Noninvasive Visualization of the Activated αvβ3 Integrin in Cancer Patients by Positron Emission Tomography and [18F]Galacto-RGD

BackgroundThe integrin αvβ3 plays an important role in angiogenesis and tumor cell metastasis, and is currently being evaluated as a target for new therapeutic approaches. Several techniques are being studied to enable noninvasive determination of αvβ3 expression. We developed [¹⁸F]Galacto-RGD, a ¹⁸F-labeled glycosylated αvβ3 antagonist, allowing monitoring of αvβ3 expression with positron emission tomography (PET).ConclusionsMolecular imaging with [¹⁸F]Galacto-RGD and PET can provide important information for planning and monitoring anti-angiogenic therapies targeting the αvβ3 integrins and can reveal the involvement and role of this integrin in metastatic and angiogenic processes in various diseases.     

Discovery and optimisation of 1-hydroxyimino-3,3-diphenylpropanes,a new class of orally active GPBAR1 (TGR5) agonists

A series of non-steroidal GPBAR1 (TGR5) agonists was developed from a hit in a high-throughput screening campaign. Lead identification efforts produced biphenyl-4-carboxylic acid derivative (R)-22, which displayed a robust secretion of PYY after oral administration in a degree that can be correlated with the unbound plasma concentration. Further optimisation work focusing on reduction of the lipophilicity provided the 1-phenylpiperidine-4-carboxylic acid derivative (R)-29 (RO5527239), which showed an improved secretion of PYY and GLP-1, translating into a significant reduction of postprandial blood glucose excursion in an oral glucose tolerance test in DIO mice.     

Die Bestimmung des Umladebereiches (isoelektrischer Punkt) von Gewebselementen mit dem Fluorochrom Acridinorange

Zusammenfassung Umladebereich und mittlerer isoelektrischer Punkt (IEP_M) verschiedener menschlicher Gewebe wurden mit Hilfe von gestuften Reihen gepufferter Lösungen vom Fluorochrom Acridinorange auf fluoreszenzmikroskopischem Wege bestimmt. Zum Vergleich wurde an Schnittpräparaten der gleichen Gewebe die Bestimmung von Umladebereich und IEP_M mit den Diachromen Methylenblau und Rubin S durchgeführt. Es zeigte sich, daß das Fluorochrom Acridinorange in der Bestimmung des Umladebereiches und IEP_M den Diachromen überlegen ist. Infolge der höheren Nachweisempfindlichkeit von Acridinorange und seiner Fluoreszenzmetachromasie läßt sich der Umladebereich viel leichter als bei den Hellfeldfarbstoffen einengen und somit genauer bestimmen. Während man bei Verwendung von Diachromen zur exakten Bestimmung des IEP_M zwei Farbstoffe benötigt, genügt bei der Acridinorange Methode ein Farbstoff. Die mit Acridinorange bestimmten Werte vom Umladebereich und IEP_M der einzelnen Gewebselemente liegen meist weiter im sauren Bereich als bei Bestimmung mit Diachromen.Bei dem Vergleich unfixierter, in Alkohol oder Formalin gehärteter Gewebe ergab sich, daß nach Fixierung die untere Grenze vom Umladebereich gegenüber der an unfixiertem Gewebe bestimmten weiter in den sauren Bereich verschoben ist.Kurze Originalmitteilung: Naturwiss. 42, 442 (1955). Für die Durchführung der Untersuchungen standen Mittel der Deutschen Forschungsgemeinschaft zur Verfügung.     

The PI3K/Akt/mTOR Pathway Is Implicated in the Premature Senescence of Primary Human Endothelial Cells Exposed to Chronic Radiation

The etiology of radiation-induced cardiovascular disease (CVD) after chronic exposure to low doses of ionizing radiation is only marginally understood. We have previously shown that a chronic low-dose rate exposure (4.1 mGy/h) causes human umbilical vein endothelial cells (HUVECs) to prematurely senesce. We now show that a dose rate of 2.4 mGy/h is also able to trigger premature senescence in HUVECs, primarily indicated by a loss of growth potential and the appearance of the senescence-associated markers ß-galactosidase (SA-ß-gal) and p21. In contrast, a lower dose rate of 1.4 mGy/h was not sufficient to inhibit cellular growth or increase SA-ß-gal-staining despite an increased expression of p21. We used reverse phase protein arrays and triplex Isotope Coded Protein Labeling with LC-ESI-MS/MS to study the proteomic changes associated with chronic radiation-induced senescence. Both technologies identified inactivation of the PI3K/Akt/mTOR pathway accompanying premature senescence. In addition, expression of proteins involved in cytoskeletal structure and EIF2 signaling was reduced. Age-related diseases such as CVD have been previously associated with increased endothelial cell senescence. We postulate that a similar endothelial aging may contribute to the increased rate of CVD seen in populations chronically exposed to low-dose-rate radiation.     

