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Biomedical subjects

W Zauner

Publications and source records attributed to W Zauner.

At least 19 recordsLinked to original sources
PubMed2002

Improvement of the enzymatic stability of a cytotoxic T-lymphocyte-epitope model peptide for its oral administration.

Oral administration of peptide antigens, to provide proper mucosal and/or systemic immunity, is largely ineffective. This is mainly due to the very small quantity of antigen that survives degradation in the intestine and that crosses the intestinal absorption membrane. The present study focuses on the improvement of the enzymatic stability of a 13 amino acid long peptide containing a cytotoxic T-lymphocytes (CTL)-epitope. Within this study, it is shown, that simple chemical modification at the N- and C-terminus of the peptide can provide significant stability towards enzymatic attack by intestinal exopeptidases. Around 50% of the modified peptide resisted enzymatic attack on native porcine intestinal mucosa within 3h of incubation at pH 6.8 and 37 degrees C, whereas unmodified control peptide was almost completely degraded within the same time period. Additionally, a mucoadhesive drug carrier matrix with specific inhibitory properties towards luminally secreted endopeptidases has been generated. The incorporation of the simply modified peptide in this delivery system should enhance the amount of biologically active antigen being available at the mucosal site for further presentation to immunomodulating systems. This might open the door for a successful oral immunotherapy.

Administration, Oral↗
PubMed2002

[Diabetes Infobus 1999. General facts].

In Austria in 1999, a Diabetes screening program was carried out by the Austrian Diabetes Association and by Novo Nordisk and Roche Diagnostics with a special equipped bus touring through more than 90 cities. The population was invited to a screening of blood glucose (random), blood pressure, body weight und serum-cholesterol. The diabetes risk was evaluated by history and familiar burden with a questionnaire. The results were handed over to the visitors and their general practitioners, and a copy was used for anonymously analysis.

Adult↗
PubMed2001

In vitro uptake of polystyrene microspheres: effect of particle size, cell line and cell density.

Uptake of polycation-DNA particles is the first step in achieving gene delivery with non-viral vehicles. One of the important characteristics determining uptake of DNA particles is their size. Here we have characterized the ability of several cell lines to internalise labelled polystyrene microspheres of different sizes. All the cell lines tested ingested 20-nm microspheres avidly. With larger microspheres (93, 220, 560 and 1010 nm) cell type as well as growth related differences were observed. Whereas some cell lines (HUVEC, ECV 304 and HNX 14C) took up microspheres up to 1010 nm even when the cells were confluent, others did not take up many microspheres larger than 93 nm (Hepa 1-6 and HepG2). In one cell line (KLN 205), uptake of 93-, 220- and 560-nm microspheres was avid in growing cells, but not detectable when they were confluent. In KLN 205 cells, a good correlation was found between the uptake of 560-nm microspheres and the uptake of a peptide-DNA polyplex formulation, when it was prepared under conditions leading to small particle sizes. Little correlation was found when the polyplex formulation was allowed to aggregate.

Amino Acid Sequence↗
PubMed2001

CL22 - a novel cationic peptide for efficient transfection of mammalian cells.

Condensing peptide-DNA complexes have great potential as nonviral agents for gene delivery. To date, however, such complexes have given transfection activities greatly inferior to adenovirus and somewhat inferior to cationic lipid-DNA complexes, even for cell lines and primary cells in vitro. We report here the identification of a novel condensing peptide, CL22, which forms DNA complexes that efficiently transfect many cell lines, as well as primary dendritic and endothelial cells. We report studies with sequence and structure variants that define some properties of the peptide that contribute to efficient transfection. We demonstrate that the superior transfection activity of CL22 compared with other DNA condensing peptides is conferred at a step after uptake of the complexes into cells. We show that CL22-DNA complexes have transfection activity that is at least equivalent to the best available nonviral agents.

Amino Acid Sequence↗
PubMed2001

Defined synthetic vaccines.

Although vaccines have proven very successful in preventing certain infectious diseases, progress in the field has been slowed by the tediousness of developing classical vaccines consisting of whole pathogens. Thus, there is great need for improvement in several areas: firstly, the range of diseases which can be treated has to be expanded. Secondly, antigens have to be defined to make the use of whole pathogens as antigen obsolete. And thirdly, new adjuvants have to be developed which show low toxicity, high potency and are also able to drive the immune response in the desired direction. Ideally, a vaccine would only consist of well-characterized, synthetic materials. This review summarizes the different approaches for the development of completely defined synthetic vaccines.

Adjuvants, Immunologic↗
PubMed1999

The glucocorticoid receptor cooperates with the erythropoietin receptor and c-Kit to enhance and sustain proliferation of erythroid progenitors in vitro.

