Biomedical subjects

Jürgen Schlegel

Publications and source records attributed to Jürgen Schlegel.

17 recordsLinked to original sources

PubMed2006

A functional polymorphism within plasminogen activator urokinase (PLAU) is associated with Alzheimer's disease.

A number of susceptibility loci for Alzheimer's disease (AD) have been identified including a region on Chromosome 10q21-q22. Within this region the plasminogen activator urokinase gene (PLAU) was considered as a reasonable candidate from its functional implication in plasmin generation, a serine protease capable of degrading beta-Amyloid (Abeta) protein. We screened 56 single nucleotide polymorphisms (SNPs) around PLAU using 1751 individuals from four independent case-control samples (Munich, N=679; Bonn N=282; Brescia (Italy) N=219; Perth (Australia) N=557 and one discordant sib-pair sample (Munich N=622). In brain tissue samples of neuropathologically confirmed cases with AD (N=33) we analyzed plaque counts according to the risk allele. We identified that one functional exonic SNP (rs2227564) is associated with development of AD using the four independent case-control samples (Munich, P=0.02; Bonn, P=0.005; Brescia (Italy), P=0.001; Perth (Australia), P=0.03) and the discordant sib-pair sample (P=0.001). In brain tissue, from neuropathologically confirmed cases with AD, we identified significantly higher plaque counts in carriers of the risk allele (N=6; 60.3+/-16.9) compared with non-carriers (N=9; 26.3+/-8.8; P=0.007). This study provides compelling evidence of a genetic and functional involvement of a common PLAU variant into the pathogenesis of AD. Further functional investigations are warranted to elucidate the specific role of PLAU, respectively, PLAU variants in the metabolism of Abeta proteins.

Age of Onset↗

PubMed2006

Regional vulnerability of mesencephalic dopaminergic neurons prone to degenerate in Parkinson's disease: a post-mortem study in human control subjects.

Parkinson's disease (PD) is characterized by loss of dopaminergic (DA) neurons in the human midbrain, which varies greatly among mesencephalic subregions. The genetic expression profiles of mesencephalic DA neurons particularly prone to degenerate during PD (nigrosome 1 within the substantia nigra pars compacta-SNpc) and those particularly resistant in the disease course (central grey substance-CGS) were compared in five control subjects by immuno-laser capture microdissection followed by RNA arbitrarily primed PCR. 8 ESTs of interest were selected for analysis by real time quantitative reverse transcription PCR. DA neurons in the CGS preferentially expressed implicated in cell survival (7 out of 8 genes selected), whereas SNpc DA neurons preferentially expressed one gene making them potentially susceptible to undergo cell death in PD. We propose that factors making CGS DA neurons more resistant may be helpful in protecting SNpc DA neurons against a pathological insult.

Aged↗

PubMed2005

Gene expression profiling of Lewy body-bearing neurons in Parkinson's disease.

Lewy bodies (LB) are a pathological hallmark of Parkinson's disease (PD). Whether LBs are neuroprotective, cytotoxic, or an age-related epiphenomenon is still debated. In the present study, the genetic fingerprints of mesencephalic dopaminergic (DA) neurons containing LBs versus mesencephalic DA neurons not containing LBs were compared in five PD patients. Total RNA from single neurons of both neuronal subpopulations was obtained by immuno-laser capture microdissection. Subsequently, RNA arbitrarily primed PCR was employed to generate expression profiles from the extracted RNA. Differentially displayed polymorphic fragments were dissected from silver-stained polyacrylamide gels. Most of these expressed sequence tags (ESTs) were homologous to known human sequences (56/64, 87.5%). Based on the potential significance of individual ESTs in neurodegenerative disorders, 5 ESTs of interest were selected for further quantitative expression analysis by real-time quantitative reverse transcription PCR (rtq RT-PCR). DA neurons without LBs preferentially expressed molecules beneficial for cell survival, whereas genes preferentially expressed in DA neurons containing LBs may support a cytotoxic role of LBs. Thus, we favor the view that LB-positive DA neurons are sicker than their LB-negative counterparts, and that inhibition of LB formation may indeed represent a therapeutic strategy in PD.

