIGNIFICANCE ; TUMOR-ASSOCIATED MACROPHAGES ; REGULATORY T-CELLS
    Abstract: Pancreatic ductal adenocarcinoma (PDAC) represents one of the deadliest cancers in the world. PDAC cells activate tumor-specific immune responses but simultaneously trigger a strong immunosuppression. We showed that PDAC cells produce high amount of chronic inflammatory mediators and PDAC tumors build an immunosuppressive cytokine milieu, which correlates with tumor progression. We observed a low frequency of dendritic cells (DC) and a pronounced accumulation of macrophages and myeloid-derived suppressor cells (MDSC) in murine PDAC tumors. A strong accumulation of MDSC has also been demonstrated in the peripheral blood of resected PDAC patients. While DC and macrophages seem not to play a significant role in this PDAC model in the context of immunosuppression, MDSC are highly suppressive, and their accumulation is associated with an increase in intratumoral VEGF concentration during the PDAC progression. Application of the phosphodiesterase-5 inhibitor sildenafil led to a prolonged survival of PDAC-bearing female mice, which was due to the decrease in MDSC frequencies and in the systemic VEGF level. This led to a restoration of anticancer immune responses, manifested in the recovery of T lymphocyte functions and in an increase in the frequency of conventional CD4(+) T cells in tumors and IFN gamma level in serum of PDAC-bearing mice. Thus, MDSC are strongly involved in the PDAC-associated immunosuppression and that their depletion could create new approaches for therapy of PDAC.
    Type of Publication: Journal article published
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  • 6
    Keywords: EXPRESSION ; IN-VIVO ; MESSENGER-RNA ; MUTATIONS ; SONIC HEDGEHOG ; MOUSE MODEL ; PROTEASE NEXIN-1 ; GENETIC PROFILES ; HEDGEHOG PATHWAY ; NEURAL-TUBE
    Abstract: Background Medulloblastomas are malignant childhood brain tumors that arise due to the aberrant activity of developmental pathways during postnatal cerebellar development and in adult humans. Transcriptome analysis has identified four major medulloblastoma subgroups. One of them, the Sonic hedgehog (SHH) subgroup, is caused by aberrant Hedgehog signal transduction due to mutations in the Patched1 (PTCH1) receptor or downstream effectors. Mice carrying a Patched-1 null allele (Ptch1(Delta/+)) are a good model to study the alterations underlying medulloblastoma development as a consequence of aberrant Hedgehog pathway activity. Results Transcriptome analysis of human medulloblastomas shows that SERPINE2, also called Protease Nexin-1 (PN-1) is overexpressed in most medulloblastomas, in particular in the SHH and WNT subgroups. As siRNA-mediated lowering of SERPINE2/PN-1 in human medulloblastoma DAOY cells reduces cell proliferation, we analyzed its potential involvement in medulloblastoma development using the Ptch1(Delta/+) mouse model. In Ptch1(Delta/+) mice, medulloblastomas arise as a consequence of aberrant Hedgehog pathway activity. Genetic reduction of Serpine2/Pn-1 interferes with medulloblastoma development in Ptch1(Delta/+) mice, as similar to 60% of the pre-neoplastic lesions (PNLs) fail to develop into medulloblastomas and remain as small cerebellar nodules. In particular the transcription factor Atoh1, whose expression is essential for development of SHH subgroup medulloblastomas is lost. Comparative molecular analysis reveals the distinct nature of the PNLs in young Ptch1(Delta/+)Pn-1(Delta/+) mice. The remaining wild-type Ptch1 allele escapes transcriptional silencing in most cases and the aberrant Hedgehog pathway activity is normalized. Furthermore, cell proliferation and the expression of the cell-cycle regulators Mycn and Cdk6 are significantly reduced in PNLs of Ptch1(Delta/+)Pn-1(Delta/+) mice. Conclusions Our analysis provides genetic evidence that aberrant Serpine2/Pn-1 is required for proliferation of human and mouse medulloblastoma cells. In summary, our analysis shows that Serpine2/PN-1 boosts malignant progression of PNLs to medulloblastomas, in which the Hedgehog pathway is activated in a SHH ligand-independent manner.
    Type of Publication: Journal article published
    PubMed ID: 25901736
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  • 7
    Keywords: RECEPTOR ; CANCER ; IN-VIVO ; CRE RECOMBINASE ; GTPASE-ACTIVATING PROTEIN ; POLARITY ; THERAPEUTIC TARGETS ; ACUTE KIDNEY INJURY ; ORIENTED CELL-DIVISION ; CDC42 GEF
    Abstract: Morphogenesis, homeostasis, and regeneration of epithelial tissues rely on the accurate orientation of cell divisions, which is specified by the mitotic spindle axis. To remain in the epithelial plane, symmetrically dividing epithelial cells align their mitotic spindle axis with the plane. Here, we show that this alignment depends on epithelial cell-cell communication via semaphorin-plexin signaling. During kidney morphogenesis and repair, renal tubular epithelial cells lacking the transmembrane receptor Plexin-B2 or its semaphorin ligands fail to correctly orient the mitotic spindle, leading to severe defects in epithelial architecture and function. Analyses of a series of transgenic and knockout mice indicate that Plexin-B2 controls the cell division axis by signaling through its GTPase-activating protein (GAP) domain and Cdc42. Our data uncover semaphorin-plexin signaling as a central regulatory mechanism of mitotic spindle orientation necessary for the alignment of epithelial cell divisions with the epithelial plane.
    Type of Publication: Journal article published
    PubMed ID: 25892012
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  • 8
    Keywords: IN-VIVO ; DENDRITIC CELLS ; SELF-ANTIGENS ; T-CELLS ; antigen presentation ; ADAPTIVE IMMUNITY ; GENE FAMILY ; DYING CELLS ; FORMYL PEPTIDE RECEPTOR ; BONE-MARROW CULTURES
    Abstract: Immunological tolerance is constantly being maintained in the periphery by dendritic cells processing material from apoptotic cells (ACs) in the steady-state. Although research has focused on the uptake of ACs by phagocytes, tolerogenic signals exposed by the ACs are much less well defined. In this article, we show that the annexin (Anx) family members AnxA5 and AnxA13 translocate to the surface of ACs to function as redundant tolerogenic signals in vitro and in vivo. Exposure of bone marrow-derived dendritic cells to AnxA5 or AnxA13 in vitro resulted in the inhibition of both proinflammatory cytokine secretion and the upregulation of costimulatory molecules upon TLR stimulation. The highly conserved Anx core domain was sufficient to mediate these effects, whereas recognition by N-formyl peptide receptor family members was dispensable. In vivo, coinjection of OVA-expressing and Anx-expressing ACs prevented induction of Ag-specific CD8(+) T cells. Moreover, mice immunized with Anx-expressing ACs became refractory to an antigenic challenge. These results suggest that several Anxs contribute to AC-induced suppression of dendritic cell activation. Therefore, manipulating Anx-mediated immunosuppression may prove beneficial for patients with cancer or autoimmune diseases and chronic inflammatory disorders.
    Type of Publication: Journal article published
    PubMed ID: 25917090
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  • 9
    Keywords: carcinoma ; IN-VIVO ; THERAPY ; TRIAL ; FEASIBILITY ; PIG MODEL ; rectum ; TISSUE ABLATION ; ELECTRIC-FIELD ; NERVES
    Abstract: BACKGROUND: Irreversible electroporation (IRE), a new tissue ablation procedure available since 2007, could meet the requirements for ideal focal therapy (FT) with its postulated features, especially the absence of a thermal ablative effect. Thus far, there is no adequate tumor-entity-specific proof of its effectiveness, and its clinical application has hitherto been confined to very small patient cohorts. This also holds true for prostate cancer (PCA). Nevertheless, it is now being increasingly applied outside clinical trials-to a certain extent due to active advertising in the lay press. AIM OF THE STUDY: In this study, current discrepancies between the clinical application and study situation and the approval and market implementation of the procedure are described. The media portrayal of IRE is discussed from different perspectives, particularly with reference to the FT of PCA. This is followed by a final clinical assessment of IRE using the NanoKnife(R) system. DISCUSSION: Strict requirements govern new drug approvals. According to the German Drug Act (AMG), evidence of additional benefit over existing therapy must be provided through comparative clinical trials. For medicotechnical treatment procedures, on the other hand, such trial-based proof is not required according to the Medical Devices Act (MPG). The use of IRE even outside clinical trials has been actively promoted since the NanoKnife(R) system was put on the market. This has led to an increase in the number of uncontrolled IRE treatments of PCA in the last 2 years. The patients have to cover the high treatment costs themselves in these cases. If articles in the lay press advertise the procedure with promising but unverified contents, false hopes are raised in those concerned. This is disastrous if it delays the use of truly effective treatment options. CONCLUSION: IRE basically still has high potential for the treatment of malignancies; however, whether it can really be used for FT remains unclear due to the lack of data. This also holds true for the treatment of PCA. Only carefully conducted scientific research studies can clarify the unresolved issues regarding IRE of PCA. The urgently needed development of universally valid treatment standards for IRE is unnecessarily hampered by the flow commercially driven patients.
