Preferential liver gene expression with polypropylenimine dendrimers.

Previously, the lower generation (DAB 8-generation 2 and DAB 16-generation 3) polypropylenimine dendrimers have been shown to be effective gene delivery systems in vitro. In the current work, we sought to: (a) test the effect of the strength of the carrier, DNA electrostatic interaction on gene transfer and (b) to study the in vivo gene transfer activity of these low molecular weight (<1687 Da) non-amphiphilic plain and quaternary ammonium gene carriers. Towards this aim, methyl quaternary ammonium derivatives of DAB 4 (generation 1), DAB 8, DAB 16 and DAB 32 (generation 4) were synthesised to give Q4, Q8, Q16 and Q32, respectively. Quaternisation of DAB 8 proved to be critical in improving DNA binding, as evidenced by data from the ethidium bromide exclusion assay and dendrimer-DNA colloidal stability data. This improved colloidal stability had a major effect on vector tolerability, as Q8-DNA formulations were well tolerated on intravenous injection while a similar DAB 8-DNA dose was lethally toxic by the same route. Quaternisation also improved the in vitro cell biocompatibility of DAB 16-DNA and DAB 32-DNA dendrimer complexes by about 4-fold but not that of the lower generation DAB 4-DNA and DAB 8-DNA formulations. In contrast to previous reports with non-viral gene delivery systems, the intravenous administration of DAB 16-DNA and Q8-DNA formulations resulted in liver targeted gene expression as opposed to the lung targeted gene expression obtained with the control polymer-Exgen 500 [linear poly(ethylenimine)] and a lung avoidance hypothesis is postulated. We conclude that the polypropylenimine dendrimers are promising gene delivery systems which may be used to target the liver and avoid the lung and also that molecular modifications conferring colloidal stability on gene delivery formulations have a profound effect on their tolerability on intravenous administration.     

Review of novel particulate antigen delivery systems with special focus on treatment of type I allergy.

For the treatment of infectious diseases, cancer and allergy, the directed induction of an appropriate immune response is the ultimate goal. Therefore, with the development of pure, often very small proteins, peptides or DNA by molecular biology techniques, the research for suitable adjuvants or delivery systems became increasingly important. Particle formulations are made of a variety of materials, including lipids, proteins or amino acids, polysaccharides, polyacrylic substances or organic acids. Microparticles serve as vehicles and provide a depot for the entrapped or coupled antigen. The release occurs in a pulsatile or continuous manner, a feature, which is well controllable for many particulate systems. Particles attract antigen presenting cells to the administration site, thereby guaranteeing the efficient presentation of the antigen to the immune system. Importantly, particles also protect the entrapped substance. This is especially necessary after oral application to avoid gastric or tryptic breakdown. In this article, the design and construction of different antigen delivery systems and their immune effects, with special focus on the suitability for allergy treatment, are discussed.     

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Das Jodoform-Lapisätzmittel in der Hauttherapie

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Detergent fractionation with subsequent subtractive suppression hybridization as a tool for identifying genes coding for plasma membrane proteins

Abstract:  The identification of tumor-specific proteins located at the plasma membrane is hampered by numerous methodological pitfalls many of which are associated with the post-translational modification of such proteins. Here, we present a new combination of detergent fractionation of cells and of subtractive suppression hybridization (SSH) to gain overexpressed genes coding for membrane-associated or secreted proteins. Fractionation of subcellular components by digitonin allowed sequestering mRNA of the rough Endoplasmatic reticulum and thereby increasing the percentage of sequences coding for membrane-bound proteins. Fractionated mRNAs from the cutaneous T-cell lymphoma (CTCL) cell line HuT78 and from normal peripheral blood monocytes were used for SSH leading to the enrichment of sequences overexpressed in the tumor cells. We identified some 21 overexpressed genes, among them are GPR137B, FAM62A, NOMO1, HSP90, SLIT1, IBP2, CLIF, IRAK and ARC. mRNA expression was tested for selected genes in CTCL cell lines, skin specimens and peripheral blood samples from CTCL patients and healthy donors. Several of the detected sequences are clearly related to cancer, but have not yet been associated with CTCL. qPCR confirmed an enrichment of these mRNAs in the rough endoplasmic reticulum fraction. RT-PCR confirmed the expression of these genes in skin specimens and peripheral blood of CTCL patients. Western blotting verified protein expression of HSP90 and IBP2 in HuT78. GPR137B could be detected by immunohistology in HuT78 and in keratinocytes of dysplastic epidermis, but also in sweat glands of healthy skin. In summary, we developed a new technique, which allows identifying overexpressed genes coding preferentially for membrane-associated proteins.     