Although erythropoietin (Epo) is essential for the production of mature red blood cells, the cooperation with other factors is required for a proper balance between progenitor proliferation and differentiation. In avian erythroid progenitors, steroid hormones cooperate with tyrosine kinase receptors to induce renewal of erythroid progenitors. We examined the role of corticosteroids in the in vitro expansion of primary human erythroid cells in liquid cultures and colony assays. Dexamethasone (Dex), a synthetic glucocorticoid hormone, cooperated with Epo and stem cell factor to induce erythroid progenitors to undergo 15 to 22 cell divisions, corresponding to a 10(5)- to 10(6)-fold amplification of erythroid cells. Dex acted directly on erythroid progenitors and maintained the colony-forming capacity of the progenitor cells expanded in liquid cultures. The hormone delayed terminal differentiation into erythrocytes, which was assayed by morphology, hemoglobin accumulation, and the expression of genes characteristic for immature cells. Sustained proliferation of erythroid progenitors could be induced equally well from purified erythroid burst-forming units (BFU-E), from CD34(+) blast cells, and from bone marrow depleted from CD34(+) cells.

Adult↗
PubMed1999

Differential behaviour of lipid based and polycation based gene transfer systems in transfecting primary human fibroblasts: a potential role of polylysine in nuclear transport.

DNA delivery systems for gene therapy applications have to be able to trigger the uptake of plasmid DNA into the nucleus. We have tested two types of non-viral vector systems, lipofection (cationic lipid-based, using Lipofectamine) and polyfection (cationic polymer-based, using glycerol enhanced transferrinfection), for their ability to transfect confluent, contact inhibited primary human fibroblasts. While both systems worked well with growing fibroblasts, polyfection was superior with confluent cells. A slight reduction in cell associated plasmid DNA was observed with resting cells, but it was similar for both types of complexes. Lipofectamine showed a prevalence for transfecting cycling cells as judged by costaining transfected cells with cell cycle markers. No such bias was observed when glycerol enhanced transferrinfection was used. Microinjection of plasmid DNA/polylysine complexes into the cytoplasm of fibroblasts resulted in a higher percentage of expressing cells than injection of plasmid DNA, offering an explanation for the higher transfection levels obtained with transferrinfection in non-growing cells.

Biological Transport↗
PubMed1998

Application of membrane-active peptides for drug and gene delivery across cellular membranes.

Naturally occurring peptides and protein domains with amphipathic sequences play a dominant role in physiological, lipid membrane-reorganizing processes like fusion, disruption, or pore formation. More recently this capacity to modulate membrane integrity has been exploited for drug delivery into cells. Incorporation of synthetic membrane-active peptides into delivery systems has been found to enhance intracellular delivery of drugs including oligonucleotides, peptides, or plasmid DNA. In the majority of applications, the amphipathic peptides are designed to act after uptake by endocytosis, releasing the delivered agent from intracellular vesicles to the cytoplasm. Alternatively, peptides might mediate direct drug transfer across the plasma membrane. Although encouraging results have been obtained with the use of synthetic peptides to enhance cellular delivery of various compounds, the naturally evolved mechanisms observed in the entry of viruses or protein toxins are still far more efficient. For the development of improved synthetic peptides and carrier systems a better understanding of the molecular details of membrane-destabilization and reorganization will be essential.

Journal Article↗
PubMed1998

The mouse and rat MAP1B genes: genomic organization and alternative transcription.

We report the genomic organization of the mouse and rat genes coding for the 2460-amino-acid microtubule-associated protein (MAP) 1B. In addition to seven exons that encode full-length MAP1B, we have identified two alternative exons, exon 3A and the novel exon 3U. We demonstrate that alternative MAP1B transcripts containing either exon 3A or exon 3U are expressed in a variety of mouse and rat tissues at about 1 to 10% of the level of regular transcripts. The alternative transcripts, if translated, would give rise to MAP1B isoforms truncated at the N-terminus. The exon/intron organization underlying the alternative transcripts and the N-terminal amino acid sequence of the putative truncated MAP1B isoforms resemble those of MAP1A, providing further evidence for an evolutionary relationship. The detection of alternative transcripts has implications for the interpretation of conflicting results recently obtained in MAP1B knockout mice.

Alternative Splicing↗
PubMed1998

Polylysine-based transfection systems utilizing receptor-mediated delivery.