    Type of Publication: Journal article published
    PubMed ID: 26024649
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  • 10
    Keywords: IN-VIVO ; MOUSE MODEL ; WHITE-MATTER ; MULTIPLE-SCLEROSIS ; SPINAL-CORD ; CARPAL-TUNNEL-SYNDROME ; OPTIC-NERVE ; MEDIAN NERVE ; MR NEUROGRAPHY ; RETINAL ISCHEMIA
    Abstract: PURPOSE: To investigate the potential of diffusion tensor imaging (DTI) parameters as in-vivo biomarkers of axon and myelin sheath integrity of the median nerve in the carpal tunnel as validated by correlation with electrophysiology. METHODS: MRI examinations at 3T including DTI were conducted on wrists in 30 healthy subjects. After manual segmentation of the median nerve quantitative analysis of fractional anisotropy (FA) as well as axial, radial and mean diffusivity (AD, RD, and MD) was carried out. Pairwise Pearson correlations with electrophysiological parameters comprising sensory nerve action potential (SNAP) and compound muscle action potential (CMAP) as markers of axon integrity, and distal motor latency (dml) and sensory nerve conduction velocity (sNCV) as markers of myelin sheath integrity were computed. The significance criterion was set at P=0.05, Bonferroni corrected for multiple comparisons. RESULTS: DTI parameters showed a distinct proximal-to-distal profile with FA, MD, and RD extrema coinciding in the center of the carpal tunnel. AD correlated with CMAP (r=0.50, p=0.04, Bonf. corr.) but not with markers of myelin sheath integrity. RD correlated with sNCV (r=-0.53, p=0.02, Bonf. corr.) but not with markers of axon integrity. FA correlated with dml (r=-0.63, p=0.002, Bonf. corr.) and sNCV (r=0.68, p=0.001, Bonf. corr.) but not with markers of axon integrity. CONCLUSION: AD reflects axon integrity, while RD (and FA) reflect myelin sheath integrity as validated by correlation with electrophysiology. DTI parameters consistently indicate a slight decrease of structural integrity in the carpal tunnel as a physiological site of median nerve entrapment. DTI is particularly sensitive, since these findings are observed in healthy participants. Our results encourage future studies to evaluate the potential of DTI in differentiating axon from myelin sheath injury in patients with manifest peripheral neuropathies.
    Type of Publication: Journal article published
    PubMed ID: 26114630
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  • 11
    Keywords: EXPRESSION ; GROWTH-FACTOR ; IN-VIVO ; PHENOTYPE ; HUMAN EPIDERMIS ; REPLICATIVE SENESCENCE ; HUMAN-CELLS ; CELLULAR SENESCENCE ; INDUCED PREMATURE SENESCENCE ; INTERACTION NETWORKS
    Abstract: Most molecular hallmarks of cellular senescence have been identified in studies of cells aged in vitro by driving them into replicative or stress-induced senescence. Comparatively, less is known about the characteristic features of cells that have aged in vivo. Here we provide a systematic molecular analysis of normal human dermal fibroblasts (NHDFs) that were isolated from intrinsically aged human skin of young versus middle aged versus old donors. Intrinsically aged NHDFs in culture exhibited more frequently nuclear foci positive for p53 binding protein 1 and promyelocytic leukemia protein reminiscent of 'DNA segments with chromatin alterations reinforcing senescence (DNA-SCARS)'. Formation of such foci was neither accompanied by increased DNA double strand breaks, nor decreased cell viability, nor telomere shortening. However, it was associated with the development of a secretory phenotype, indicating incipient cell senescence. By quantitative analysis of the entire secretome present in conditioned cell culture supernatant, combined with a multiplex cytokine determination, we identified 998 proteins secreted by intrinsically aged NHDFs in culture. Seventy of these proteins exhibited an age-dependent secretion pattern and were accordingly denoted 'skin aging-associated secreted proteins (SAASP)'. Systematic comparison of SAASP with the classical senescence-associated secretory phenotype (SASP) revealed that matrix degradation as well as proinflammatory processes are common aspects of both conditions. However, secretion of 27 proteins involved in the biological processes of 'metabolism' and 'adherens junction interactions' was unique for NHDFs isolated from intrinsically aged skin. In conclusion, fibroblasts isolated from intrinsically aged skin exhibit some, but not all, molecular hallmarks of cellular senescence. Most importantly, they secrete a unique pattern of proteins that is distinct from the canonical SASP and might reflect specific processes of skin aging.
    Type of Publication: Journal article published
    PubMed ID: 25815425
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  • 12
    Keywords: IN-VIVO ; CYCLE PROGRESSION ; DNA-REPLICATION ; HEMATOPOIETIC STEM-CELLS ; UBIQUITIN LIGASE ; TRANSGENE EXPRESSION ; EARLY XENOPUS-EMBRYOS ; MAJOR DEVELOPMENTAL TRANSITION ; LICENSING FACTOR CDT1 ; TARGETED GENE-EXPRESSION
    Abstract: Visualizing the cell cycle behavior of individual cells within living organisms can facilitate the understanding of developmental processes such as pattern formation, morphogenesis, cell differentiation, growth, cell migration, and cell death. Fluorescence Ubiquitin Cell Cycle Indicator (FUCCI) technology offers an accurate, versatile, and universally applicable means of achieving this end. In recent years, the FUCCI system has been adapted to several model systems including flies, fish, mice, and plants, making this technology available to a wide range of researchers for studies of diverse biological problems. Moreover, a broad range of FUCCI-expressing cell lines originating from diverse cell types have been generated, hence enabling the design of advanced studies that combine in vivo experiments and cell-based methods such as high-content screening. Although only a short time has passed since its introduction, the FUCCI technology has already provided fundamental insight into how cells establish quiescence and how G1 phase length impacts the balance between pluripotency and stem cell differentiation. Further discoveries using the FUCCI technology are sure to come. WIREs Dev Biol 2015, 4:469-487. doi: 10.1002/wdev.189 For further resources related to this article, please visit the WIREs website.
    Type of Publication: Journal article published
    PubMed ID: 25827130
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  • 13
    Keywords: EXPRESSION ; IN-VIVO ; MODEL ; SITES ; STEM-CELLS ; SAFETY ; INTEGRATION ; INFUSION ; LENTIVIRAL VECTOR ; ENHANCE
    Abstract: "Ex vivo" regional gene therapy using lentiviral (LV) vectors to over-express bone morphogenetic protein 2 (BMP-2) is an effective way to enhance bone healing in animal models. Here, we evaluated two different "ex vivo" approaches using either "same day" rat bone marrow cells (SDRBMCs) or cultured rat bone marrow cells (C-RBMCs), both transduced with a LV based two-step transcriptional activation system overexpressing GFP (LV-TSTA-EGFP), to assess the fate of the transduced cells and the safety of this approach. The transduced cells were implanted in femoral defects of syngeneic rats. Animals were sacrificed at 4, 14, 28 and 56 days after surgery (n=5 per group). Viral copies were detectable in the defect site of SD-RBMC group and gradually declined at 8w (5 log decrease compared to 4d). In the C-RBMC animals, there was a 2-4 log decline in the viral copy numbers at 2w and 4w, but at 8w there was a relative rise (about 100 fold) in the number of the viral vectors in the defect site of 4 (out of 5) animals compared to the previous time points. For both gene transfer approaches, the pattern of tissue distribution was non-specific and no histological abnormalities were noted in either group. In summary, we demonstrated that the LV-TSTA transduced cells remain in the defect site for at least 56 days, though the numbers decreased over time. There were no consistent findings of viral copies in internal organs which is encouraging with respect to the development of this strategy for use in humans.