Aged↗

PubMed2005

Migration of iron oxide-labeled human hematopoietic progenitor cells in a mouse model: in vivo monitoring with 1.5-T MR imaging equipment.

PURPOSE: To evaluate the use of clinical 1.5-T magnetic resonance (MR) imaging equipment to depict the in vivo distribution of iron oxide-labeled human hematopoietic progenitor cells in athymic mice. MATERIALS AND METHODS: This study was approved by the ethical committee, and all women had given consent to donate umbilical cord blood for research. Twenty athymic female Balb/c mice underwent MR imaging before and 1, 4, 24, and 48 hours after intravenous injection of (1-3) x 10(7) human hematopoietic progenitor cells labeled with the superparamagnetic iron oxide particles ferumoxides through simple incubation (n = 10) or P7228 through lipofection (n = 10). Fifteen female Balb/c control mice were examined after intravenous injection of the pure contrast agents (n = 6 for both probes) or nonlabeled cells (n = 3). Signal intensities of liver, spleen, and bone marrow on MR images obtained before and after injection were measured and compared for significant differences by using the t test. MR imaging data were compared with the results of immunostaining against human CD31(+) cells and against the coating of the contrast agents; these results served as the standard of reference. RESULTS: Ferumoxides was internalized into more mature CD34(-) cells but not into CD34(+) stem cells, while P7228 liposomes were internalized into both CD34(-) and CD34(+) cells. After injection of iron oxide-labeled hematopoietic cells, a significant decrease in MR signal intensity was observed in liver and spleen at 1, 4, 24, and 48 hours after injection (P < .05) and in the bone marrow at 24 and 48 hours after injection (P < .05). The signal intensity decrease in bone marrow was significantly stronger after injection of iron oxide-labeled cells compared to controls that received injections of the pure contrast agent (P < .05). Results of histopathologic examination confirmed homing of iron oxide-labeled human progenitor cells in the murine recipient organs. CONCLUSION: The in vivo distribution of intravenously administered iron oxide-labeled hematopoietic progenitor cells can be monitored with 1.5-T MR imaging equipment.

Animals↗

PubMed2004

In vivo tracking of genetically engineered, anti-HER2/neu directed natural killer cells to HER2/neu positive mammary tumors with magnetic resonance imaging.

The purpose of this study is to optimize labeling of the human natural killer (NK) cell line NK-92 with iron-oxide-based contrast agents and to monitor the in vivo distribution of genetically engineered NK-92 cells, which are directed against HER2/neu receptors, to HER2/neu positive mammary tumors with magnetic resonance (MR) imaging. Parental NK-92 cells and genetically modified HER2/neu specific NK-92-scFv(FRP5)-zeta cells, expressing a chimeric antigen receptor specific to the tumor-associated ErbB2 (HER2/neu) antigen, were labeled with ferumoxides and ferucarbotran using simple incubation, lipofection and electroporation techniques. Labeling efficiency was evaluated by MR imaging, Prussian blue stains and spectrometry. Subsequently, ferucarbotran-labeled NK-92-scFv(FRP5)-zeta (n=3) or parental NK-92 cells were intravenously injected into the tail vein of six mice with HER2/neu-positive NIH 3T3 mammary tumors, implanted in the mammary fat pad. The accumulation of the cells in the tumors was monitored by MR imaging before and 12 and 24 h after cell injection (p.i.). MR data were correlated with histopathology. Both the parental NK-92 and the genetically modified NK-92-scFv(FRP5)-zeta cells could be labeled with ferucarbotran and ferumoxides by lipofection and electroporation, but not by simple incubation. The intracellular cytoplasmatic iron-oxide uptake was significantly higher after labeling with ferucarbotran than ferumoxides (P<0.05). After intravenous injection of 5 x 10(6) NK-92-scFv(FRP5)-zeta cells into tumor-bearing mice, MR showed a progressive signal decline in HER2/neu-positive mammary tumors at 12 and 24 h (p.i.). Conversely, injection of 5 x 10(6) parental NK-92 control cells, not directed against HER2/neu receptors, did not cause significant signal intensity changes of the tumors. Histopathology confirmed an accumulation of the former, but not the latter cells in tumor tissue. The human natural killer cell line NK-92 can be efficiently labeled with clinically applicable iron-oxide contrast agents, and the accumulation of these labeled cells in murine tumors can be monitored in vivo with MR imaging. This MR cell tracking technique may be applied to monitor NK-cell based immunotherapies in patients in order to assess the presence and extent of NK-cell tumor accumulations and, thus, to determine therapy response early and non-invasively.