A novel GalR2-specific peptide agonist

The galanin peptide family and its three receptors have with compelling evidence been implicated in several high-order physiological disorders. The co-localization with other neuromodulators and the distinct up-regulation during and after pathological disturbances has drawn attention to this neuropeptide family. In the current study we present data on receptor binding and functional response for a novel galanin receptor type 2 (GalR2) selective chimeric peptide, M1145 [(RG)₂-N-galanin(2-13)-VL-(P)₃-(AL)₂-A-amide]. The M1145 peptide shows more than 90-fold higher affinity for GalR2 over GalR1 and a 76-fold higher affinity over GalR3. Furthermore, the peptide yields an agonistic effect in vitro, seen as an increase in inositol phosphate (IP) accumulation, both in the absence or the presence of galanin. The peptide design with a N-terminal extension of galanin(2-13), prevails new insights in the assembly of novel subtype specific ligands for the galanin receptor family and opens new possibilities to apply the galanin system as a putative drug target.     

Involvement of ADAM10 in axonal outgrowth and myelination of the peripheral nerve

The disintegrin and metalloproteinase 10 (ADAM10) is a membrane‐anchored metalloproteinase with both proteolytic and disintegrin characteristics. Here, we investigate the expression, regulation, and functional role of ADAM10 in axonal outgrowth and myelination of the peripheral nerve. Expression pattern analysis of 11 ADAM family members in co‐cultures of rat dorsal root ganglia (DRG) neurons and Schwann cells (SCs) demonstrated the most pronounced mRNA expression for ADAM10. In further studies, ADAM10 was found to be consistently upregulated in DRG‐SC co‐cultures before the induction of myelination. Neurons as well as SCs widely expressed ADAM10 at the protein level. In neurons, the expression of ADAM10 was exclusively limited to the axons before the induction of myelination. Inhibition of ADAM10 activity by the hydroxamate‐based inhibitors GI254023X and GW280264X resulted in a significant decrease in the mean axonal length. These data suggest that ADAM10 represents a prerequisite for myelination, although its activity is not required during the process of myelination itself as demonstrated by expression analysis of myelin protein zero (P0) and Sudan black staining. Hence, during the process of myelin formation, ADAM10 is highly upregulated and appears to be critically involved in axonal outgrowth that is a requirement for myelination in the peripheral nerve. © 2009 Wiley‐Liss, Inc.     

Development and evaluation of a case group concept for inpatients with mental disorders in Germany: Using self-report and expert-rated instruments

The aim of this study was to evaluate a case-mix system to classify inpatients with mental disorders in Germany by means of self-report and expert-rated instruments. The use of case-mix systems enhances the transparency of performance and cost structure and can thus improve the quality of mental health care. We analysed a consecutive sample of 1677 inpatients with mental disorders from 11 hospitals using regression tree analysis. The model assigns patients to 17 groups, accounting for 17% of the variance for duration of stay. Patients with eating disorders had a longer duration of stay than patients with anxiety disorder, duration of mental illness of less than 3–5 years, lower levels of interpersonal problems and higher occupational position. The results showed that besides diagnosis, variables such as duration of illness and interpersonal problems are important for classifying inpatients with mental disorders. The results of the study should be critically reviewed regarding the empirical results of other studies and the appropriateness of case group concepts for inpatients with mental disorders.