    Type of Publication: Journal article published
    PubMed ID: 26264707
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  • 14
    Keywords: RECEPTOR ; IN-VIVO ; TRANSDUCTION ; GENE-TRANSFER ; DENDRITIC CELLS ; GAG ; HUMAN MONOCYTES ; HIV-1 INFECTION ; RESTRICTION ; MEASLES-VIRUS
    Abstract: To induce and trigger innate and adaptive immune responses, antigen-presenting cells (APCs) take up and process antigens. Retroviral particles are capable of transferring not only genetic information but also foreign cargo proteins when they are genetically fused to viral structural proteins. Here, we demonstrate the capacity of lentiviral protein transfer vectors (PTVs) for targeted antigen transfer directly into APCs and thereby induction of cytotoxic T cell responses. Targeting of lentiviral PTVs to APCs can be achieved analogously to gene transfer vectors by pseudotyping the particles with truncated wild-type measles virus (MV) glycoproteins (GPs), which use human SLAM (signaling lymphocyte activation molecule) as a main entry receptor. SLAM is expressed on stimulated lymphocytes and APCs, including dendritic cells. SLAM-targeted PTVs transferred the reporter protein green fluorescent protein (GFP) or Cre recombinase with strict receptor specificity into SLAM-expressing CHO and B cell lines, in contrast to broadly transducing vesicular stomatitis virus G protein (VSV-G) pseudotyped PTVs. Primary myeloid dendritic cells (mDCs) incubated with targeted or nontargeted ovalbumin (Ova)-transferring PTVs stimulated Ova-specific T lymphocytes, especially CD8(+) T cells. Administration of Ova-PTVs into SLAM-transgenic and control mice confirmed the observed predominant induction of antigen-specific CD8(+) T cells and demonstrated the capacity of protein transfer vectors as suitable vaccines for the induction of antigen-specific immune responses. IMPORTANCE: This study demonstrates the specificity and efficacy of antigen transfer by SLAM-targeted and nontargeted lentiviral protein transfer vectors into antigen-presenting cells to trigger antigen-specific immune responses in vitro and in vivo. The observed predominant activation of antigen-specific CD8(+) T cells indicates the suitability of SLAM-targeted and also nontargeted PTVs as a vaccine for the induction of cytotoxic immune responses. Since cytotoxic CD8(+) T lymphocytes are a mainstay of antitumoral immune responses, PTVs could be engineered for the transfer of specific tumor antigens provoking tailored antitumoral immunity. Therefore, PTVs can be used as safe and efficient alternatives to gene transfer vectors or live attenuated replicating vector platforms, avoiding genotoxicity or general toxicity in highly immunocompromised patients, respectively. Thereby, the potential for easy envelope exchange allows the circumventing of neutralizing antibodies, e.g., during repeated boost immunizations.
    Type of Publication: Journal article published
    PubMed ID: 26085166
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  • 15
    Keywords: IN-VIVO ; MODEL ; TISSUE ; antibodies ; MOUSE ; IDENTIFICATION ; GENE-THERAPY ; TROPISM ; DISPLAY PEPTIDE LIBRARIES ; VIRUS TYPE-2
    Abstract: Adeno-associated viral (AAV) vectors yield high potential for clinical gene therapy but, like for other vectors systems, they frequently do not sufficiently transduce the target tissue and their unspecific tropism prevents their application for multifocal diseases such as disseminated cancer. Targeted AAV vectors have been obtained from random AAV display peptide libraries but so far, all vector variants selected from AAV libraries upon systemic administration in vivo retained some collateral tropism, frequently the heart. Here we explored, if this impediment can be overcome by microRNA-regulated transgene cassettes as the combination of library-derived capsid targeting and micro-RNA control has not been evaluated so far. We used a tumor-targeted AAV capsid variant (ESGLSQS) selected from random AAV-display peptide libraries in vivo with remaining off-target tropism toward the heart and regulated targeted transgene expression in vivo by complementary target elements for heart-specific microRNA (miRT-1d). Although this vector still maintained its strong transduction capacity for tumor target tissue after intravenous injection, transgene expression in the heart was almost completely abrogated. This strong and completely tumor-specific transgene expression was used for therapeutic gene transfer in an aggressive multifocal, transgenic, polyoma middle T-induced, murine breast cancer model. A therapeutic suicide gene, delivered systemically by this dual-targeted AAV vector to multifocal breast cancer, significantly inhibited tumor growth after one single vector administration while avoiding side effects compared with untargeted vectors.
    Type of Publication: Journal article published
    PubMed ID: 26446851
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  • 16
    Keywords: IN-VIVO ; MODEL ; TISSUE ; antibodies ; MOUSE ; IDENTIFICATION ; GENE-THERAPY ; TROPISM ; DISPLAY PEPTIDE LIBRARIES ; VIRUS TYPE-2
    Abstract: Adeno-associated viral (AAV) vectors yield high potential for clinical gene therapy but, like for other vectors systems, they frequently do not sufficiently transduce the target tissue and their unspecific tropism prevents their application for multifocal diseases such as disseminated cancer. Targeted AAV vectors have been obtained from random AAV display peptide libraries but so far, all vector variants selected from AAV libraries upon systemic administration in vivo retained some collateral tropism, frequently the heart. Here we explored, if this impediment can be overcome by microRNA-regulated transgene cassettes as the combination of library-derived capsid targeting and micro-RNA control has not been evaluated so far. We used a tumor-targeted AAV capsid variant (ESGLSQS) selected from random AAV-display peptide libraries in vivo with remaining off-target tropism toward the heart and regulated targeted transgene expression in vivo by complementary target elements for heart-specific microRNA (miRT-1d). Although this vector still maintained its strong transduction capacity for tumor target tissue after intravenous injection, transgene expression in the heart was almost completely abrogated. This strong and completely tumor-specific transgene expression was used for therapeutic gene transfer in an aggressive multifocal, transgenic, polyoma middle T-induced, murine breast cancer model. A therapeutic suicide gene, delivered systemically by this dual-targeted AAV vector to multifocal breast cancer, significantly inhibited tumor growth after one single vector administration while avoiding side effects compared with untargeted vectors.
    Type of Publication: Journal article published
    PubMed ID: 26034897
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  • 17
    Keywords: IN-VIVO ; RESTRICTED DIFFUSION ; SPIN-ECHO ; LAPLACIAN EIGENFUNCTIONS ; POROUS-MEDIA ; SELF-DIFFUSION ; MAGNETIC-FIELD GRADIENT ; NUCLEAR MAGNETIZATION ; SURFACE RELAXATION ; CPMG SEQUENCE
    Abstract: The time-dependent apparent diffusion coefficient as measured by pulsed gradient NMR can be used to estimate parameters of porous structures including the surface-to-volume ratio and the mean curvature of pores. In this work, the short-time diffusion limit and in particular the influence of the temporal profile of diffusion gradients on the expansion as proposed by Mitra et al. (1993) is investigated. It is shown that flow-compensated waveforms, i.e. those whose first moment is zero, are blind to the term linear in observation time, which is the term that is proportional to mean curvature and surface permeability. A gradient waveform that smoothly interpolates between flow-compensated and bipolar waveform is proposed and the degree of flow-compensation is used as a second experimental dimension. This two-dimensional ansatz is shown to yield an improved precision when characterizing the confining domain. This technique is demonstrated with simulations and in experiments performed with cylindrical capillaries of 100mum radius.
    Type of Publication: Journal article published
    PubMed ID: 26254733
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  • 18
    Keywords: IN-VIVO ; GENERATION ; EXPRESSION ANALYSIS ; PROTEIN-SYNTHESIS ; neurogenesis ; SUBVENTRICULAR ZONE ; OLFACTORY-BULB ; Niche ; RNA-SEQ ; ADULT SUBEPENDYMAL ZONE
    Abstract: Heterogeneous pools of adult neural stem cells (NSCs) contribute to brain maintenance and regeneration after injury. The balance of NSC activation and quiescence, as well as the induction of lineage-specific transcription factors, may contribute to diversity of neuronal and glial fates. To identify molecular hallmarks governing these characteristics, we performed single-cell sequencing of an unbiased pool of adult subventricular zone NSCs. This analysis identified a discrete, dormant NSC subpopulation that already expresses distinct combinations of lineage-specific transcription factors during homeostasis. Dormant NSCs enter a primed-quiescent state before activation, which is accompanied by downregulation of glycolytic metabolism, Notch, and BMP signaling and a concomitant upregulation of lineage-specific transcription factors and protein synthesis. In response to brain ischemia, interferon gamma signaling induces dormant NSC subpopulations to enter the primed-quiescent state. This study unveils general principles underlying NSC activation and lineage priming and opens potential avenues for regenerative medicine in the brain.
    Type of Publication: Journal article published
    PubMed ID: 26235341
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  • 19
    Keywords: IN-VIVO ; DIVERSITY ; WORKING-MEMORY ; GAMMA OSCILLATIONS ; PARVALBUMIN-POSITIVE INTERNEURONS ; PLACE CELLS ; BEHAVING RAT ; GABAERGIC NEURONS ; INHIBITORY INTERNEURONS ; NETWORK OSCILLATIONS
    Abstract: GABAergic interneurons in the hippocampus coordinate neuronal activity that gives rise to network oscillations. In this article we present advances in our understanding of how different classes of hippocampal interneurons contribute to distinct hippocampal network oscillations. We focus on two main experimental approaches that were recently applied in awake animals. Firstly, we review experiments in which selective activation or inhibition of subgroups of GABAergic interneurons was used to asses their contribution to network oscillations. Secondly, we present work that provides a first characterization of the firing activity of anatomically identified interneurons in the hippocampus of behaving animals.