Functional study of mammalian Neph proteins in Drosophila melanogaster

Neph molecules are highly conserved immunoglobulin superfamily proteins (IgSF) which are essential for multiple morphogenetic processes, including glomerular development in mammals and neuronal as well as nephrocyte development in D. melanogaster. While D. melanogaster expresses two Neph-like proteins (Kirre and IrreC/Rst), three Neph proteins (Neph1-3) are expressed in the mammalian system. However, although these molecules are highly abundant, their molecular functions are still poorly understood. Here we report on a fly system in which we overexpress and replace endogenous Neph homologs with mammalian Neph1-3 proteins to identify functional Neph protein networks required for neuronal and nephrocyte development. Misexpression of Neph1, but neither Neph2 nor Neph3, phenocopies the overexpression of endogenous Neph molecules suggesting a functional diversity of mammalian Neph family proteins. Moreover, structure-function analysis identified a conserved and specific Neph1 protein motif that appears to be required for the functional replacement of Kirre. Hereby, we establish D. melanogaster as a genetic system to specifically model molecular Neph1 functions in vivo and identify a conserved amino acid motif linking Neph1 to Drosophila Kirre function.     

Profiling signalling pathways in formalin-fixed and paraffin-embedded breast cancer tissues reveals cross-talk between EGFR, HER2, HER3 and uPAR

In the last few years, new approaches and developments in patient-tailored cancer therapies have raised the need to select, more precisely, those patients who will respond to personalized treatments. Therefore, the most efficient way for optimal therapy and patient selection is to provide a tumour-specific protein network portrait prior to treatment. The aim of our study was to monitor protein networks in formalin-fixed and paraffin-embedded (FFPE) breast cancer tissues, with special emphasis on epidermal growth factor receptor 2 (HER2)-mediated signalling pathways, to identify and validate new disease markers. For this purpose we used a recently developed technology to extract full-length proteins from FFPE tissues and analysed 23 molecules involved in HER2-related signalling by reverse phase protein microarray (RPPA) in a series of 106 FFPE breast cancer tissue samples. We found a significant correlation of HER2 with human epidermal growth factor receptor 3 (HER3/erbB3), epidermal growth factor receptor 1 (EGFR/HER1/erbB1) and urokinase plasminogen receptor (uPAR) in routinely used FFPE breast cancer tissues. Thus, targeting HER2, EGFR, HER3 and uPAR together may offer a more efficient treatment option for patients with breast cancer.     

Therapy of Pelizaeus-Merzbacher disease in mice by feeding a cholesterol-enriched diet

Duplication of PLP1 (proteolipid protein gene 1) and the subsequent overexpression of the myelin protein PLP (also known as DM20) in oligodendrocytes is the most frequent cause of Pelizaeus-Merzbacher disease (PMD), a fatal leukodystrophy without therapeutic options. PLP binds cholesterol and is contained within membrane lipid raft microdomains. Cholesterol availability is the rate-limiting factor of central nervous system myelin synthesis. Transgenic mice with extra copies of the Plp1 gene are accurate models of PMD. Dysmyelination followed by demyelination, secondary inflammation and axon damage contribute to the severe motor impairment in these mice. The finding that in Plp1-transgenic oligodendrocytes, PLP and cholesterol accumulate in late endosomes and lysosomes (endo/lysosomes), prompted us to further investigate the role of cholesterol in PMD. Here we show that cholesterol itself promotes normal PLP trafficking and that dietary cholesterol influences PMD pathology. In a preclinical trial, PMD mice were fed a cholesterol-enriched diet. This restored oligodendrocyte numbers and ameliorated intracellular PLP accumulation. Moreover, myelin content increased, inflammation and gliosis were reduced and motor defects improved. Even after onset of clinical symptoms, cholesterol treatment prevented disease progression. Dietary cholesterol did not reduce Plp1 overexpression but facilitated incorporation of PLP into myelin membranes. These findings may have implications for therapeutic interventions in patients with PMD.     

Isolation,identification and quantitative evaluation of specific cell types from the mammalian gastric mucosa

Functional in vitro studies with isolated gastric mucosal cells require cytological identification of different cell types in suspension or primary culture. Since suitable techniques have not been well established, different staining methods for the discrimination of dispersed pig and guinea pig gastric cells have been developed on the basis of modified previous protocols for enzymatic cell dispersion. Chief and parietal cells were visualized by combined periodic acid-Schiff stains. Surface mucous and mucous neck cells were identified by affinity-labelling, using lectins with selective staining properties in situ. Two of the lectins were found to be specific markers for gastric polymorphonuclear cells. The following vital tests were found to be useful: succinic dehydrogenase for parietal cells, Nile blue/brilliant cresyl blue stains for chief cells, and different phagocytosis assays for endothelial cells and gastric phagocytes. Endocrine cells were characterized by immunocytochemistry using specific antibodies against gastrin, somatostatin, histamine and serotonin. The same technique using a vimentin antibody was performed for the identification of fibroblasts. Proliferation of mucosal cells in primary culture was monitored by the incorporation of bromo-deoxyuridine, which was subsequently detected by a monoclonal antibody.Some of these results were presented at the 7th International Conference on Experimental Ulcer, Berlin, Germany 1991 (see Giebel and Schwenk 1991)