Search

International audience

Objectives: The epidermal growth factor receptor (EGFR) is overexpressed in many types of human epithelial cancers. Because of its important role in the initiation and progression of tumors, it is an interesting molecular target for new radiotracers. [1] Recently, two short peptides for targeting the EGFR have been reported - the D4 peptide (sequence: LARLLT) and the GE11 peptide (sequence: YHWYGYTPQNVI).[2,3] These short oligopeptides should provide a high specific binding to EGFR-positive tumor cells as well as a rapid tumor uptake and fast blood clearance. We investigated the chemical modification and radiolabeling of the two peptides and examined the application of the D4 peptide as a possible agent for diagnosis and therapy of cancer. Methods: The D4 and the GE11 peptide haven been conjugated with several chelators, e.g. NOTA, DOTA and HYNIC, to allow radiolabeling with suitable radionuclides for diagnostic (e.g. 99mTc, 64Cu) or for therapeutic applications (e.g. 90Y). The properties of the conjugates were modified by the previous insertion of the newly developed hydrophilic, heterobifunctional linker 1. Toxicity tests have been made using a cell viability assay MTS on the EGFR-expressing cell lines A431 (epidermoid carcinoma cells) and FaDu (squamous carcinoma cells). Binding assays were performed on membranes of A431-, FaDu- and non-EGFR-expressing MDA-MB435S cells. Kinetics and saturation assays were conducted using the 64Cu-labeled conjugate 2. First biodistribution PET studies in nude mice have been performed. Results: Conjugation of the two peptides with the new linker 1 followed by the reaction with different chelators gave overall yields of 40-65%. The GE11 peptide itself, as well as all of its conjugates which did not contain the hydrophilic linker 1, were not soluble in water and therefore inapplicable for further experiments. The remaining compounds were labeled with 64Cu and 68Ga (NOTA conjugates), 99mTc (HYNIC conjugates) and 90Y (DOTA conjugates) with specific activities of up to 980 GBq/μmol for 64Cu-2. No change of cell viability was observed after treating A431 and FaDu cells with plain D4 and D4-1 for 4 days at concentrations of up to 125 μM. Binding association with 64Cu-2 on A431 membranes reached equilibrium after one hour, on FaDu membranes equilibrium was not complete after two hours. TDiss½ was less than 5 min. In saturation assays, the affinity determination of 64Cu-2 on A431 and FaDu membranes gave Kd values of 28 ± 6 nM and 72 ± 18 nM, respectively. No specific binding of 64Cu-2 to MDA-MB453S cell membranes was observed. First PET studies with healthy nude mice showed a favorable biodistribution. Conclusions: We successfully prepared and radiolabeled several conjugates of the EGFR-affine peptides D4 and GE11 with satisfying yields and high specific activities. The insertion of the new hydrophilic heterobifunctional linker 1 was necessary to achieve high solubility in water. The preservation of cell viability after D4 and D4-1 treatment indicates no toxic effect of the conjugates. The affinity of 64Cu-2 to the high EGFR-expressing A431 cells and the moderate EGFR-expressing FaDu cells was in the expected range. Binding assays with radiolabeled GE11-conjugates as well as PET studies of GE11-conjugates and 64Cu-2 with tumor-bearing nude mice are currently under investigation. References: [1] Cardo-Vila M, et al. (2010) Proc Natl Acad Sci USA, 107, 5118-23. [2] Song S, et al. (2009) FASEB, 23, 1396-404. [3] Li Z, et al. (2005) FASEB, 19, 1978-85.