    Type of Publication: Journal article published
    PubMed ID: 25240150
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  • 20
    Keywords: IN-VIVO ; validation ; MAGNETIC-RESONANCE ; T-1 ; INFARCTION ; HEART-FAILURE ; HYPERTROPHIC CARDIOMYOPATHY ; RELAXATION-TIME ; LOOK-LOCKER ; MOLLI
    Abstract: Purpose To develop and validate a fast cardiac magnetic resonance imaging T1 mapping technique with high spatial resolution based on a radial inversion-recovery ( IR inversion recovery ) spoiled gradient-echo acquisition. Materials and Methods Approval for the study was granted by the local institutional review board, and all subjects gave written informed consent. An electrocardiographically triggered radial single-shot IR inversion recovery ( TRASSI ECG-triggered radial single-shot IR ) sequence was developed in conjunction with a custom-written fitting algorithm. The proposed imaging technique was validated in phantom measurements and then used for cardiac T1 mapping in 62 subjects with or without cardiac disease. The study population included 51 healthy subjects, three patients with arrhythmia, and eight patients with myocardial infarction. The potential heart rate dependency of the TRASSI ECG-triggered radial single-shot IR method was tested by using linear regression analysis. Statistically significant differences between the sexes and various section orientations were analyzed with a Student t test for independent groups and a repeated-measures analysis of variance for dependent groups. Results High-spatial-resolution T1 maps (1.17 x 1.17 mm) without motion artifacts and without heart rate dependency (slope = -0.0303, R2 = 0.0000887, P = .899) were acquired with an acquisition time of less than 6 seconds in all subjects. The mean T1 of healthy left ventricular myocardium across all examined subjects was 1031 msec +/- 33 (standard deviation). Testing for reproducibility in three individuals with 34 repetitive measurements revealed a mean standard deviation of 4.1 msec (0.412%). Subacute and chronic myocardial infarction could be detected in all eight patients. T1 disturbances due to arrhythmia proved to be minimal in three patients (standard deviation, 〈1.2%). Conclusion Fast and accurate cardiac T1 mapping is feasible within a single-shot IR inversion recovery experiment. (c) RSNA, 2014 Online supplemental material is available for this article.
    Type of Publication: Journal article published
    PubMed ID: 25393945
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  • 21
    Keywords: IRRADIATION ; IN-VIVO ; MAGNETIC-RESONANCE ; PH ; human brain ; CARTILAGE ; PROTON-EXCHANGE ; 7 T ; CHEMICAL-EXCHANGE ; NONINVASIVE DETECTION
    Abstract: Off-resonant spinlock (SL) enables an NMR imaging technique that can detect dilute metabolites similar to chemical exchange saturation transfer. However, in clinical MR scanners, RF pulse widths are restricted due to recommended specific absorption rate limits. Therefore, trains of short RF pulses that provide effective saturation during the required irradiation period are commonly employed. Quantitative evaluation of spectra obtained by pulsed saturation schemes is harder to achieve, since the theory of continuous wave saturation cannot be applied directly. In this paper we demonstrate the general feasibility of quantifying proton exchange rates from data obtained in pulsed SL experiments on a clinical 3 T MR scanner. We also propose a theoretical treatment of pulsed SL in the presence of chemical exchange using an interleaved saturation-relaxation approach. We show that modeling magnetization transfer during the pauses between the RF pulses is crucial, especially in the case of exchange rates that are small with respect to the delay times. The dynamics is still governed by a monoexponential decay towards steady state, for which we give the effective rate constant. The derived analytical model agrees well with the full numerical simulation of the Bloch-McConnell equations for a broad range of values of the system parameters. Copyright (c) 2014 John Wiley & Sons, Ltd.
    Type of Publication: Journal article published
    PubMed ID: 25328046
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  • 22
    Keywords: IN-VIVO ; THERAPY ; ACTIVATION ; GENOME-WIDE ANALYSIS ; SEVERE COMBINED IMMUNODEFICIENCY ; RETROVIRAL DNA-INTEGRATION ; VECTOR INTEGRATION ; BETA-THALASSEMIA ; VIRUS TYPE-1 ; CONSEQUENT
    Abstract: Gene therapy utilizing lentiviral-vectors (LVs) is postulated as a dynamic therapeutic alternative for monogenic diseases. However, retroviral gene transfer may cause insertional mutagenesis. Although, such risks had been originally estimated as extremely low, several reports of leukemias or clonal dominance, have led to a re-evaluation of the mechanisms operating in insertional mutagenesis. Therefore, unraveling the mechanism of retroviral integration is mandatory towards safer gene therapy applications. In the present study, we undertook an experimental approach which enabled direct correlation of the cell cycle stage of the target cell with the integration profile of LVs. CD34+ cells arrested at different stages of cell cycle, were transduced with a GFP-LV. LAM-PCR was employed for integration site detection, followed by microarray analysis to correlate transcribed genes with integration sites. The results indicate that approximately 10% of integration events occurred in actively transcribed genes and that the cell cycle stage of target cells affects integration pattern. Specifically, use of thymine promoted a safer profile, since it significantly reduced integration within cell cycle-related genes, while we observed increased possibility for integration into genes related to development, and decreased possibility for integration within cell cycle and cancer-related genes, when transduction occurs during mitosis.Molecular Therapy (2014); doi:10.1038/mt.2014.246.
    Type of Publication: Journal article published
    PubMed ID: 25523760
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  • 23
    Keywords: IN-VIVO ; DNA-REPAIR ; HUMAN GENOME ; HEMATOPOIETIC STEM-CELLS ; DOUBLE-STRAND BREAKS ; cord blood ; VECTOR INTEGRATION ; ZINC-FINGER NUCLEASES ; HOMOLOGY-DIRECTED REPAIR ; DONOR BONE-MARROW
    Abstract: Genome engineering with designer nucleases is a rapidly progressing field, and the ability to correct human gene mutations in situ is highly desirable. We employed fibroblasts derived from a patient with Fanconi anemia as a model to test the ability of the clustered regularly interspaced short palindromic repeats/Cas9 nuclease system to mediate gene correction. We show that the Cas9 nuclease and nickase each resulted in gene correction, but the nickase, due to its ability to preferentially mediate homology-directed repair, resulted in a higher frequency of corrected clonal isolates. To assess the off-target effects, we used both a predictive software platform to identify intragenic sequences of homology as well as a genome-wide screen utilizing linear amplification-mediated PCR. We observed no off-target activity and show RNA-guided endonuclease candidate sites that do not possess low sequence complexity function in a highly specific manner. Collectively, we provide proof of principle for precision genome editing in Fanconi anemia, a DNA repair-deficient human disorder.
    Type of Publication: Journal article published
    PubMed ID: 25545896
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  • 24
    Keywords: RECEPTOR ; IN-VIVO ; METASTATIC MELANOMA ; TRAFFICKING ; CANCER-THERAPY ; adoptive immunotherapy ; NANOPARTICLES ; PHOTODYNAMIC THERAPY ; RICIN ; TUMOR-LOCALIZATION
    Abstract: Recently conducted clinical trials have provided impressive evidence that chemotherapy resistant metastatic melanoma and several hematological malignancies can be cured using adoptive T cell therapy or T cell-recruiting bispecific antibodies. However, a significant fraction of patients did not benefit from these treatments. Here we have evaluated the feasibility of a novel combination therapy which aims to further enhance the killing potential of bispecific antibody-redirected T lymphocytes by using these cells as targeted delivery system for photosensitizing agents. For a first in vitro proof-of-concept study, ex vivo activated human donor T cells were loaded with a poly(styrene sulfonate) (PSS)-complex of the model photosensitizer 5,10,15,20-tetrakis(3-hydroxyphenyl)porphyrin (mTHPP). In the absence of light and when loading with the water-soluble PSS/mTHPP-complex occurred at a tolerable concentration, viability and cytotoxic function of loaded T lymphocytes were not impaired. When "drug-enhanced" T cells were co-cultivated with EpCAM-expressing human carcinoma cells, mTHPP was transferred to target cells. Notably, in the presence of a bispecific antibody, which cross-links effector and target cells thereby inducing the cytolytic activity of cytotoxic T lymphocytes, significantly more photosensitizer was transferred. Consequently, upon irradiation of co-cultures, redirected drug-loaded T cells were more effective in killing A549 lung and SKOV-3 ovarian carcinoma cells than retargeted unloaded T lymphocytes. Particularly, the additive approach using redirected unloaded T cells in combination with appropriate amounts of separately applied PSS/mTHPP was less efficient as well. Thus, by loading T lymphocytes with a stimulus-sensitive anti-cancer drug, we were able to enhance the cytotoxic capacity of carrier cells. Photosensitizer boosted T cells could open new perspectives for adoptive T cell therapy as well as targeted photodynamic therapy.