Analysis of Variance↗

PubMed2004

Serial induction of mutations by ethylnitrosourea in PC12 cells: a new model for a phenotypical characterization of the neurotoxic response to 6-hydroxydopamine.

Here we show that the serial generation of allelic mutations by treatment with the mutagen ethylnitrosourea (ENU) in PC12 cells provides a new model for the phenotypical dissection of biological properties. We tested this approach in the neurotoxic 6-OHDA model of Parkinson's disease in PC12 cells which has been widely used as an in vitro model for the investigation of the molecular pathogenesis of neuronal cell death and for novel treatment approaches. ENU treatment at doses of 0.2 and 0.3 mg/ml for 1 h resulted in 35 and 25% surviving PC12 cells, respectively, which showed mutation frequencies of approximately 10 mutations per genome. Clones derived from single ENU treated PC12 cells showed marked differences in their resistance against 6-OHDA. The phenotypical analysis of resistant and sensitive clones showed a differential transcriptional regulation of multiple genes. The applicability of this approach could be demonstrated by the identification of the rat TM9SF1 gene coding for a transmembrane protein of the nonaspanin superfamily as a regulated gene in PC12 clones resistant against 6-OHDA. Our data demonstrate the suitability of this model for the investigation of the molecular pathogenesis of neurodegeneration and for high-throughput analysis, e.g. for drug discovery.

Animals↗

PubMed2004

Cell tracking with gadophrin-2: a bifunctional contrast agent for MR imaging, optical imaging, and fluorescence microscopy.

The purpose of this study was to assess the feasibility of use of gadophrin-2 to trace intravenously injected human hematopoietic cells in athymic mice, employing magnetic resonance (MR) imaging, optical imaging (OI), and fluorescence microscopy. Mononuclear peripheral blood cells from GCSF-primed patients were labeled with gadophrin-2 (Schering AG, Berlin, Germany), a paramagnetic and fluorescent metalloporphyrin, using established transfection techniques with cationic liposomes. The labeled cells were evaluated in vitro with electron microscopy and inductively coupled plasma atomic emission spectrometry. Then, 1x10(6)-3x10(8) labeled cells were injected into 14 nude Balb/c mice and the in vivo cell distribution was evaluated with MR imaging and OI before and 4, 24, and 48 h after intravenous injection (p.i.). Five additional mice served as controls: three mice were untreated controls and two mice were investigated after injection of unlabeled cells. The contrast agent effect was determined quantitatively for MR imaging by calculating signal-to-noise-ratio (SNR) data. After completion of in vivo imaging studies, fluorescence microscopy of excised organs was performed. Intracellular cytoplasmatic uptake of gadophrin-2 was confirmed by electron microscopy. Spectrometry determined an uptake of 31.56 nmol Gd per 10(6) cells. After intravenous injection, the distribution of gadophrin-2 labeled cells in nude mice could be visualized by MR, OI, and fluorescence microscopy. At 4 h p.i., the transplanted cells mainly distributed to lung, liver, and spleen, and 24 h p.i. they also distributed to the bone marrow. Fluorescence microscopy confirmed the distribution of gadophrin-2 labeled cells to these target organs. Gadophrin-2 is suited as a bifunctional contrast agent for MR imaging, OI, and fluorescence microscopy and may be used to combine the advantages of each individual imaging modality for in vivo tracking of intravenously injected hematopoietic cells.

Animals↗

PubMed2004

Gene expression profiles derived from single cells in human postmortem brain.

