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Biomedical subjects

E Wagner

Publications and source records attributed to E Wagner.

At least 73 records · Page 4Linked to original sources
PubMed2000

A retinoic acid synthesizing enzyme in ventral retina and telencephalon of the embryonic mouse.

Most retinoic acid (RA) in the embryonic mouse is generated by three retinaldehyde dehydrogenases (RALDHs). RALDH1 (also called E1, AHD2 or ALDH1) is expressed in the dorsal retina, and RALDH2 (V2, ALDH11) generates most RA in the embryonic trunk. The third one, RALDH3 (V1), synthesizes the bulk of RA in the head of the early embryo. We show here that RALDH3 is a mouse homologue to ALDH6, an aldehyde dehydrogenase cloned from adult human salivary gland (Hsu, L.C., Chang, W.-C., Hiraoka, L., Hsien, C.-L., 1994. Molecular cloning, genomic organization, and chromosomal localization of an additional human aldehyde dehydrogenase gene, ALDH6. Genomics 24, 333-341), which was recently reported to act as a RALDH (Yoshida, A., Rzhetsky, A., Hsu, L.C., Chang, C., 1998. Human aldehyde dehydrogenase gene family. Eur. J. Biochem. 251, 549-557). RALDH3 expression begins in the surface ectoderm over the optic recess. In rapidly changing expression patterns it labels the appearance of several ectodermal structures: it marks the formation of the lens and the olfactory organ from ectodermal placodes, and it delineates the beginning eyelid field. Within the optic vesicle, RALDH3 is expressed in the ventral retina and the dorsal pigment epithelium. In the telencephalon, RALDH3 is expressed at high levels in the lateral part of the ganglionic eminence. From here it extends via the piriform cortex into the lower part of the septum. Of the three RALDHs, RALDH3 shows the strongest predilection for epithelia.

Aldehyde Dehydrogenase↗
PubMed2000

Liposomes containing interferon-gamma as adjuvant in tumor cell vaccines.

PURPOSE: Liposomal systems may be useful as a cytokine supplement in tumor cell vaccines by providing a cytokine reservoir at the antigen presentation site. Here, we examined the effect of liposome incorporation of mIFNgamma on its potency as adjuvant in an established tumor cell vaccination protocol in the murine B16 melanoma model. Adjuvanticity of the mIFNgamma-liposomes was compared to that achieved by mIFNgamma-gene transfection of the B16 tumor cells. Furthermore, we studied whether liposomal incorporation of mIFNgamma indeed increases the residence time of the cytokine at the vaccination site. METHODS: C57B1/6 mice were immunized with i) irradiated IFNgamma-gene transfected B16 melanoma cells or ii) irradiated wild type B16 cells supplemented with (liposomal) mIFNgamma, followed by a challenge with viable B16 cells. The residence time of the (liposomal) cytokine at the subcutaneous (s.c.) vaccination site was monitored using radiolabeled mIFNgamma and liposomes. RESULTS: Immunization with irradiated tumor cells admixed with liposomal mIFNgamma generated comparable protection against B16 challenge as immunization with mIFNgamma-gene modified tumor cells. Irradiated tumor cells admixed with soluble mIFNgamma did not generate any protective responses. Radiolabeling studies indicated that free mIFNgamma rapidly cleared from the s.c. injection site. Association of [125I]-mIFNgamma with liposomes increased the local residence time substantially: liposomal association of mIFNgamma resulted in a prolonged local residence time of the cytokine as reflected by a 4-fold increase of the area under the curve. The amount of released cytokine in the optimal dose range corresponds to the amount released by the gene-transfected cells. Moderate but significant CTL-activity against B16 cells was found for mice immunized with irradiated cells supplemented with mIFNgamma-liposomes compared to untreated control animals. CONCLUSIONS: Prolonged presence of mIFNgamma at the site of antigen presentation is crucial for the generation of systemic immune responses in the B16 melanoma model. These studies show that liposomal encapsulation of cytokines is an attractive strategy for paracrine cytokine delivery in tumor vaccine development.

Adjuvants, Immunologic↗
PubMed2000

Interleukin-2 gene-modified allogeneic melanoma cell vaccines can induce cross-protection against syngeneic tumors in mice.