    Type of Publication: Journal article published
    PubMed ID: 25449805
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  • 25
    Keywords: CANCER ; IN-VIVO ; TRIAL ; NK cells ; MALIGNANT-MELANOMA ; RECEPTORS ; ADOPTIVE TRANSFER ; NKG2D ligands ; VIVO EXPANSION ; LINE NK-92
    Abstract: During the recent years, immunotherapy has obtained substantial impact on the clinical treatment of melanoma. Besides promising approaches based on T lymphocytes, natural killer (NK) cells have gained more and more attention as anti-melanoma effector cells. NK cell activation is inhibited by HLA class I molecules expressed by target cells, so they preferentially attack tumor cells that express low levels of HLA class I. Partial or complete loss of HLA class I expression is a frequent event during the development of melanoma. In parallel, ligands for activating NK cell receptors become induced upon malignant transformation. Thus, melanoma cells are often efficiently recognized and lysed by NK cells at least in vitro. In vivo, however, melanomas have developed multiple sophisticated strategies to escape from NK cell mediated attack. Several novel approaches aim at harnessing NK cells to treat melanoma patients and to counteract existing tumor escape mechanisms. This review summarizes the most recent advances in the field.
    Type of Publication: Journal article published
    PubMed ID: 25640488
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  • 26
    Keywords: IN-VIVO ; NUDE-MICE ; COLON-CANCER ; MULTIDRUG-RESISTANCE ; P53 PATHWAY ; ANTHELMINTIC DRUG MEBENDAZOLE ; RESISTANT CANCER-CELLS ; ONTOLOGY LEGO VECTORS ; BINDING AGENTS ; ALBENDAZOLE
    Abstract: Flubendazole was shown to exert anti-leukaemia and anti-myeloma activity through inhibition of microtubule function. Here, flubendazole was tested for its effects on the viability of in total 461 cancer cell lines. Neuroblastoma was identified as highly flubendazole-sensitive cancer entity in a screen of 321 cell lines from 26 cancer entities. Flubendazole also reduced the viability of five primary neuroblastoma samples in nanomolar concentrations thought to be achievable in humans and inhibited vessel formation and neuroblastoma tumour growth in the chick chorioallantoic membrane assay. Resistance acquisition is a major problem in high-risk neuroblastoma. 119 cell lines from a panel of 140 neuroblastoma cell lines with acquired resistance to various anti-cancer drugs were sensitive to flubendazole in nanomolar concentrations. Tubulin-binding agent-resistant cell lines displayed the highest flubendazole IC50 and IC90 values but differences between drug classes did not reach statistical significance. Flubendazole induced p53-mediated apoptosis. The siRNA-mediated depletion of the p53 targets p21, BAX, or PUMA reduced the neuroblastoma cell sensitivity to flubendazole with PUMA depletion resulting in the most pronounced effects. The MDM2 inhibitor and p53 activator nutlin-3 increased flubendazole efficacy while RNAi-mediated p53-depletion reduced its activity. In conclusion, flubendazole represents a potential treatment option for neuroblastoma including therapy-refractory cells.
    Type of Publication: Journal article published
    PubMed ID: 25644037
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  • 27
    Keywords: IN-VIVO ; QUALITY ; BONE ; CONE-BEAM CT ; COMPUTED-TOMOGRAPHY ANGIOGRAPHY ; DECOMPOSITION ; TO-NOISE RATIO ; RENAL STONE COMPOSITION ; MATERIAL-SELECTIVE CT ; TIN-FILTER
    Abstract: PURPOSE: Dual Energy CT (DECT) provides so-called monoenergetic images based on a linear combination of the original polychromatic images. At certain patient-specific energy levels, corresponding to certain patient- and slice-dependent linear combination weights, e.g., E = 160 keV corresponds to alpha = 1.57, a significant reduction of metal artifacts may be observed. The authors aimed at analyzing the method for its artifact reduction capabilities to identify its limitations. The results are compared with raw data-based processing. METHODS: Clinical DECT uses a simplified version of monochromatic imaging by linearly combining the low and the high kV images and by assigning an energy to that linear combination. Those pseudo-monochromatic images can be used by radiologists to obtain images with reduced metal artifacts. The authors analyzed the underlying physics and carried out a series expansion of the polychromatic attenuation equations. The resulting nonlinear terms are responsible for the artifacts, but they are not linearly related between the low and the high kV scan: A linear combination of both images cannot eliminate the nonlinearities, it can only reduce their impact. Scattered radiation yields additional noncanceling nonlinearities. This method is compared to raw data-based artifact correction methods. To quantify the artifact reduction potential of pseudo-monochromatic images, they simulated the FORBILD abdomen phantom with metal implants, and they assessed patient data sets of a clinical dual source CT system (100, 140 kV Sn) containing artifacts induced by a highly concentrated contrast agent bolus and by metal. In each case, they manually selected an optimal alpha and compared it to a raw data-based material decomposition in case of simulation, to raw data-based material decomposition of inconsistent rays in case of the patient data set containing contrast agent, and to the frequency split normalized metal artifact reduction in case of the metal implant. For each case, the contrast-to-noise ratio (CNR) was assessed. RESULTS: In the simulation, the pseudo-monochromatic images yielded acceptable artifact reduction results. However, the CNR in the artifact-reduced images was more than 60% lower than in the original polychromatic images. In contrast, the raw data-based material decomposition did not significantly reduce the CNR in the virtual monochromatic images. Regarding the patient data with beam hardening artifacts and with metal artifacts from small implants the pseudo-monochromatic method was able to reduce the artifacts, again with the downside of a significant CNR reduction. More intense metal artifacts, e.g., as those caused by an artificial hip joint, could not be suppressed. CONCLUSIONS: Pseudo-monochromatic imaging is able to reduce beam hardening, scatter, and metal artifacts in some cases but it cannot remove them. In all cases, the CNR is significantly reduced, thereby rendering the method questionable, unless special post processing algorithms are implemented to restore the high CNR from the original images (e.g., by using a frequency split technique). Raw data-based dual energy decomposition methods should be preferred, in particular, because the CNR penalty is almost negligible.
    Type of Publication: Journal article published
    PubMed ID: 25652515
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  • 28
    Keywords: IN-VIVO ; POLYCYCLIC AROMATIC-HYDROCARBONS ; CANCER-CELLS ; aristolochic acid ; ADDUCT FORMATION ; HISTONE DEACETYLASE INHIBITORS ; CELL-CYCLE ARREST ; PROSTAGLANDIN-H SYNTHASE ; P450 1A1 ; TARGETED CHEMOTHERAPEUTICS
    Abstract: Ellipticine is a DNA-damaging agent acting as a prodrug whose pharmacological efficiencies and genotoxic side effects are dictated by activation with cytochrome P450 (CYP). Over the last decade we have gained extensive experience in using pure enzymes and various animal models that helped to identify CYPs metabolizing ellipticine. In this review we focus on comparison between the in vitro and in vivo studies and show a necessity of both approaches to obtain valid information on CYP enzymes contributing to ellipticine metabolism. Discrepancies were found between the CYP enzymes activating ellipticine to 13-hydroxy- and 12-hydroxyellipticine generating covalent DNA adducts and those detoxifying this drug to 9-hydroxy- and 7-hydroellipticine in vitro and in vivo. In vivo, formation of ellipticine-DNA adducts is dependent not only on expression levels of CYP3A, catalyzing ellipticine activation in vitro, but also on those of CYP1A that oxidize ellipticine in vitro mainly to the detoxification products. The finding showing that cytochrome b5 alters the ratio of ellipticine metabolites generated by CYP1A1/2 and 3A4 explained this paradox. Whereas the detoxification of ellipticine by CYP1A and 3A is either decreased or not changed by cytochrome b5, activation leading to ellipticine-DNA adducts increased considerably. We show that (I) the pharmacological effects of ellipticine mediated by covalent ellipticine-derived DNA adducts are dictated by expression levels of CYP1A, 3A and cytochrome b5, and its own potency to induce these enzymes in tumor tissues, (II) animal models, where levels of CYPs are either knocked out or induced are appropriate to identify CYPs metabolizing ellipticine in vivo, and (III) extrapolation from in vitro data to the situation in vivo is not always possible, confirming the need for these animal models.