Ziel/Aim: Der EGFR wird oft überexprimiert in verschiedenen Tumorarten. Daher werden geeignete radionuklidmarkierte EGFR-Liganden für eine gezielte Tumordiagnostik mittels bildgebender Verfahren, z.B. PET, entwickelt. Zwei kürzlich beschriebene EGFR-spezifische Peptide GE11 (1) und D4 (2) sollten mit Chelatoren sowie Spacern so konjugiert werden, dass sie mit dem PET-Nuklid Kupfer-64 markiert werden können. Mit einem der markierten Peptid-Konjugate sollte das Bindungsverhalten (Zeitabhängigkeit und Affinität) an Zellmembranen charakterisiert werden. Methodik/Methods: Die Peptide wurden am N-Terminus jeweils mit verschiedenen Spacern sowie mit dem Chelator NOTA verknüpft und anschließend mit Kupfer-64 radiomarkiert. Für Bindunggsassays wurden Membranen der EGFR-exprimierenden A431-Zellen (epidermoides Karzinom) und FaDu-Zellen (Plattenepithelkarzinom) untersucht. Kinetik- und Sättigungs-Assays wurden mit einem Kupfer-64 markierten NOTA-D4-Konjugat ([64Cu]Cu-D4-JM) durchgeführt. Ergebnisse/Results: Die Peptidkonjugate konnten mit einer Ausbeute von 75-91% hergestellt werden. Bei der Radiomarkierung mit Kupfer-64 konnten spezifische Aktivitäten von bis zu 1000 GBq/µmol erreicht werden, jedoch wurden diese für Bindungsassays auf etwa 30 GBq/µmol eingestellt. Für die Bindung mit [64Cu]Cu-D4-JM an A431-Membranen war nach 1 Stunde Assoziation das Bindungsgleichgewicht erreicht, an FaDu-Membranen nach 2 Stunden nicht vollständig. TDiss½ lag unter 5 min. Die Affinität mittels Sättigung ergab für A431- und für FaDu-Membranen Kd-Werte von 19 bzw. 42 nM. Schlussfolgerungen/Conclusions: Mit GE11 und D4 konnten verschiedene EGFR-spezifische Peptid-Konjugate erfolgreich hergestellt werden. Nachdem [64Cu]Cu-D4-JM an Zellmembranen untersucht wurde, sind weiterführende Assays an intakten Zellen sowie Tumoraufnahme an Xenograft tragenden Mäusen geplant. Literatur/References: (1) Li Z, Zhao R, Wu X et al. FASEB J. 2005, 19(14), 1978-85 (2) Song S, Liu D, Peng JL, et al. Intl. Conf. on Biomedical and Pharmaceutical Engineering 2006, 290-3

In many insects, mate finding relies on female-released sex pheromones, which have to be deciphered by the male olfactory system within an odorous background of plant volatiles present in the environment of a calling female. With respect to pheromone-mediated mate localization, plant odorants may be neutral, favorable, or disturbing. Here we examined the impact of plant odorants on detection and coding of the major sex pheromone component, (Z)-11-hexadecenal (Z11-16:Ald) in the noctuid moth Heliothis virescens. By in vivo imaging the activity in the male antennal lobe (AL), we monitored the interference at the level of olfactory sensory neurons (OSN) to illuminate mixture interactions. The results show that stimulating the male antenna with Z11-16:Ald and distinct plant-related odorants simultaneously suppressed pheromone-evoked activity in the region of the macroglomerular complex (MGC), where Z11-16:Ald-specific OSNs terminate. Based on our previous findings that antennal detection of Z11-16:Ald involves an interplay of the pheromone binding protein (PBP) HvirPBP2 and the pheromone receptor (PR) HR13, we asked if the plant odorants may interfere with any of the elements involved in pheromone detection. Using a competitive fluorescence binding assay, we found that the plant odorants neither bind to HvirPBP2 nor affect the binding of Z11-16:Ald to the protein. However, imaging experiments analyzing a cell line that expressed the receptor HR13 revealed that plant odorants significantly inhibited the Z11-16:Ald-evoked calcium responses. Together the results indicate that plant odorants can interfere with the signaling process of the major sex pheromone component at the receptor level. Consequently, it can be assumed that plant odorants in the environment may reduce the firing activity of pheromone-specific OSNs in H. virescens and thus affect mate localization.

The aim of the present study was to determine what impact phylogeny and life history might have on the coding of odours in the brain. Using three species of hawk moths (Sphingidae) and two species of owlet moths (Noctuidae), we visualized neural activity patterns in the antennal lobe, the first olfactory neuropil in insects, evoked by a set of ecologically relevant plant volatiles. Our results suggest that even between the two phylogenetically distant moth families, basic olfactory coding features are similar. But we also found different coding strategies in the moths' antennal lobe; namely, more specific patterns for chemically similar odorants in the two noctuid species than in the three sphingid species tested. This difference demonstrates the impact of the phylogenetic distance between species from different families despite some parallel life history traits found in both families. Furthermore, pronounced differences in larval and adult diet among the sphingids did not translate into differences in the olfactory code; instead, the three species had almost identical coding patterns.

