Efficiency of different selection strategies against boar taint in pigs.

The breeding scheme of a Swiss sire line was modeled to compare different target traits and information sources for selection against boar taint. The impact of selection against boar taint on production traits was assessed for different economic weights of boar taint compounds. Genetic gain and breeding costs were evaluated using ZPlan+, a software based on selection index theory, gene flow method and economic modeling. Scenario I reflected the currently practiced breeding strategy as a reference scenario without selection against boar taint. Scenario II incorporated selection against the chemical compounds of boar taint, androstenone (AND), skatole (SKA) and indole (IND) with economic weights of -2.74, -1.69 and -0.99 Euro per unit of the log transformed trait, respectively. As information sources, biopsy-based performance testing of live boars (BPT) was compared with genomic selection (GS) and a combination of both. Scenario III included selection against the subjectively assessed human nose score (HNS) of boar taint. Information sources were either station testing of full and half sibs of the selection candidate or GS against HNS of boar taint compounds. In scenario I, annual genetic gain of log-transformed AND (SKA; IND) was 0.06 (0.09; 0.02) Euro, which was because of favorable genetic correlations with lean meat percentage and meat surface. In scenario II, genetic gain increased to 0.28 (0.20; 0.09) Euro per year when conducting BPT. Compared with BPT, genetic gain was smaller with GS. A combination of BPT and GS only marginally increased annual genetic gain, whereas variable costs per selection candidate augmented from 230 Euro (BPT) to 330 Euro (GS) or 380 Euro (both). The potential of GS was found to be higher when selecting against HNS, which has a low heritability. Annual genetic gain from GS was higher than from station testing of 4 full sibs and 76 half sibs with one or two measurements. The most effective strategy to reduce HNS was selecting against chemical compounds by conducting BPT. Because of heritabilities higher than 0.45 for AND, SKA and IND and high genetic correlations to HNS, the (correlated) response in units of the trait could be increased by 62% compared with scenario III with GS and even by 79% compared with scenario III, with station testing of siblings with two measurements. Increasing the economic weights of boar taint compounds amplified negative effects on average daily gain, drip loss and intramuscular fat percentage.

Interplay between heritability, genetic correlation and economic weighting in a selection index with and without genomic information.

The availability of genomic information demands proper evaluation on how the kind (phenotypic versus genomic) and the amount of information influences the interplay of heritability (h(2)), genetic correlation (r(GiGj)) and economic weighting of traits with regard to the standard deviation of the index (σI). As σI is directly proportional to response to selection, it was the chosen parameter for comparing the indices. Three selection indices incorporating conventional and genomic information for a two trait (i and j) breeding goal were compared. Information sources were chosen corresponding to pig breeding applications. Index I incorporating an own performance in trait j served as reference scenario. In index II, additional information in both traits was contributed by a varying number of full-sibs (2, 7, 50). In index III, the conventional own performance in trait j was combined with genomic information for both traits. The number of animals in the reference population (NP = 1000, 5000, 10,000) and thus the accuracy of GBVs were varied. With more information included in the index, σI became more independent of r(GiGj), h(j)(2) and relative economic weighting. This applied for index II (more full-sibs) and for index III (more accurate GBVs). Standard deviations of index II with seven full-sibs and index III with NP = 1000 were similar when both traits had the same heritability. If the heritability of trait j was reduced (h(j)(2) = 0.1), σI of index III with NP = 1000 was clearly higher than for index II with seven full-sibs. When enhancing the relative economic weight of trait j, the decrease in σI of the conventional full-sib index was much stronger than for index III. Our results imply that NP = 1000 can be considered a minimum size for a reference population in pig breeding. These conclusions also hold for comparing the accuracies of the indices.

Integration of genomic information into sport horse breeding programs for optimization of accuracy of selection.

Reliable selection criteria are required for young riding horses to increase genetic gain by increasing accuracy of selection and decreasing generation intervals. In this study, selection strategies incorporating genomic breeding values (GEBVs) were evaluated. Relevant stages of selection in sport horse breeding programs were analyzed by applying selection index theory. Results in terms of accuracies of indices (r(TI) ) and relative selection response indicated that information on single nucleotide polymorphism (SNP) genotypes considerably increases the accuracy of breeding values estimated for young horses without own or progeny performance. In a first scenario, the correlation between the breeding value estimated from the SNP genotype and the true breeding value (= accuracy of GEBV) was fixed to a relatively low value of r(mg) = 0.5. For a low heritability trait (h(2) = 0.15), and an index for a young horse based only on information from both parents, additional genomic information doubles r(TI) from 0.27 to 0.54. Including the conventional information source 'own performance' into the before mentioned index, additional SNP information increases r(TI) by 40%. Thus, particularly with regard to traits of low heritability, genomic information can provide a tool for well-founded selection decisions early in life. In a further approach, different sources of breeding values (e.g. GEBV and estimated breeding values (EBVs) from different countries) were combined into an overall index when altering accuracies of EBVs and correlations between traits. In summary, we showed that genomic selection strategies have the potential to contribute to a substantial reduction in generation intervals in horse breeding programs.