    Type of Publication: Journal article published
    PubMed ID: 25547492
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  • 29
    Keywords: EXPRESSION ; IN-VIVO ; LIGAND ; FEATURES ; BLOOD-BRAIN-BARRIER ; NOTCH ; CCM1 ; INHIBITS SPROUTING ANGIOGENESIS ; MUSCLE-CELL FORMATION ; VASCULAR INTEGRITY
    Abstract: Background and Purpose-Cerebral cavernous malformation (CCM) is a neurovascular dysplasia characterized by conglomerates of enlarged endothelial channels in the central nervous system, which are almost devoid of pericytes or smooth muscle cells. This disease is caused by loss-of-function mutations in CCM1, CCM2, or CCM3 genes in endothelial cells, making blood vessels highly susceptible to angiogenic stimuli. CCM1- and CCM3-silenced endothelial cells have a reduced expression of the Notch ligand Delta-like 4 (DLL4) resulting in impaired Notch signaling and irregular sprouting angiogenesis. This study aimed to address if DLL4, which is exclusively expressed on endothelial cells, may influence interactions of endothelial cells with pericytes, which express Notch3 as the predominant Notch receptor. Methods-Genetic manipulation of primary human endothelial cells and brain pericytes. Transgenic mouse models were also used. Results-Endothelial cell-specific ablation of Ccm1 and Ccm2 in different mouse models led to the formation of CCM-like lesions, which were poorly covered by periendothelial cells. CCM1 silencing in endothelial cells caused decreased Notch3 activity in cocultured pericytes. DLL4 proteins stimulated Notch3 receptors on human brain pericytes. Active Notch3 induced expression of PDGFRB2, N-Cadherin, HBEGF, TGFB1, NG2, and S1P genes. Notch3 signaling in pericytes enhanced the adhesion strength of pericytes to endothelial cells, limited their migratory and invasive behavior, and enhanced their antiangiogenic function. Pericytes silenced for Notch3 expression were more motile and could not efficiently repress angiogenesis. Conclusions-The data suggest that Notch signaling in pericytes is important to maintain the quiescent vascular phenotype. Deregulated Notch signaling may, therefore, contribute to the pathogenesis of CCM.
    Type of Publication: Journal article published
    PubMed ID: 25791711
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  • 30
    Keywords: IN-VIVO ; MICE ; MUTATIONS ; MOUSE MODEL ; TRANSGENE EXPRESSION ; ONCOGENESIS ; VECTOR INTEGRATION ; ENDOGENOUS MICRORNA ; HUMAN-FACTOR-IX ; LOW GENOTOXICITY
    Abstract: We investigated the efficacy of liver-directed gene therapy using lentiviral vectors in a large animal model of hemophilia B and evaluated the risk of insertional mutagenesis in tumor-prone mouse models. We showed that gene therapy using lentiviral vectors targeting the expression of a canine factor IX transgene in hepatocytes was well tolerated and provided a stable long-term production of coagulation factor IX in dogs with hemophilia B. By exploiting three different mouse models designed to amplify the consequences of insertional mutagenesis, we showed that no genotoxicity was detected with these lentiviral vectors. Our findings suggest that lentiviral vectors may be an attractive candidate for gene therapy targeted to the liver and may be potentially useful for the treatment of hemophilia.
    Type of Publication: Journal article published
    PubMed ID: 25739762
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  • 31
    Keywords: IN-VIVO ; TRANSGENIC MICE ; VASCULATURE ; ORIGIN ; lymphangiogenesis ; BLOOD-BRAIN-BARRIER ; MOUSE EMBRYOS ; GROWTH-FACTOR-C ; PHENOTYPIC HETEROGENEITY ; VEGF RECEPTOR-3
    Abstract: Pathological lymphatic diseases mostly affect vessels in specific tissues, yet little is known about organ-specific regulation of the lymphatic vasculature. Here, we show that the vascular endothelial growth factor receptor 3 (VEGFR-3)/p110alpha PI3-kinase signaling pathway is selectively required for the formation of mesenteric lymphatic vasculature. Using genetic lineage tracing, we demonstrate that part of the mesenteric lymphatic vasculature develops from cKit lineage cells of hemogenic endothelial origin through a process we define as lymphvasculogenesis. This is contrary to the current dogma that all mammalian lymphatic vessels form by sprouting from veins. Our results reveal vascular-bed-specific differences in the origin and mechanisms of vessel formation, which may critically underlie organ-specific manifestation of lymphatic dysfunction in disease. The progenitor cells identified in this study may be exploited to restore lymphatic function following cancer surgery, lymphedema, or tissue trauma.
    Type of Publication: Journal article published
    PubMed ID: 25772358
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  • 32
    Keywords: EXPRESSION ; IN-VIVO ; EMPHYSEMA ; NF-KAPPA-B ; TRANSPORT ; PHENOTYPE ; OBSTRUCTIVE PULMONARY-DISEASE ; IL-1-BETA ; OVEREXPRESSING MICE ; TRANSMEMBRANE CONDUCTANCE REGULATOR
    Abstract: RATIONALE: In many organs, hypoxic cell death triggers sterile neutrophilic inflammation via IL-1R signaling. Although hypoxia is common in airways from patients with cystic fibrosis (CF), its role in neutrophilic inflammation remains unknown. We recently demonstrated that hypoxic epithelial necrosis caused by airway mucus obstruction precedes neutrophilic inflammation in Scnn1b-transgenic (Scnn1b-Tg) mice with CF-like lung disease. OBJECTIVES: To determine the role of epithelial necrosis and IL-1R signaling in the development of neutrophilic airway inflammation, mucus obstruction, and structural lung damage in CF lung disease. METHODS: We used genetic deletion and pharmacologic inhibition of IL-1R in Scnn1b-Tg mice and determined effects on airway epithelial necrosis; levels of IL-1alpha, keratinocyte chemoattractant, and neutrophils in bronchoalveolar lavage; and mortality, mucus obstruction, and structural lung damage. Furthermore, we analyzed lung tissues from 21 patients with CF and chronic obstructive pulmonary disease and 19 control subjects for the presence of epithelial necrosis. MEASUREMENTS AND MAIN RESULTS: Lack of IL-1R had no effect on epithelial necrosis and elevated IL-1alpha, but abrogated airway neutrophilia and reduced mortality, mucus obstruction, and emphysema in Scnn1b-Tg mice. Treatment of adult Scnn1b-Tg mice with the IL-1R antagonist anakinra had protective effects on neutrophilic inflammation and emphysema. Numbers of necrotic airway epithelial cells were elevated and correlated with mucus obstruction in patients with CF and chronic obstructive pulmonary disease. CONCLUSIONS: Our results support an important role of hypoxic epithelial necrosis in the pathogenesis of neutrophilic inflammation independent of bacterial infection and suggest IL-1R as a novel target for antiinflammatory therapy in CF and potentially other mucoobstructive airway diseases.
    Type of Publication: Journal article published
    PubMed ID: 25607238
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  • 33
    Keywords: RECEPTOR ; IN-VIVO ; DYNAMICS ; c-Fos ; PRODUCT ; LIVING CELLS ; PROTEIN COMPLEXES ; DIFFUSION ; DNA-BINDING ACTIVITY ; AP-1 SITE
    Abstract: We collected mobility and interaction maps of c-Fos-eGFP and c-Jun-mRFP1 transcription factors within living cell nuclei. c-Fos dimerizes with c-Jun to form the transcription activator protein-1 (AP-1) which binds to the specific recognition site. To monitor this process, we used fluorescence cross-correlation spectroscopy on a single plane illumination microscope (SPIM-FCCS), which provides diffusion coefficient and protein-protein interaction data in the whole image plane simultaneously, instead of just one point on conventional confocal FCS. We find a strong correlation between diffusional mobility and interaction: regions of strong interaction show slow mobility. Controls containing either an eGFP-mRFP dimer, separately expressing eGFP and mRPF, or c-Fos-eGFP and c-Jun-mRFP1 mutants lacking dimerization and DNA-binding domains, showed no such correlation. These results extend our earlier findings from confocal FCCS to include spatial information.
    Type of Publication: Journal article published
    PubMed ID: 25875593
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  • 34
    Keywords: IN-VIVO ; B-CELLS ; TERMINAL DIFFERENTIATION ; NUCLEAR ANTIGEN-2 ; EBV INFECTION ; GASTRIC-CARCINOMA ; ORAL HAIRY LEUKOPLAKIA ; NASOPHARYNGEAL EPITHELIAL-CELLS ; MEMBRANE-PROTEIN 1 ; STABLE REPLICATION
    Abstract: The Epstein-Barr virus (EBV) is transmitted from host-to-host via saliva and is associated with epithelial malignancies including nasopharyngeal carcinoma (NPC) and some forms of gastric carcinoma (GC). Nevertheless, EBV does not transform epithelial cells in vitro where it is rapidly lost from infected primary epithelial cells or epithelial tumor cells. Long-term infection by EBV, however, can be established in hTERT-immortalized nasopharyngeal epithelial cells. Here, we hypothesized that increased telomerase activity in epithelial cells enhances their susceptibility to infection by EBV. Using HONE-1, AGS and HEK293 cells we generated epithelial model cell lines with increased or suppressed telomerase activity by stable ectopic expression of hTERT or of a catalytically inactive, dominant negative hTERT mutant. Infection experiments with recombinant prototypic EBV (rB95.8), recombinant NPC EBV (rM81) with increased epithelial cell tropism compared to B95.8, or recombinant B95.8 EBV with BZLF1-knockout that is not able to undergo lytic replication, revealed that infection frequencies positively correlate with telomerase activity in AGS cells but also partly depend on the cellular background. AGS cells with increased telomerase activity showed increased expression mainly of latent EBV genes, suggesting that increased telomerase activity directly acts on the EBV infection of epithelial cells by facilitating latent EBV gene expression early upon virus inoculation. Thus, our results indicate that infection of epithelial cells by EBV is a very selective process involving, among others, telomerase activity and cellular background to allow for optimized host-to-host transmission via saliva.