This study investigated the discharge practice of a Community Mental Health Team (CMHT) by examining records (electronic and file) of clients discharged between April 2005 and March 2006. Out of a total of 211 discharged clients a random sample of 20 clients was selected to examine the extent to which records and reasons for discharge adhere to current CMHT policies and guidelines. In addition, a sample of clients who had been engaged by the CMHT for 6 months or less was compared to a sample of clients who have been engaged for 1 year or longer to establish whether these differed in sociodemographic characteristics, diagnoses and extent of service provision. The majority of clients discharged during the specified period consisted of clients engaged for 6 months or less. The sampling process revealed that a proportion of these included clients seen for one-off assessments or duty calls, indicating that there is room for improvement to clarify referral criteria (e.g. to GPs) and the role of the CMHT. Similarly, the examination of recording practice also revealed room for improvement in the closing of care packages electronically and inclusion of required information in discharge letters. Almost 50% of clients in the sample were discharged following a decline of any further intervention the reasons for which it will be important to investigate in the form of an audit or survey of service user’s views. Clients engaged for 6 months or less and 1 year or longer seemed to differ mostly in terms of employment rates, diagnosis and previous inpatient admission and mental health act sections. The findings are discussed in relation to the limitations of this study, implications for the service and further research.

A well defined surface layer of a ceramic substrate can be modified by introducing a selected second phase into a melt pool generated locally by a laser beam. CuO, WO3 powders with nano-sized particles were used to alloy alumina and a glass ceramic LTCC (Low Temperature Co-fired Ceramic). Depending on the process parameters the nano-particles were melted during the laser process and solidified during cooling in the ceramic matrix. As a result a composite with complex multiphase microstructure was developed with particle agglomerates, small crystals as well as grains covered with reaction phase, in parts with typical length scales down to the submicron range. Also the geometry of the modified area could be controlled by the process parameters. A significant change of properties could be established for the laser alloyed tracks. Especially the thermal and electrical properties were changed in comparison to that of the ceramic substrate. The developed composites showed a measurable electrical conductivity with a negative temperature coefficient for the resistivity. Therefore, the resistivity decreases with increasing temperature, which is typical for a thermally activated conduction mechanism as in semiconductors. The thermal conductivity could be increased to about 20% for CuO- and up to 70% for WO3-powder compared to the unmodified LTCC-substrate.

14 Group Report: Olfactory Microcircuits R. W. F RIEDRICH , Rapporteur S. F IRESTEIN ,C.G.G ALIZIA ,C.A.G REER ,G.L AURENT , P. - M . L LEDO ,P.M OMBAERTS ,andS.S ACHSE OVERVIEW Most organisms rely on an olfactory system to detect and analyze chemical cues in the environment in the context of essential behaviors. The basic layout of the first processing centers in the olfactory nervous system is remarkably similar in diverse phylogenetic classes, including insects and vertebrates. Chemicals are detected by odorant receptor proteins expressed by olfactory sensory neurons (OSNs), which send an axon to the first processing center in the brain, the olfac- tory bulb in vertebrates and the antennal lobe in insects. OSNs terminate in ana- tomically distinct input modules, the olfactory glomeruli. In all vertebrate and invertebrate species investigated to date, each OSN expresses only one or a few odorant receptors, and each glomerulus receives convergent input from only one type of OSN. Glomeruli are, therefore, considered functional units integrat - ing sensory input from idiotypic afferents. Even simple odors stimulate multiple odorant receptors and thus evoke odor-specific patterns of afferent activity across the array of glomeruli. Within glomeruli, OSNs make excitatory synap - ses onto the output neurons, the mitral cells in vertebrates, and projection neu - rons in insects, as well as with local inhibitory interneurons. As a result of synap - tic interactions within this network, the output of a given projection neuron is not simply determined by the sensory input to the glomeruli it innervates, but also by the activity of inputs channeled through other glomeruli. In addition, synaptic interactions temporally pattern olfactory bulb/antennal lobe output ac - tivity on at least two timescales. It is currently debated how odor information is encoded in the olfactory bulb/antennal lobe, and how neuronal circuits process odor information conveyed by sensory afferents. Moreover, the development and plasticity of olfactory circuits are only beginning to be elucidated. These issues are of particular interest because OSNs and interneurons in the olfactory bulb undergo continuous turnover throughout life in vertebrates