Dequalinium vesicles form stable complexes with plasmid DNA which are protected from DNase attack.

Upon sonication, the antimicrobial and antineoplastic compound dequalinium forms vesicles (DQAsomes, Weissig et al., 1998). Dequalinium (1,1'-(1,10-decamethylene-bis-[aminoquinaldinium])-chloride) was shown to be a fluorophore with an emission maximum at 366 nm. Addition of DNA results in a characteristic quenching of its intrinsic fluorescence. After density gradient centrifugation a band of dequalinium (DQA) tightly associated with DNA is located between the DNA and DQA bands. DQA/DNA-complexes containing plasmid DNA at a molar ratio of DQA/DNA 6:1 are completely protected against DNase activity. Addition of negatively-charged lipids release intact DNA in the same manner as from cationic lipid/DNA complexes. As regards biological effects, DQAsomes show a differential cytotoxicity for normal and sarcoma cell lines. In vitro incubation with fluorescein-labeled oligodeoxynucleotides (5'-fluorescein-[GATC]5) showed an increased uptake of the tagged oligodeoxynucleotide if complexed with dequalinium. We hypothesize that the DQA/DNA complexes are well-suited for 'DQAsomal gene transfer' in vitro and in vivo. Noteworthy, they display an intrinsic antitumor activity manifested by differential cytotoxicity for normal and sarcoma cells.

Interaction of UV light-induced alpha-tocopherol radicals with lipids detected by an electron spin resonance prooxidation effect.

The reaction rate constants of the interaction between light-induced alpha-tocopherol radicals with unsaturated lipids in a heterogeneous system compared to a homogeneous system are of the same order of magnitude. The decay rates of compartmentalized alpha-tocopherol radicals were significantly reduced by using negatively charged sodium dodecyl sulfate (SDS) micelles. A partially resolved electron spin resonance (ESR) hyperfine structure was observed under the conditions of both high lipid concentrations in comparison to the alpha-tocopherol concentration and of a regular distribution of alpha-tocopherol molecules inside the heterogeneous lipid structures. Alpha-tocopherol radicals have a considerable prooxidation potential at higher concentrations. Ascorbic acid dissolved in the aqueous medium provokes very fast alpha-tocopherol radical recycling through the boundary layer between the aqueous medium and micelles. By contrast, very slow reactions such as those of alpha-tocopherol radicals with glutathione through this boundary layer are measurable. Despite using the heterogeneous SDS micellar system, the decay kinetics of the alpha-tocopherol radical ESR signal is simply compounded. In addition to the known stabilization effect of cholesterol in membrane systems, cholesterol itself acts as a target molecule attacked by free radicals, e.g. alpha-tocopherol radicals. Using stratum corneum extracts that contain unsaturated lipids and cholesterol the alpha-tocopherol radical can prooxidatively react with these compounds. Using focused UV light generates a high radical yield in a relatively short time compared to the lifetime of the alpha-tocopherol radicals. The decay processes after radical induction can be characterized as consecutive reactions. The compartmentalization of radicals induced in SDS micelles and the close proximity of target molecules are essential if very slow one-electron reductions are to be measured.

DQAsomes: a novel potential drug and gene delivery system made from Dequalinium.

PURPOSE: Dequalinium, a drug known for over 30 years, is a dicationic amphiphile compound resembling bolaform electrolytes. The purpose of our work was to determine the state of aggregation of dequalinium in aqueous medium and to investigate both, its ability to bind DNA and its potential to serve as a novel non-viral transfection vector. METHODS: The form of aggregation was determined employing electron microscopic techniques. The DNA binding capacity of dequalinium was assayed using SYBR Green I stain. For in vitro cell transfection experiments plasmid DNA encoding for firefly luciferase was used. RESULTS: Dequalinium forms in aqueous medium liposome-like aggregates, which we term DQAsomes. These dequalinium vesicles bind DNA and they are able to transfect cells in vitro with an efficiency comparable to Lipofectin. CONCLUSIONS: Based on the intrinsic properties of dequalinium such as the in vivo selectivity for carcinoma cells and selective accumulation in mitochondria we propose DQAsomes as a novel and unique drug and gene delivery system.

[Liposomal DNA transfection of human sarcoma cells with p53 alterations]. / Liposomale DNA-Transfektion humaner Sarkomzellen mit p53-Alterationen.