    Type of Publication: Journal article published
    PubMed ID: 25856387
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  • 35
    Keywords: IN-VIVO ; CELL-DEATH ; CALCIUM ; OXIDATIVE STRESS ; NEURONS ; INTRACELLULAR HYDROGEN-PEROXIDE ; SUPEROXIDE FLASHES ; YELLOW FLUORESCENT PROTEIN ; MITOCHONDRIAL OXIDANT STRESS ; CA2+ INDICATORS
    Abstract: Redox signals have emerged as important regulators of cellular physiology and pathology. The advent of redox imaging in vertebrate systems now provides the opportunity to dynamically visualize redox signaling during development and disease. In this review, we summarize recent advances in the generation of genetically encoded redox indicators (GERIs), introduce new redox imaging strategies, and highlight key publications in the field of vertebrate redox imaging. We also discuss the limitations and future potential of in vivo redox imaging in zebrafish and mice.
    Type of Publication: Journal article published
    PubMed ID: 25720068
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  • 36
    Keywords: IN-VIVO ; TRANSCRIPTION FACTOR ; ESCHERICHIA-COLI ; DNA-BINDING ; DISULFIDE BOND FORMATION ; HYDROGEN-PEROXIDE ; REDOX REGULATION ; 3-DIMENSIONAL STRUCTURE ; REVERSIBLE OXIDATION ; SOLVENT ACCESSIBILITY
    Abstract: A few small-molecule oxidants, most notably hydrogen peroxide, can act as messengers in signal transduction. They trigger so-called 'thiol switches', cysteine residues that are reversibly oxidized to transiently change the functional properties of their host proteins. The proteome-wide identification of functionally relevant 'thiol switches' is of significant interest. Unfortunately, prediction of redox-active cysteine residues on the basis of surface accessibility and other computational parameters appears to be of limited use. Proteomic thiol labeling approaches remain the most reliable strategy to discover new thiol switches in a hypothesis-free manner. We discuss if and how genomic knock-in strategies can help establish the physiological relevance of a 'thiol switch' on the organismal level. We conclude that surprisingly few attempts have been made to thoroughly verify the physiological relevance of thiol-based redox switches in mammalian model organisms.
    Type of Publication: Journal article published
    PubMed ID: 25719318
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  • 37
    Keywords: ANGIOGENESIS ; tumor ; IN-VIVO ; MODEL ; CHORIOALLANTOIC MEMBRANE ASSAY
    Abstract: Fertilized chicken eggs are suggested as an alternative to mammalian models. The chorioallantoic membrane (CAM) of the chick embryo is widely used for examination of angiogenesis, xenotransplants and for virus production. Unfortunately, it is mostly not taken into account, that the chick embryo's ability to experience pain starts to develop at day 7 of breeding. In our view, this model is only in accordance with the 3 R principles, if an appropriate anesthesia of the chick embryo in potentially painful procedures is provided. Although many experimental approaches are performed on the none-innervated CAM, the euthanasia of the embryo strongly requires a more human technique than the usually used freezing at -20 degrees C, decapitation or in ovo fixation with paraformaldehyde without prior anesthesia. However, protocols regarding feasible and ethical methods for anesthesia and euthanasia of avian embryos are currently not available. Therefore, we established an easy and reliable method for the euthanasia and short-term anesthesia of the chick embryo.
    Type of Publication: Journal article published
    PubMed ID: 25592390
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  • 38
    Keywords: IN-VIVO ; MODEL ; GENE ; ACTIVATION ; IMMUNE-RESPONSES ; ACUTE LUNG INJURY ; SPINAL-CORD ; HYPERSENSITIVITY ; NERVE INJURY ; HUMAN NEUTROPHIL ELASTASE
    Abstract: Neuropathic pain is a major, intractable clinical problem and its pathophysiology is not well understood. Although recent gene expression profiling studies have enabled the identification of novel targets for pain therapy, classical study designs provide unclear results owing to the differential expression of hundreds of genes across sham and nerve-injured groups, which can be difficult to validate, particularly with respect to the specificity of pain modulation. To circumvent this, we used two outbred lines of rats, which are genetically similar except for being genetically segregated as a result of selective breeding for differences in neuropathic pain hypersensitivity. SerpinA3N, a serine protease inhibitor, was upregulated in the dorsal root ganglia (DRG) after nerve injury, which was further validated for its mouse homolog. Mice lacking SerpinA3N developed more neuropathic mechanical allodynia than wild-type (WT) mice, and exogenous delivery of SerpinA3N attenuated mechanical allodynia in WT mice. T lymphocytes infiltrate the DRG after nerve injury and release leukocyte elastase (LE), which was inhibited by SerpinA3N derived from DRG neurons. Genetic loss of LE or exogenous application of a LE inhibitor (Sivelastat) in WT mice attenuated neuropathic mechanical allodynia. Overall, we reveal a novel and clinically relevant role for a member of the serpin superfamily and a leukocyte elastase and crosstalk between neurons and T cells in the modulation of neuropathic pain.
    Type of Publication: Journal article published
    PubMed ID: 25915831
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  • 39
    Keywords: IN-VIVO ; SYSTEM ; MOUSE ; MUTANT MICE ; ADIPOSE-TISSUE ; C-KIT ; STABILIZATION ; immune cells ; KIT(W-SH/W-SH) MICE ; W-WV
    Abstract: Obesity, insulin resistance, and related pathologies are associated with immune-mediated chronic inflammation. Kit mutant mice are protected from diet-induced obesity and associated co-morbidities, and this phenotype has previously been attributed to their lack of mast cells. We performed a comprehensive metabolic analysis of Kit-dependent Kit(W/Wv) and Kit-independent Cpa3(Cre/+) mast-cell-deficient mouse strains, employing diet-induced or genetic (Lep(Ob/Ob) background) models of obesity. Our results show that mast cell deficiency, in the absence of Kit mutations, plays no role in the regulation of weight gain or insulin resistance. Moreover, we provide evidence that the metabolic phenotype observed in Kit mutant mice, while independent of mast cells, is immune regulated. Our data underscore the value of definitive mast cell deficiency models to conclusively test the involvement of this enigmatic cell in immune-mediated pathologies and identify Kit as a key hematopoietic factor in the pathogenesis of metabolic syndrome.
    Type of Publication: Journal article published
    PubMed ID: 25955205
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  • 40
    Keywords: IN-VIVO ; GENE-EXPRESSION ; MAMMALIAN-CELLS ; ADAPTIVE IMMUNITY ; FUNCTIONAL GENOMICS ; HUMAN-CELLS ; CAS SYSTEMS ; SEQUENCE-SPECIFIC CONTROL ; ONE-STEP GENERATION ; GUIDE RNA
    Abstract: The discovery that the machinery of the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 bacterial immune system can be re-purposed to easily create deletions, insertions and replacements in the mammalian genome has revolutionized the field of genome engineering and re-invigorated the field of gene therapy. Many parallels have been drawn between the newly discovered CRISPR-Cas9 system and the RNA interference (RNAi) pathway in terms of their utility for understanding and interrogating gene function in mammalian cells. Given this similarity, the CRISPR-Cas9 field stands to benefit immensely from lessons learned during the development of RNAi technology. We examine how the history of RNAi can inform today's challenges in CRISPR-Cas9 genome engineering such as efficiency, specificity, high-throughput screening and delivery for in vivo and therapeutic applications.
    Type of Publication: Journal article published
    PubMed ID: 25800748
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  • 41
    Keywords: IN-VIVO ; TUMORS ; SUPPRESSION ; SPECTROSCOPY ; Coherence ; SEL-MQC ; SINGLE-SCAN ; RESONANCES
    Abstract: Selective detection of lactate signals in in vivo MR spectroscopy with spectral editing techniques is necessary in situations where strong lipid or signals from other molecules overlap the desired lactate resonance in the spectrum. Several pulse sequences have been proposed for this task. The double-quantum filter SSel-MQC provides very good lipid and water signal suppression in a single scan. As a major drawback, it suffers from significant signal loss due to incomplete refocussing in situations where long evolution periods are required. Here we present a refocused version of the SSel-MQC technique that uses only one additional refocussing pulse and regains the full refocused lactate signal at the end of the sequence.