Vaccination using well-characterized allogeneic tumor cell lines expressing standardized doses of immunostimulatory cytokines is an attractive alternative for autologous gene-transfected tumor cell vaccines. In the present study, we show that vaccination with irradiated allogeneic K1 735 (H-2k) or B16F10 (H-2b) melanoma cells induces a moderate degree of cross-protection against the M-3 melanoma (H-2d) in DBA/2 mice. Cross-protection against the syngeneic tumor was markedly improved when the allogeneic vaccines were transfected with the interleukin-2 (IL-2) gene. The IL-2 gene-modified allogeneic vaccines were effective for prophylactic vaccination against subsequent tumor challenge and for therapeutic vaccination against pre-existing tumor deposits, with efficacies that were comparable with that of the IL-2 gene-modified syngeneic vaccines. Cross-protection correlated with the cytotoxic activity of splenocytes against M-3 targets. Allogeneic vaccination was not effective in another model, against the B16F10 melanoma in C57BL/6 mice, irrespective of genetic modification with the IL-2 or granulocyte-macrophage colony-stimulating factor genes.

Animals↗
PubMed2000

Cell cycle dependence of gene transfer by lipoplex, polyplex and recombinant adenovirus.

The aim of this study was to investigate the influence of cell cycle on transfection efficiency. Counterflow centrifugal elutriation was used which avoids possible side-effects from chemical treatment of cells. With this method, cell populations were fractionated by means of size and density, and fractions corresponding to discrete cell cycle phase-specific populations were transfected with various nonviral methods (Lipofectamine, TfpLys and TfPEI), adenovirus-enhanced transferrinfection (AVET system) and recombinant adenovirus. Transfection efficiency was found to be strongly dependent on the cell cycle stage at the time of transfection. Luciferase activity from cells transfected with polycation- or lipid-based transfection systems was 30- to more than 500-fold higher when transfection was performed during S or G2 phase compared with cells in G1 phase which have the lowest expression levels. In contrast, this effect was not observed with recombinant adenovirus which varied only four-fold. Our results indicate that mitotic activity enhances transfection not only by lipoplexes but also by polyplexes, but not a viral system which has an efficient nuclear entry machinery, suggesting that transfection close to M phase is facilitated perhaps by nuclear membrane breakdown. Furthermore, low transfection success into G1 cells indicates that DNA complexes deposited in G1 cells are probably not retained long enough to take advantage of mitosis effects or that passage of transfected cells through S phase is inhibitory. Gene Therapy (2000) 7, 401-407.

Adenoviridae↗
PubMed2000

The role of lipoprotein lipase in adipose tissue development and metabolism.

Lipoprotein lipase (LPL) is essential for the hydrolysis and distribution of triglyceride-rich lipoprotein-associated fatty acids among extrahepatic tissues. Additionally, the enzyme facilitates several non-lipolysis associated functions including the cellular uptake of whole lipoprotein particles and lipophilic vitamins. The tissue-specific variations of LPL expression have been implicated in the pathogenesis of various lipid disorders, obesity and atherosclerosis. Transgenic technology provided the means to study the physiological response to the overexpression or absence of the enzyme in adipose tissue, muscle and macrophages. The effects of varying LPL expression in adipose tissue and muscle are summarized in this article.

Adipose Tissue↗
PubMed2000

Efficient in vitro transfection of human keratinocytes with an adenovirus-enhanced receptor-mediated system.

Efficient gene delivery to the skin is important for gene therapy of skin diseases and in-depth biologic studies of epidermis. In this report, we investigated three nonviral transfection systems for gene transfer in cultured human keratinocytes and organotypic cultures. SuperFect is a highly branched polycationic transfection reagent, PrimeFector a polycationic liposome compound, and the AVET (adenovirus-enhanced transferrin-mediated) system consists of a ternary complex of biotinylated chemically inactivated adenovirus noncovalently complexed with plasmid DNA and polylysine-transferrin. After AVET transfection of cultured keratinocytes with pCIbetagal, a CMV/beta-galactosidase reporter plasmid, 28.8% +/- 1.4% of the cells were stained blue. SuperFect was about 2-fold less efficient, whereas Primefector did not transfect keratinocytes. Similar results were obtained when transfection efficiencies were measured by enzyme assays. Addition of holotransferrin to the culture medium or replacement of polylysine-transferrin by polylysine in the ternary complex did not affect the transfection efficiency. Using AVET complexes without adenovirus, however, strongly diminished gene delivery. This indicates that the AVET complex is taken up by an adenovirus receptor. Separation of AVET/pCIbetagal transfected keratinocytes by adhesion to collagen IV into two fractions (rapidly and slowly adhering cells) showed that the latter were transfected at a 3-fold higher level. Therefore, it seems that putative stem cells adhering rapidly to collagen IV are not efficiently transfected by AVET. AVET-transfected keratinocytes derived from keratinocyte trans- glutaminase negative lamellar ichthyosis patients with a CMV-TGK expression plasmid showed that it is possible to reach a level of total enzyme activity similar to that found in cultured keratinocytes from normal individuals. In organotypic cultures from outer root sheath cells AVET transfection was not successful, which might be due to the presence of the cornified layer or inaccessibility of the adenovirus receptor. In summary, the AVET system provides a powerful tool for transient in vitro transfection of keratinocytes.