The impact of skin viability on drug metabolism and permeation -- BSA toxicity on primary keratinocytes.

For testing cutaneous absorption of drugs, ingredients of cosmetics and also for risk assessment of industrial compounds predictable in vitro test protocols are under investigation using excised skin or reconstructed human epidermis. Since the metabolizing enzymes expressed by viable skin can influence the absorption behaviour of substances by changing their structure and thereby their physicochemical characteristics, the metabolic capacity should be considered in the design of the test protocols of compounds susceptible to metabolism. Then data, generated using viable reconstructed epidermis may reflect the in vivo situation. Interestingly, bovine serum albumin (BSA) commonly used in receptor media in permeation studies to facilitate solubility of highly lipophilic substances strongly inhibited the metabolism of topically applied prednicarbate in reconstructed epidermis. Here, we show that 5% BSA is toxic to reconstructed epidermis and keratinocytes which was consistent with the earlier findings. While media toxicity (deficiency media) was at least partly the cause of both apoptotic and necrotic processes in keratinocytes, BSA only slightly increased the rate of necrotic cells. Moreover, caspase inhibitors did not reduce BSA toxicity. Yet, the results show that BSA toxicity on keratinocytes has to be carefully considered if this protein is used in permeation studies with reconstructed epidermis.

Reconstructed epidermis versus human and animal skin in skin absorption studies.

European chemical policy in general and the REACH initiative in particular will increase the number of chemical substances submitted to toxicological evaluation by several orders of magnitude compared to the current status. To limit animal exposure the resulting enormous increase in testing, however, asks for validated in vitro test systems. While the OECD favours in vitro testing for cutaneous absorption using viable human and animal skin (Guideline 428) the availability of viable human skin is already limited today. We present a comparison of various in vitro techniques suitable for routine skin absorption studies including commercially available reconstructed human epidermis which may be a reliable alternative to excised human and animal skin. In order to develop a protocol for the subsequent transfer to partner laboratories the experimental set-up was analysed stepwise using the OECD reference compounds caffeine and testosterone. Franz cell type, the donor and receptor media for hydrophilic/lipophilic substances, albumin and tensid addition, and storage conditions of the excised skins were systematically varied. A protocol has been developed which now allows to proceed to the pre-validation process.

RU 58841-myristate--prodrug development for topical treatment of acne and androgenetic alopecia.

Acne and androgenetic alopecia are linked to androgen effects and therefore should improve following topical application of antiandrogens. We present a new antiandrogen prodrug, RU 58841-myristate (RUM) for topical therapy. Almost devoid of affinity to the androgen receptor, as derived from investigations in the mouse fibroblast cell line 29 +/GR +, RUM is rapidly metabolised to the potent antiandrogen RU 58841 by cultured human foreskin fibroblasts and keratinocytes, male occipital scalp skin dermal papilla cells, and by cells of the sebaceous gland cell line SZ95. In order to improve a specific targeting of the hair follicle, RUM was loaded on solid lipid nanoparticles (SLN), which are already known to support dermal targeting effects. Physically stable RUM loaded SLN were produced by hot homogenization. Penetration/permeation studies carried out using the Franz diffusion cell proved only negligible permeation of reconstructed epidermis and excised porcine skin within 6 h, implying a more topical action of the drug. Targeting to the hair follicle using SLN was visualised by fluorescence microscopy, following the application of Nile Red labelled SLN to human scalp skin. Transmission electron microscopy (TEM) allowed to detect intact silver labelled SLN in porcine hair follicles of preparations applied to the skin for 24 h. RUM loaded SLN should be considered for topical antiandrogen therapy of acne and androgenetic alopecia.

Cutaneous estradiol permeation, penetration and metabolism in pig and man.

AIM AND METHODS: Drug development in dermatotherapy and also development of transdermal therapeutic systems (TTS) demand high-predictive in vitro models to estimate drug levels in skin and systemic uptake. Here we compare three ready-to-use models, reconstructed human epidermis, split porcine skin and the perfused porcine forelimb. 17beta-Estradiol (E(2)), which is highly metabolized by skin cells, serves as model drug since E(2) application is of high relevance in hormone replacement therapy while topical E(2) may promote wound healing. E(2) TTS, gel and an ethanolic solution were investigated for cutaneous penetration, permeation and metabolism. RESULTS: E(2) TTS enabled an E(2) uptake of 42.9% of the applied dose accompanied by a high percentage of E(2) metabolism (30% of the penetrated dose) in the perfused porcine forelimb. In Franz cell experiments with reconstructed human epidermis and split porcine skin, the gel allowed an E(2) uptake of 41.7 and 22.9% of the applied dose accompanied by a high E(2) metabolism (42.6 and 28.6% of the penetrated dose). Due to toxic effects of the vehicle, this was not true with an ethanolic solution, then E(2) permeation and metabolism were clearly diminished. Most importantly, the in vitro models proved to be predictive with respect to the E(2)/estrone ratio in female plasma under transdermal hormone replacement therapy. CONCLUSION: In vitro tests should reduce the need for both animal and human studies for cutaneous uptake and metabolism in the future.