Receptor-mediated gene transfer is a promising gene delivery technique. It employs a DNA-binding polycation, such as polylysine, to compact plasmid DNA to a size that can be taken up by cells (<100-200 nm). To allow internalization by receptor-mediated endocytosis, cell binding ligands, such as asialoglycoproteins or galactose for hepatocytes, anti-CD3 and anti-CD5 for T-cells, and transferrin, have been covalently attached to polylysine. Intracellular barriers for successful gene transfer include release of DNA complexes from endosomes or lysosomes, nuclear import of DNA complexes, and disassembly of the DNA-polylysine particles. Release of particles from internal vesicles has been achieved by the addition of lysosomotropic agents or glycerol to the transfection medium, or by the incorporation of endosomolytic compounds, such as viruses or membrane active peptides. This technique has already been used to transfect certain organs in vivo, including liver and lung.

Journal Article↗
PubMed1998

Influence of the DNA complexation medium on the transfection efficiency of lipospermine/DNA particles.

Dioctadecylamidoglycylspermine (DOGS, Transfectam) is a cationic lipid able to interact with DNA to form complexes that mediate efficient gene transfer into various eukaryotic cells. The state of condensation of the plasmid changes with the medium composition. We therefore investigated to what extent the DNA condensation buffer influences the transfection efficiency of Transfectam/DNA particles. Our results show that in a variety of cell lines, a greater than 100-fold difference in luciferase gene expression is observed with Transfectam/DNA complexes at a +/- charge ratio of 0.75 depending on the conditions of complex formation. The best transfection conditions consisted of particles formed in RPMI medium, NaHCO3/Na2HPO4 or sodium citrate solutions. Mixing in a 150 mM sodium chloride solution (as recommended) resulted in lower gene expression. When the helper lipid 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) was present in the DNA/cationic lipid formulation, the increase in reporter activity was also observed, although to a lower extent. Thus, choosing the optimal conditions for formulating DNA/lipid complexes considerably reduces the amount of lipid and DNA needed to obtain maximum gene transfer.

Animals↗
PubMed1997

Glycerol and polylysine synergize in their ability to rupture vesicular membranes: a mechanism for increased transferrin-polylysine-mediated gene transfer.

The presence of about 1.2 M glycerol during transfection with DNA/transferrin-polylysine and DNA/polylysine complexes dramatically increases transgene expression in a variety of cell types, provided that the complexes have an excess of polylysine. We have characterized this phenomenon using a human melanoma cell line (H225). The addition of 1.2 M glycerol to the transfection medium has no influence on the internalization of DNA complexes or on the promoter activity used to direct reporter gene expression. Neither prenor postincubation of the cells with glycerol results in a notable increase in transgene expression. Bafilomycin A1 and chloroquine, two drugs affecting the endosomal pathway, both influenced transgene expression, indicating that glycerol acts on internal vesicles. Glycerol and polylysine synergized in their ability to lyse erythrocytes as well as internal vesicles (microsomes) isolated from H225 cells, indicating that the glycerol effect is due to a labilization of vesicular membranes, which facilitates membrane disruption by polylysine. Our current model suggests that the excess of polylysine in the DNA complexes disrupts vesicular membranes in the presence of glycerol, thus allowing the release of DNA complexes into the cytoplasm.

Anti-Bacterial Agents↗
PubMed1997

Transloading of tumor antigen-derived peptides into antigen-presenting cells.

The discovery of a steadily growing number of tumor antigens (TAs) has made generic, cell-free, peptide-based cancer vaccines a possible alternative to cytokine-transfected autologous cellular cancer vaccines. The major drawback of peptide vaccines, however, is the poor immunogenicity of peptides. It is commonly thought that for the induction of an effective anticancer immune response, antigen-presenting cells (APCs) have to display TA-derived peptides to T lymphocytes. Polycationic amino acids have been employed in the past to enhance transport of proteins into cells. In a systematic study, the ability of different cationic polymers to transfer fluorescence-tagged peptides to APCs was investigated. We were able to show that several compounds enhance uptake of fluorescence-labeled peptides by APCs to different degrees. The most efficient compound identified, polyarginine (pArg), enhanced peptide delivery by more than 2 logs as compared with cells treated with peptide alone, whereas polylysine (pLys) treatment resulted in approximately 10-fold increased levels of fluorescence. Augmentation of peptide uptake was concentration-dependent, and the molecular weight of pArg or pLys also influenced peptide delivery. Furthermore, highly negatively charged peptides appear to be delivered with higher efficiency, although neutral peptides were also taken up at enhanced rates. Whereas peptide uptake mediated by pLys appears to be due to an at least transient permeabilization of cell membranes, peptide delivery in the presence of pArg may rely on endocytic processes. TA-derived peptides applied as cancer vaccines in conjunction with polycations afforded antitumor protection in animal models.