    Type of Publication: Journal article published
    PubMed ID: 25909643
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  • 42
    Keywords: brain ; IN-VIVO ; NEURITE OUTGROWTH ; INDEPENDENT PROGNOSTIC MARKER ; STEM-CELL ; DISTINCT SUBGROUPS ; Pilocytic astrocytoma ; GENETIC PROFILES ; POSTERIOR-FOSSA EPENDYMOMA ; IMMUNOHISTOCHEMICAL EXPRESSION
    Abstract: The CD24 glycoprotein is a mediator of neuronal proliferation, differentiation and immune suppression in the normal CNS, and a proposed cancer biomarker in multiple peripheral tumor types. We performed a comparative analysis of CD24 gene expression in a large cohort of pediatric and adult brain tumors (n = 813), and further characterized protein expression in tissue sections (n = 39), primary brain tumor cultures (n = 12) and a novel orthotopic group 3 medulloblastoma xenograft model. Increased CD24 gene expression was demonstrated in ependymomas, medulloblastomas, anaplastic astrocytomas and glioblastomas, although medulloblastomas displayed higher expression than all other tumor entities. Preferential expression of CD24 in medulloblastomas was confirmed at protein level by immunostaining and computerized image analysis of cryosections. Morphologies and immunophenotyping of CD24(+) cells in tissue sections tentatively suggested disparate functions in different tumor subsets. Notably, protein staining of medulloblastoma cells was associated with prominent cytoplasmic and membranous granules, enabling rapid and robust identification of medulloblastoma cells in clinical tissue samples, as well as in experimental model systems. In conclusion, our results implicate CD24 as a clinically and experimentally useful medulloblastoma immunomarker. Although our results encourage further functional studies of CD24 as a potential molecular target in subsets of brain tumors, the promiscuous expression of CD24 in vivo highlights the importance of specificity in the future design of such targeted treatment.
    Type of Publication: Journal article published
    PubMed ID: 25820321
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  • 43
    Keywords: IN-VIVO ; ANTITUMOR-ACTIVITY ; STEM-CELLS ; PROGENITOR CELLS ; GENE-THERAPY ; PANCREATIC-CANCER CELLS ; REVERSE-TRANSCRIPTASE PROMOTER ; CELLULAR VEHICLES ; TRAIL RESISTANCE ; MODIFIED FIBERS
    Abstract: Oncolytic viruses have demonstrated in pre-clinical and clinical studies safety and a unique pleiotropic activity profile of tumor destruction. Yet, their delivery suffers from virus inactivation by blood components and sequestration to healthy tissues. Therefore, mesenchymal stromal cells (MSCs) have been applied as carrier cells for shielded virus delivery to tumors after ex vivo infection with oncolytic viruses. However, infection and particle production by MSCs have remained unsatisfying. Here, we report engineered oncolytic adenoviruses (OAds) for improved virus production and delivery by MSCs. OAds are uniquely amenable to molecular engineering, which has facilitated improved tumor cell destruction. But for MSC-mediated regimens, OAd engineering needs to achieve efficient infection and replication in both MSCs and tumor cells. We show that an Ad5/3 chimeric OAd capsid, containing the adenovirus serotype 3 cell-binding domain, strongly increases the entry into human bone marrow-derived MSCs and into established and primary pancreatic cancer cells. Further, we reveal that OAd with engineered post-entry functions-by deletion of the anti-apoptotic viral gene E1B19K or expression of the death ligand TRAIL-markedly increased virus titers released from MSCs, while MSC migration was not hampered. Finally, these virus modifications, or viral expression of FCU1 for local 5-FC prodrug activation, improved tumor cell killing implementing complementary cytotoxicity profiles in a panel of pancreatic cancer cell cultures. Together, our study establishes post-entry modification of OAd replication for improving virus delivery by carrier cells and suggests a panel of optimized OAds for future clinical development in personalized treatment of pancreatic cancer.
    Type of Publication: Journal article published
    PubMed ID: 25604186
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  • 44
    Keywords: IN-VIVO ; DIFFERENTIATION ; METHYLATION ; 5-methylcytosine ; SINGLE-CELL ; SELF-RENEWAL ; DNA demethylation ; PRIMORDIAL GERM-CELLS ; GROUND-STATE PLURIPOTENCY ; NAIVE PLURIPOTENCY
    Abstract: Embryonic stem cell (ESC) cultures display a heterogeneous gene expression profile, ranging from a pristine naive pluripotent state to a primed epiblast state. Addition of inhibitors of GSK3beta and MEK (so-called 2i conditions) pushes ESC cultures toward a more homogeneous naive pluripotent state, but the molecular underpinnings of this naive transition are not completely understood. Here, we demonstrate that DAZL, an RNA-binding protein known to play a key role in germ-cell development, marks a subpopulation of ESCs that is actively transitioning toward naive pluripotency. Moreover, DAZL plays an essential role in the active reprogramming of cytosine methylation. We demonstrate that DAZL associates with mRNA of Tet1, a catalyst of 5-hydroxylation of methyl-cytosine, and enhances Tet1 mRNA translation. Overexpression of DAZL in heterogeneous ESC cultures results in elevated TET1 protein levels as well as increased global hydroxymethylation. Conversely, null mutation of Dazl severely stunts 2i-mediated TET1 induction and hydroxymethylation. Our results provide insight into the regulation of the acquisition of naive pluripotency and demonstrate that DAZL enhances TET1-mediated cytosine hydroxymethylation in ESCs that are actively reprogramming to a pluripotent ground state.
    Type of Publication: Journal article published
    PubMed ID: 26077710
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  • 45
    Keywords: IN-VIVO ; CONTRAST AGENT ; MAGNETIC-RESONANCE-SPECTROSCOPY ; MALIGNANT GLIOMAS ; CEREBRAL BLOOD-VOLUME ; GLIOBLASTOMA-MULTIFORME ; HIGH-GRADE GLIOMAS ; RECURRENT GLIOBLASTOMA ; RESPONSE ASSESSMENT ; INTRATUMORAL-SUSCEPTIBILITY-SIGNALS
    Abstract: Due to the introduction of advanced functional and spectroscopic magnetic resonance (MR) sequences, MR imaging has gained significant importance in neuro-oncology. In contrast to recent years when neuro-oncological imaging was mostly limited to contrast-enhanced T1-weighted images, advanced MR methods provide direct visualization and assessment of tumor pathophysiology. This article summarizes the most relevant MR methods for neuro-oncological imaging and highlights the pathophysiological background as well as potential clinical applications. Ultimately, this article gives a glimpse into the future and introduces potential applications of ultra-high field MRI.
    Type of Publication: Journal article published
    PubMed ID: 26017379
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  • 46
    Keywords: carcinoma ; IN-VIVO ; TISSUE ; SPECTROSCOPY ; PROGRESSION ; sensitivity ; MALIGNANCY ; CANCER DIAGNOSIS ; histopathology ; SCATTERING MICROSCOPY
    Abstract: At present, tumor diagnostic imaging is commonly based on hematoxylin and eosin or immunohistochemical staining of biopsies, which requires tissue excision, fixation, and staining with exogenous marker molecules. Here, we report on label-free tumor imaging using confocal spontaneous Raman scattering microspectroscopy, which exploits the intrinsic vibrational contrast of endogenous biomolecular species. We present a chemically specific and quantitative approach to monitoring normal human skin cells (keratinocytes and fibroblasts) as well as the human HaCaT in vitro skin carcinogenesis model and the tumor-derived MET in vivo skin cancer progression model. Mapping the amplitudes of two spectrally well isolated Raman bands at 752 and 785 cm(-1) allowed for direct visualization of the distributions representative of tryptophan-rich proteins and nucleic acids, respectively, with subcellular spatial resolution. Using these Raman markers, it was feasible to discriminate between normal human epidermal keratinocytes (NHEK) and dermal fibroblasts (NHDF) and to confine all tumorigenic cells from both the NHEK and NHDF. First evidence for the successful application of the proposed intracellular nucleic acid and tryptophan Raman signatures for skin cancer diagnosis was further demonstrated in an organotypic cutaneous squamous cell carcinomas model, allowing for the identification of tumor cells and their surrounding stroma in the tissue context.
    Type of Publication: Journal article published
    PubMed ID: 25984831
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  • 47
    Keywords: carcinoma ; IN-VIVO ; PATHWAY ; GENES ; MUTATIONS ; MOUSE MODEL ; C-KIT ; CYCLE ARREST ; NEUROENDOCRINE TUMORS ; NOTCH
    Abstract: We have sequenced the genomes of 110 small cell lung cancers (SCLC), one of the deadliest human cancers. In nearly all the tumours analysed we found bi-allelic inactivation of TP53 and RB1, sometimes by complex genomic rearrangements. Two tumours with wild-type RB1 had evidence of chromothripsis leading to overexpression of cyclin D1 (encoded by the CCND1 gene), revealing an alternative mechan