The study of postmortem human brain tissue remains the basis for the understanding of many CNS disorders and to verify data obtained in experimental studies. So far, however, gene expression profiling in cellular sub-populations derived from human postmortem brain was hampered by several technical drawbacks. Here, we describe a method that allows the generation of mRNA expression profiles from single neurons. Dopaminergic neurons from different midbrain areas including substantia nigra, central gray substance and ventral tegmental area were identified and isolated by immuno-laser capture microscopy (LCM). Expression profiles were generated from microdissected cells using a modified RNA fingerprinting protocol. Using this approach, we were able to generate specific RNA fingerprints at a high resolution from phenotype-specific single neurons. Polymorphic fragments were isolated from gels and differential gene expression was confirmed by real-time PCR using gene-specific primer pairs and hybridization probes. The method described here is easy to use and reliable for profiling gene expression at the single cell level in human postmortem brain. It could therefore be valuable to open new insights into the molecular pathogenesis of CNS disorders.

Aged↗

PubMed2004

Transcriptional up-regulation of Gadd45a in Patched-associated medulloblastoma.

Medulloblastoma (MB) is a highly malignant embryonal tumor of the cerebellum with a preferential manifestation in children. Although the majority of MBs occur sporadically, this tumor is also associated with familial cancer syndromes including the nevoid basal cell carcinoma or Gorlin syndrome. Mutations in the tumor suppressor gene PATCHED 1 (PTCH1) have been described in both familial and sporadic cases and inactivation of one Patched 1 (Ptch1) allele in mice promotes development of MB. In order to determine candidate genes involved in tumorigenesis of MB, we have screened tumors of heterozygous Ptch1 mice for differentially expressed genes by means of cDNA microarray technology. Our data show that genes involved in cell cycle, signal transduction and metastasis are transcriptionally up-regulated in MB compared to normal cerebellum. Gene ontology analysis reveals cell cycle regulators to be the predominant functional gene class altered in MB of Ptch1 mutants, including D-type cyclins and cyclin-dependent kinase 4. We furthermore describe that overexpression of the growth arrest and DNA-damage-inducible gene Gadd45a is common in Ptch1-associated tumors and Ptch1 null embryos. These results suggest that cDNA microarray technology is a useful tool to discover genes involved in the development of MB that arise in response to a persistent activation of sonic hedgehog (Shh) signaling. This approach may provide novel data for diagnosis, treatment and prevention of human PTCH1-related malignancies.

Animals↗

PubMed2003

Targeting of hematopoietic progenitor cells with MR contrast agents.

PURPOSE: To label human hematopoietic progenitor cells with various magnetic resonance (MR) imaging contrast agents and to obtain 1.5-T MR images of them. MATERIALS AND METHODS: Hematopoietic progenitor cells, labeled with ferumoxides, ferumoxtran, magnetic polysaccharide nanoparticles-transferrin, P7228 liposomes, and gadopentetate dimeglumine liposomes underwent MR imaging with T1- and T2-weighted spin-echo and fast field-echo sequences. Data were analyzed by measuring MR signal intensities and R1 and R2* relaxation rates of labeled cells and nonlabeled control cells. Mean quantitative data for the various contrast agent groups were assessed for significant differences compared with control cells by means of the Scheffe test. As a standard of reference, MR imaging data were compared with electron microscopic and spectrometric data. RESULTS: For all contrast agents, intracellular cytoplasm uptake was demonstrated with electron microscopy and was quantified with spectrometry. When compared with nonlabeled control cells, progenitor cells labeled with iron oxides showed significantly (P <.05) increased R2*. Cells labeled with gadopentetate dimeglumine liposomes showed significantly increased R1. Detection thresholds were 5 x 10(5) cells for gadopentetate dimeglumine liposomes and ferumoxtran, 2.5 x 10(5) cells for ferumoxides and P7228 liposomes, and 1 x 10(5) cells for magnetic polysaccharide nanoparticles-transferrin. CONCLUSION: Hematopoietic progenitor cells can be labeled with MR contrast agents and can be depicted with a standard 1.5-T MR imager.

Cell Survival↗

PubMed2003

Highly efficient paramagnetic labelling of embryonic and neuronal stem cells.