An vital assay allows to optimize liposomal transfection for human tumor cells via FACS. Various cationic lipids were tested to analyse the reporter gene expression (green fluorescent protein, GFP) in different soft tissue sarcoma (STS) cells with known genetic alterations. Furthermore, the cellular uptake of fluorescence-labeled oligodeoxynucleotides (ODN's) was determined. The results obtained with two self-established sarcoma cell lines (LMS6-93, US8-93) were compared with ATCC sarcoma cell lines (Saos-2, A-204, RD) and fibroblast cells. We found maximal 37% cells expressing GFP 24 h post-transfection. All mesenchymal (tumor) cells but not fibroblast cells could be transfected in a cell-specific and lipid-dependent manner. In kinetic studies highest transfection rates were determined between 24 and 48 h, whereas the GFP expression is downregulated after 72 h. Furthermore, we found transfectability is p53 mutation-independent and a relative low toxicity of the new lipids (Lipotaxi and Clonfectin) in comparison to other lipids (Lipofectin, Lipofectamine). By a cell sorting system sarcoma cell lines expressing the reporter gene could be enriched up to 84% of the living cell population. Labeled ODN's were taken up more efficiently (> 90%) when they were mixed with lipids before, but ODN's alone were incorporated into sarcoma cells only in a low percentage (< 10%) and concentration. STS cell cultures showed also a relative high ODN uptake compared with cell lines. We propose the liposomal transfection strategy as an efficient method which can be applied to adherent-growing tumor cells. The method allows simultaneously to study transfection rates, apoptosis and cell cycle alterations in vitro. Furthermore, in future, extension on ex vivo and in vivo transgene expression (xenotransplanted sarcomas) will be evaluated.

[Human herpesviruses 6 and 7. Basic principles and possible significance for dermatology]. / Die humanen Herpesviren 6 und 7. Grundlagen und mögliche Bedeutung für die Dermatologie.

Primary infection with human herpesviruses 6 and 7 (HHV-6 and HHV-7) during early childhood causes permanent latent infection, usually without any ill effects; only a small percentage of primary infections will lead to exanthem subitum. Like other herpesviruses. HHV-6 and HHV-7 can be reactivated at any time if host defence mechanisms become defective (e.g. in transplant recipients, AIDS, tumour patients). HHV-6 can be reactivated under such conditions and cause a variety of clinical problems, such as exanthems along with interstitial pneumonia or hepatitis for example. In addition, the reactivated virus may influence the course of autoimmune and proliferative diseases such as systemic lupus erythematosus and Hodgkin's disease. While, HHV-7 may be associated with similar disorder, more systematic studies are needed to clarify the clinical implications and the pathogeetic mechanisms of both viruses.

[Apoptosis and cell proliferation in HHV-6 infections. Regulatory mechanisms of p53/bcl-2/ras interactions]. / Apoptose und Zellproliferation bei HHV-6-Infektionen. Regulationsmechanismen über p53/bcl-2/ras-Interaktionen.

HHV-6 infected immature T (HSB2) and Hodgkin (HDLM2) cells and biopsy tissues from lymph nodes of patients with Hodgkin's disease (HD) and Kikuchi lymphadenitis (KL) were studied immunohistologically for virus antigen expression and for the oncogene/anti-oncogene products ras, bcl-2 and p53. Cell proliferation and cell death were tentatively monitored in tissue culture by PCNA staining, by viability testing and in situ end labeling of fragmented DNA. PCNA was also used in biopsy samples. KL is characterized by high incidences of focal cell death (i.e. histiocytic necrotizing lymphadenitis), while HD is apparently more a proliferative disease. The techniques used revealed no significant differences in the cellular expression of viral DNA or antigens among cell lines, HD or KL. The HDLM2 cell line with the superior survival after HHV-6 infection showed a significantly lower expression of p53 and PCNA than HSB2 cells. Biopsy samples from patients with KL did not express p53, and ras and PCNA were observed in fewer cells than in HD. Bcl-2, however, was significantly more frequently seen than in HD. The interpretation of the data is difficult; they suggest that there are additional regulatory influences in control of cell proliferation and cell death, such as cytokines and growth factors, which are altered after viral infection. Also, virus-induced cell death probably includes other mechanisms besides apoptosis, such as cell damage caused by oxygen radicals.