3T3 Cells↗
PubMed2000

Selection of a polyurethane membrane for the manufacture of ventricles for a totally implantable artificial heart: blood compatibility and biocompatibility studies.

Membranes made from 4 commercial poly(carbonate urethanes): Carbothane (CB), Chronoflex (CF), Corethane 80A (CT80), and Corethane 55D (CT55), and from 2 poly(ether urethanes): Tecoflex (TF) and Tecothane (TT) were prepared by solution casting and sterilized by either ethylene oxide (EO) or gamma radiation. Their biocompatibility was evaluated in vitro in terms of proliferation, cell viability, and adhesion characteristics of human umbilical veins (HUVEC), monocytes (THP-1), and skin fibroblasts, and by measuring complement activation through the generation of the C3a complex. Their hemocompatibility was determined by measuring the level of radiolabeled platelet, neutrophil, and fibrin adhesion in an ex vivo arteriovenous circuit study in piglets as well as via an in vitro hemolysis test. The results of this study showed no endothelial cell proliferation on any of the materials. The cell viability study revealed that the CB, CF, and TF membranes sterilized by EO maintained the highest percentage of monocyte viability after 72 h of incubation (>70%) while none of the gamma-sterilized membranes displayed any cell viability. The fibroblast adhesion and C3a generation assays revealed that none of the materials supported any cell adhesion or activated complement, regardless of the sterilization method. The hemolysis test also confirmed that the 4 poly(carbonate urethanes) were hemolytic while none of the poly(ether urethanes) were. Finally, the ex vivo study revealed that significantly more platelets adhered to the CB and CT55 membranes while the levels of neutrophil and fibrin deposition were observed to be similar for all 6 materials. In conclusion, the study identified the CF and TF membranes as having superior biocompatibility and hemocompatibility compared to the other polyurethanes.

Animals↗
PubMed2000

Use of co-loaded Fluo-3 and Fura Red fluorescent indicators for studying the cytosolic Ca(2+)concentrations distribution in living plant tissue.

A method for visualisation of cytosolic [Ca(2+)] distribution was applied to living plant tissue. A mixture of the fluorescent probes Fluo-3 and Fura Red was used. The emitted fluorescence was scanned simultaneously in two channels with a laser-scanning confocal microscope and rationing was performed. The homogeneity of the Fluo-3/Fura Red concentration ratio throughout the tissue after AM-ester loading was proven. In vitro calibration permitted conversion of Fluo-3/Fura Red fluorescence ratios to [Ca(2+)] values. Apparent K(D)of 286 nM, R(min)of 0.43 and R(max)of 18 were calculated. The in vivo determination of extreme ratio values was performed by permeabilizing the plasmalemma for Ca(2+)with a ionophore and manipulating the extracellular [Ca(2+)]. The resultant R(minv)of 1.33 and R(maxv)of 2.69 for vegetative apices, and R(mini)of 1.26 and R(maxi)of 3.45 for apices induced to flowering, suggested incomplete equalization of extra- and intracellular Ca(2+)levels in these experiments. In Chenopodium rubrum, the cytosolic [Ca(2+)] patterns of apical tissue obtained using Fluo-3 and Fura Red were significantly different between vegetative apices and apices after photoperiodic flower induction. This methodological approach may also be helpful for studying cytosolic [Ca(2+)] distribution in other living plant tissues.

Aniline Compounds↗
PubMed2000

VEGF, fetal liver kinase-1, and permeability increase during unilateral lung ischemia.