Amino Acid Sequence↗
PubMed1997

Cell-free tumor antigen peptide-based cancer vaccines.

The central role that tumor antigen-derived peptides play in induction of antitumor immunity makes them ideal candidates for peptide-based cancer vaccines. We have demonstrated that "transloading" is an efficient strategy for importing short peptide ligands into antigen-presenting cells in vitro. Postulating that the transloading procedure might effect peptide uptake by antigen-presenting cells in vivo as well, we tested this approach for the generation of peptide-based cancer vaccines. In the P815 mastocytoma system, we vaccinated mice by s.c. injection of a single, known natural peptide derived from JAK-1 kinase. Whereas vaccination with peptide alone or mixed with incomplete Freund's adjuvant was ineffective, application of the peptide in conjunction with the polycation poly-L-lysine protected a significant number of animals against tumor challenge. Dependent upon the type of poly-L-lysine applied, protection against tumor take was comparable to that achieved with irradiated whole-cell vaccines, genetically modified to secrete granulocyte-macrophage colony-stimulating factor. In the murine melanoma M-3, a combination of four putative tumor antigen-derived peptides was tested as a cancer vaccine. Administered in combination with polycations, these peptides evoked potent antitumor immunity that could not be obtained with the peptides alone or peptides emulsified in incomplete Freund's adjuvant. However, peptide-polycation vaccines applied to the M-3 model were not as efficient as cellular control vaccines, consisting of irradiated interleukin 2 or granulocyte-macrophage colony-stimulating factor-secreting tumor cells.

Amino Acid Sequence↗
PubMed1996

Glycerol enhancement of ligand-polylysine/DNA transfection.

Primary human fibroblasts and a series of cell lines (A549, BNL CL.2, H225, NIH 3T3 and Rat-1) are efficiently transfected by using positively charged complexes of plasmid DNA and transferrin-polylysine or polylysine in the presence of glycerol (1 molar to 1.8 molar, depending on the cell type). An increase in gene expression of up to several-hundredfold (compared to complexes without glycerol) is obtained if the transfection mixture is incubated with the cells for 3-4 h at 37 degrees C. This simple method has been used for transient expression of luciferase, beta-galactosidase and interleukin-2, and also for the generation of stably transfected cells.

Animals↗
PubMed1995

Receptor-mediated gene transfer into human T lymphocytes via binding of DNA/CD3 antibody particles to the CD3 T cell receptor complex.

Retrovirus-mediated gene transfer is currently the method of choice for the transfection of human T lymphocytes for applications in gene therapy. Use of retroviral vectors, however, is hampered by limits on the size of the genetic material to be transferred, the requirement of dividing target cells, and by potential safety questions. Synthetic peptide-enhanced or adenovirus-enhanced receptor-mediated transferrinfection of DNA (SPET and AVET, respectively) is a powerful method for the introduction of genetic material into mammalian cells. Although transferrin has proven to be a useful ligand for gene transfer in many cell types, gene expression in T cells with transferrin/DNA complexes is usually not satisfactory. To improve gene transfer to T cells, antibodies directed against the CD3-T cell receptor complex were tested for their ability to function as ligands for DNA delivery. In T cell lines, up to 50% of the cells expressed a beta-galactosidase reporter gene using anti-CD3 gene transfer complexes. Applying optimized conditions, prestimulated primary peripheral blood lymphocytes were also transfected successfully, although at a lesser efficiency (5%).

Antibodies↗
PubMed1995

Rhinovirus-mediated endosomal release of transfection complexes.

Endocytosis is an efficient method for transfer of genes into mammalian cells. Incorporation of adenovirus particles into gene transfer complexes greatly enhances gene delivery, probably by the release of endocytosed DNA into the cytoplasm. We report here that two different serotypes of human rhinovirus (HRV), HRV2 and HRV14, are also able to enhance receptor-mediated gene transfer. The effect of several compounds known to inhibit viral infection on HRV2- and HRV14-enhanced transfection was examined. WIN I(s) and WIN IV, two compounds which inhibit viral uncoating, had different effects on HRV2- and HRV14-enhanced gene transfer to NIH 3T3 cells. While HRV14-enhanced gene transfer was severely reduced in the presence of these compounds, virtually no effects were observed when HRV2 was used. The use of antiviral compounds thus allowed transfection of human cells, which are normally lysed rapidly upon infection with HRV. Viral activity could be mimicked by using a peptide derived from the N terminus of VP1 of HRV2. This peptide possesses pH-dependent membrane-disrupting activity and enhances gene transfer to NIH 3T3 and HeLa cells.

3T3 Cells↗