Recent developments in stem cell and gene therapy will require methods to monitor stem cell survival and integration repeatedly and non-invasively with a high temporal and spatial resolution in vivo. The aim of this study was to visualise embryonic and neuronal stem cells with standard contrast agents using a conventional clinical 1.5-Tesla scanner. We therefore modified standard transfection protocols including lipofection (Lipofectin and Lipofectamine) and calcium phosphate transfection for the efficient uptake of paramagnetic particles [gadolinium-diethylene triamine penta-acetic acid (Gd-DTPA)] in stem cells. Using this approach we obtained intracellular labelling efficiencies of up to 83%. Neither the proliferation capacity nor the differentiation efficiency was affected. Identical differentiation of labelled and unlabelled embryonic and neuronal cells was observed. The established labelling techniques used in this study displayed high labelling efficiencies in embryonic and neuronal stem cells without any alterations of cellular biology; therefore this approach might be a suitable method for targeting stem cells.

Animals↗

PubMed2003

Molecular characterization of Patched-associated rhabdomyosarcoma.

Mutations in the human homologue of Drosophila Patched1 (PTCH1) have been found in several common tumours including basal cell carcinoma, medulloblastoma, and rhabdomyosarcoma (RMS). Medulloblastoma and RMS are also present in the murine model for Ptch1 deficiency. Tumours in heterozygous Ptch1(neo67/+) mice consistently exhibit elevated transcript levels of the proto-oncogene Gli1, of Ptch1 itself, and of the insulin-like growth factor 2 (Igf2). The present study has investigated additional molecular changes in RMSs of Ptch1 mutant mice by means of microarray analysis and protein expression analysis. The data show activation of the cell survival-promoting Akt/protein kinase B (Pkb). Furthermore, RMSs express increased levels of the anti-apoptotic protein Bcl-2 and of genes and proteins known to inhibit cell proliferation, including Gadd45a and p27kip1. Taken together, the data suggest that the formation of RMSs in Ptch1 mutants is associated with the ability of tumour cells to resist apoptosis.

Animals↗

PubMed2003

Up-regulation of macrophage migration inhibitory factor gene and protein expression in glial tumor cells during hypoxic and hypoglycemic stress indicates a critical role for angiogenesis in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most malignant variant of human glial tumors. A prominent feature of this tumor is the occurrence of necrosis and vascular proliferation. The regulation of glial neovascularization is still poorly understood and the characterization of factors involved in this process is of major clinical interest. Macrophage migration inhibitory factor (MIF) is a pleiotropic cytokine released by leukocytes and by a variety of cells outside of the immune system. Recent work has shown that MIF may function to regulate cellular differentiation and proliferation in normal and tumor-derived cell lines, and may also contribute to the neovascularization of tumors. Our immunohistological analysis of MIF distribution in GBM tissues revealed the strong MIF protein accumulation in close association with necrotic areas and in tumor cells surrounding blood vessels. In addition, MIF expression was frequently associated with the presence of the tumor-suppressor gene p53. To substantiate the concept that MIF might be involved in the regulation of angiogenesis in GBM, we analyzed the MIF gene and protein expression under hypoxic and hypoglycemic stress conditions in vitro. Northern blot analysis showed a clear increase of MIF mRNA after hypoxia and hypoglycemia. We could also demonstrate that the increase of MIF transcripts on hypoxic stress can be explained by a profound transcriptional activation of the MIF gene. In parallel to the increase of MIF transcripts, we observed a significant rise in extracellular MIF protein on angiogenic stimulation. The data of our preliminary study suggest that the up-regulation of MIF expression during hypoxic and hypoglycemic stress might play a critical role for the neovascularization of glial tumors.

Blotting, Western↗

PubMed2002

Motility enhancement by tumor-derived mutant E-cadherin is sensitive to treatment with epidermal growth factor receptor and phosphatidylinositol 3-kinase inhibitors.