Vascular endothelial growth factor (VEGF) is a potent mediator of increased vascular permeability and an endothelial cell mitogen. Because VEGF is upregulated during ventilated ischemia of isolated lungs and may lead to both increased vascular permeability and neovascularization, we hypothesized that VEGF and kinase insert domain-containing receptor/fetal liver kinase-1 (KDR/flk-1) expression would increase acutely after unilateral pulmonary arterial (PA) ischemia in vivo in association with evidence of endothelial cell barrier dysfunction. To test this hypothesis, VEGF and KDR/flk-1 mRNA and protein expression were measured after 4, 8, and 24 h of left PA ligation in mice. Permeability was assessed at the same time points by measurement of bronchoalveolar lavage protein concentration and lung wet-to-dry weight ratios. Results were compared with those from uninstrumented and sham-operated mice. VEGF and KDR/flk-1 protein in the left lung both increased by 4 h and then returned to baseline, whereas increased VEGF and KDR/flk-1 mRNA expression was sustained throughout 24 h of unilateral ischemia. Bronchoalveolar lavage protein concentration increased transiently during ischemia, whereas wet-to-dry weight ratio of the left lung increased more slowly and remained elevated after 24 h of left PA ligation. These results suggest that increased expression of VEGF and KDR/flk-1 during unilateral PA occlusion in mice may contribute to the development of acute lung injury in this model.

Animals↗
PubMed2000

Wrapped in silence: psychotherapists and confidentiality in the courtroom.

The impact of the Austrian Psychotherapy Act, which, in contrast to legal provisions in the United States, does not provide for any exceptions to breach confidentiality, is compared with the effects of U.S. law on dealing with confidentiality. The authors investigated the impact of this law in light of three common situations in psychotherapy that may jeopardize strict confidentiality: treating potentially dangerous patients, giving testimony, and serving as a psychotherapist in prison. Under the strict provisions of the Austrian Psychotherapy Act, a breach may be excusable in the case of a highly probable danger, but Austrian psychotherapists cannot be obliged to serve as witnesses or as experts in civil or criminal cases, as American psychotherapists can. Psychotherapy in prison, where release is contingent on the success of the therapy and the divulging of information could be in the interests of the patient as well as the court and the public, requires a modified dealing with confidentiality.

Austria↗
PubMed1999

IgG and complement-mediated tissue damage in the absence of C2: evidence of a functionally active C2-bypass pathway in a guinea pig model.

In vitro complement-mediated lysis of heavily sensitized sheep erythrocytes by C4-deficient (C4D) guinea pig and C2-deficient (C2D) human sera was demonstrated some years ago. It was postulated that these "complement-bypass" pathways resulted from activation of C1 and components of the alternative pathway. We used normal, C2D, and C4D guinea pigs in a Forssman shock model to test the in vivo relevance of the C2- and C4-bypass pathways of complement activation. High concentrations of both anti-Forssman Ab and C2D or C4D guinea pig serum induced efficient lysis of sheep erythrocytes in vitro. The most efficient lysis was observed when IgG Ab and C2D guinea pig serum were used. Blocking either the classical pathway (treatments with EGTA-Mg2+ or soluble recombinant complement receptor type 1 (sCR1)) or the alternative pathway (treatment with heating at 50 degrees C, sCR1, or soluble recombinant CR1 lacking the first of the four long homologous repeat sequences (sCR1[desLHR-A])) inhibited lysis; both pathways were required for lysis of sheep erythrocytes by C2D and C4D guinea pig sera. i.v. injection of anti-Forssman Ab in normal guinea pigs resulted in rapid death from pulmonary shock, whereas C4D guinea pigs had no adverse effect. Surprisingly, C2D guinea pigs either died in a delayed fashion or had a sublethal reaction. sCR1 treatment prevented Forssman shock in both normal and C2D guinea pigs, whereas sCR1[desLHR-A] prevented Forssman shock only in C2D animals. Our results suggest that the C2-bypass pathway occurs in vivo to produce tissue damage. Activation of complement in the absence of C2 appears to be far more efficient than in the absence of C4.

Anaphylaxis↗
PubMed1999

Application of membrane-active peptides for nonviral gene delivery.

A variety of membrane-modifying agents including pH-specific fusogenic or lytic peptides, bacterial proteins, lipids, glycerol, or inactivated virus particles have been evaluated for the enhancement of DNA-polycation complex-based gene transfer. The enhancement depends on the characteristics of both the cationic carrier for DNA and the membrane-modifying agent. Peptides derived from viral sequences such as the N-terminus of influenza virus haemagglutinin HA-2, the N-terminus of rhinovirus HRV2 VP-1 protein, and other synthetic or natural sequences such as the amphipathic peptides GALA, KALA, EGLA, JTS1, or gramicidin S have been tested. Ligand-polylysine-mediated gene transfer can be improved up to more than 1000-fold by membrane-active compounds. Other polycations like dendrimers or polyethylenimines as well as several cationic lipids provide a high transfection efficiency per se. Systems based on these polymers or lipids are only slightly enhanced by endosomolytic peptides or adenoviruses. Electroneutral cationic lipid-DNA complexes however can be strongly improved by the addition of membrane-active peptides.

Journal Article↗
PubMed1999

Mannose polyethylenimine conjugates for targeted DNA delivery into dendritic cells.

Cell surface-bound receptors represent suitable entry sites for gene delivery into cells by receptor-mediated endocytosis. Here we have taken advantage of the mannose receptor that is highly expressed on antigen-presenting dendritic cells for targeted gene transfer by employing mannosylpolyethylenimine (ManPEI) conjugates. Several ManPEI conjugates were synthesized and used for formation of ManPEI/DNA transfection complexes. Conjugates differed in the linker between mannose and polyethylenimine (PEI) and in the size of the PEI moiety. We demonstrate that ManPEI transfection is effective in delivering DNA into mannose receptor-expressing cells. Uptake of ManPEI/DNA complexes is receptor-specific, since DNA delivery can be competed with mannosylated albumin. Additionally, incorporation of adenovirus particles into transfection complexes effectively enhances transgene expression. This is particularly important for primary immunocompetent dendritic cells. It is demonstrated here that dendritic cells transfected with ManPEI/DNA complexes containing adenovirus particles are effective in activating T cells of T cell receptor transgenic mice in an antigen-specific fashion.

Adenoviridae↗
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↗
PubMed1999

Liposomes as cytokine-supplement in tumor cell-based vaccines.

Subcutaneous vaccination of C57bl/6 mice with irradiated B16 melanoma cells supplemented with liposomal interleukin-2 (IL2) or murine interferon-gamma (mIFNgamma), resulted in systemic protection in 50% of the animals, against a subsequent tumor cell challenge in a dose dependent manner. The protective efficacy was comparable to the efficacy of cytokine gene-modified cells as tumor vaccine, whereas irradiated B16 cells supplemented with soluble cytokine did not result in protective responses. In vivo evidence was obtained that the beneficial effects mediated by liposome incorporation of the cytokine are the result of a depot function of the liposomal cytokine supplement at the vaccination site. In can be concluded that liposomal delivery of cytokines offers an attractive alternative to cytokine-gene transfection of tumor cells for therapeutic vaccination protocols.

Animals↗
PubMed1999

Immunotherapy of metastatic malignant melanoma by a vaccine consisting of autologous interleukin 2-transfected cancer cells: outcome of a phase I study.

We performed a phase I trial to evaluate the safety and tolerability of repeated skin injections of IL-2-transfected autologous melanoma cells into patients with advanced disease. Cell suspensions, propagated from excised metastases, were IL-2 gene transfected by adenovirus-enhanced transferrinfection and X-irradiated prior to injection. Vaccine production was successful in 54% of the patients. Fifteen patients (37%) received two to eight skin vaccinations of either 3 x 10(6) (intradermal) or 1 x 10(7) (half intradermal, half subcutaneous) transfected melanoma cells per vaccination (secreting 140-17,060 biological response modifier program units of IL-2/10(6) cells/24 hr). Analyses of safety and efficacy were carried out in 15 and 14 patients, respectively. Overall, the vaccine was well tolerated. All patients displayed modest local reactions (erythema, induration, and pruritus) and some experienced flu-like symptoms. Apart from newly appearing (4 of 14) and increasing (5 of 14) anti-adenovirus and newly detectable anti-nuclear antibody titers (1 of 15), recipients developed de novo or exhibited increased melanoma cell-specific delayed-type hypersensitivity (DTH) reactions (8 of 15) and vitiligo (3 of 15) and showed signs of tumor regression (3 of 15). This supports the idea of a vaccine-induced or -amplified anti-cancer immune response. None of the patients exhibited complete or partial regressions, but five of them experienced periods of disease stabilization. Three of these individuals received more than the four planned vaccinations and their mean survival time was 15.7 +/- 3.5 months as compared to 7.8 +/- 4.6 months for the entire patient cohort. These data indicate that IL-2-producing, autologous cancer cells can be safely administered to stage IV melanoma patients and could conceivably be of benefit to patients with less advanced disease.

Adult↗