Diffuse-type gastric and lobular breast cancers are characterized by frequent mutations in the cell adhesion molecule E-cadherin. Here we report that tumor-associated mutations of E-cadherin enhanced random cell movement of transfected MDA-MB-435S mammary carcinoma cells as compared to wild-type (wt) E-cadherin-expressing cells. The mutations included in frame deletions of exons 8 or 9 and a point mutation in exon 8 which all affect putative calcium-binding sites within the linker region of the second and third extracellular domain. Motility enhancement by mutant E-cadherin was investigated by time-lapse laser scanning microscopy. Increased cell motility stimulated by mutant E-cadherin was influenced by cell-matrix interactions. The motility-increasing activity of mutant E-cadherin was blocked by application of pharmacological inhibitors of epidermal growth factor receptor and phosphatidylinositol (PI) 3-kinase. Investigation of the activation status of PI 3-kinase and the downstream signaling molecules Akt/protein kinase B and MAP kinase p44/42 showed that these kinases are not more strongly activated in mutant E-cadherin-expressing cells than in wt E-cadherin-expressing cells. Instead, the basal level of PI 3-kinase is necessary for mutant E-cadherin-enhanced cell motility. Our data suggest a critical role of E-cadherin mutations for the fine tuning of tumor cell motility.

Breast Neoplasms↗

PubMed2002

Differential expression of Notch genes in human osteoblastic cells.

Notch receptors participate in a conserved signaling pathway that controls the development of diverse tissues and cell types. In the present study we investigated the expression of four Notch genes in primary human osteoblasts and in human osteoblastic osteosarcoma cell line SaOS-2 by RT-PCR. We found a strong constitutive expression of Notch-1 and a weak constitutive expression of Notch-2 in both cell types. After stimulation with Dexamethasone or Vitamin D(3), two factors known to induce differentiation in osteogenic cells, both Notch receptors were downregulated, however, with a different time course. Notch-1 and Notch-2 showed a transient induction after 2 days and a decrease after 7 days in osteoblasts and after 28 days in SaOS-2 cells. Notch-4 expression could only be detected after stimulation with Dexamethasone and Vitamin D(3). However, in osteoblasts a transient induction after 2 days could be detected in osteoblasts, whereas Notch-4 expression increased after 14 and 28 days in SaOS-2 cells. In contrast, Notch-3 was not expressed in human osteoblasts and SaOS-2 osteosarcoma cells. These data show, that Notch genes are expressed in human osteoblastic cells and that the expression is differentially regulated upon stimulation with osteogenic factors.

Cell Differentiation↗

PubMedDate not supplied

Activation of the anti-apoptotic Akt/protein kinase B pathway in human malignant gliomas in vivo.

In the present study we investigated two important signal transduction pathways in glioma biopsies. By Western analysis we found an overexpression of the epidermal growth factor receptor in 10 out of 27 (37%) glioblastoma multiforme (GBM), but not in astrocytomas WHO II/III which demonstrated only weak or absent expression. Only two GBM (8%) but none of the astrocytomas WHO II/III exhibited loss of PTEN expression. Activation of Akt/protein kinase B showed a close correlation with EGF receptor overexpression in human malignant gliomas since 6 out of 7 GBMs with high degrees of protein kinase B activation exhibited overexpression of the EGF receptor. In contrast, no significant differences in MAP kinase activation could be detected between individual GBMs. Our data show that EGF receptor overexpression seems to be responsible for activation of the protein kinase B whereas PTEN deletion seems to play a minor role in the dysregulation of this important pathway in human GBM in vivo.

Adult↗

PubMedDate not supplied

Serial proton MR spectroscopy and diffusion imaging findings in HIV-related herpes simplex encephalitis.

We report the case of pathologically proved atypical herpes simplex encephalitis (HSE) in a 40-year-old male patient with AIDS who was followed up by MR imaging, which included diffusion-weighted imaging and proton MR spectroscopy ((1)H-MRS). MR revealed sparing of hippocampi and limbic cortices, necrosis of both cingulate gyri, and cerebellar involvement. Increased diffusivity and severe metabolic alterations were compatible with biopsy findings of necrotizing inflammation. Clinical recovery corresponded with partial metabolite and diffusion normalization and a myo-inositol increase that indicated evolving gliosis formation further corroborated by immunohistochemistry results. (1)H-MRS and diffusion-weighted imaging may both support the diagnosis of HSE in patients with AIDS and help in the follow-up of necrotizing inflammation.

Adult↗