Drug targeting by solid lipid nanoparticles for dermal use.

Long term topical glucocorticoid treatment can induce skin atrophy by the inhibition of fibroblasts. We, therefore, looked for the newly developed drug carriers that may contribute to a reduction of this risk by an epidermal targeting. Prednicarbate (PC, 0.25%) was incorporated into solid lipid nanoparticles of various compositions. Conventional PC cream of 0.25% and ointment served for reference. Local tolerability as well as drug penetration and metabolism were studied in excised human skin and reconstructed epidermis. With the latter drug recovery from the acceptor medium was about 2% of the applied amount following PC cream and ointment but 6.65% following nanoparticle dispersion. Most interestingly, PC incorporation into nanoparticles appeared to induce a localizing effect in the epidermal layer which was pronounced at 6 h and declined later. Dilution of the PC-loaded nanoparticle preparation with cream (1:9) did not reduce the targeting effect while adding drug-free nanoparticles to PC cream did not induce PC targeting. Therefore, the targeting effect is closely related to the PC-nanoparticles and not a result of either the specific lipid or PC adsorbance to the surface of the formerly drug free nanoparticles. Lipid nanoparticle-induced epidermal targeting may increase the benefit/risk ratio of topical therapy.

Consequences of long-term selenium-deficient diet on the prostacyclin and thromboxane release from rat aorta.

It is known that peroxides, which are increased during Se deficiency because of reduced glutathione peroxidase (GSH-Px) activity, can influence the prostacyclin I2/thromboxane A2 (PGI2/TXA2) ratio. In this study we analyzed the PGI2 and TXA2 formation of aortas of long-term Se-deficient rats. Despite low GSH-Px activity in the Se-deficient group, the basal PGI2 and TXA2 formation was not different versus control animals (PGI2: 2295+/-1134 pg/mg vs 2940+/-1134 pg/mg; TXA2: 3.83+/-1.06 pg/mg vs 5.67+/-2.99 pg/mg). However, we checked the capacity of the aortas of Se-deficient rats to compensate for a suddenly increased peroxide concentration. After peroxide stimulation, the PGI2 release was significantly lower in the Se-deficient group compared to the control group (PGI2: 3507+/-1829 pg/mg vs 7986+/-2636 pg/mg). Again, the TXA2 release did not show any differences. The release ratio of PGI2/TXA2 decreased under peroxide stress in Se-deficient animals. Although long-term Se deficiency showed a relatively well-balanced metabolism under resting conditions, sudden stress, accompanied by an excessive radical production, cannot be compensated.

Structure of transfection-active histone H1/DNA complexes.

Relationships between the structure of transfecting complexes of histone H1 and DNA and their transfection efficiency were studied. Transfection activity proved to be connected to complex aggregates. Low speed centrifugation of the complexes resulted in loss of the transfection activity. The complexes/aggregates were active with high efficiency in a broad range of weight input ratios r(i) (0.1 < r(i) < 30). Using atomic force microscopy (AFM), the complexes were imaged at negative, nearly electroneutral and positive charge conditions. Electroneutral complexes at r(i) = 1 showed a multitude of different complex forms. Fibrillar, network-like and branched structures were frequently present in one complex. Strongly positive charged complexes had a toroidal appearance. All these different forms contributed to the high transfection efficiency. Cellular uptake is supposed to be by phagocytosis.

Oxygen radical generation of neutrophils: a reason for oxidative stress during marathon running?

Hematological parameters and blood markers that indicate oxidative stress, such as lipid peroxides (LPO), reduced and oxidized glutathione (GSH, GSSG), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), were measured in 18 marathon runners before, immediately after the race, and after 8 days of rest. In parallel, the oxygen radical generation of neutrophils (PMN) was measured by chemiluminescence in six randomly selected runners. After the race, a 4.4-fold enhanced PMN count and a 1.4-fold increased capacity to generate oxygen radicals of the PMN (2.20+/-0.38 vs. 3.12+/-0.69 arb. unit/10(6) cells) were found. Consequently, a 6.25-fold increased capacity to generate oxygen radicals of the post-run blood (7.26+/-1.3 vs. 45.40+/-10.3 arb. unit/ml blood) was calculated. This points to PMN as an important oxygen radical source established in the runners' blood, which could contribute to the oxidative stress indicated in the post-run blood by increased LPO (11.46+/-3.09 vs. 13.09+/-3.14 micromol/l plasma), GSSG (0.038+/-0.003 vs. 0.045+/-0. 005 mmol/l blood) and GSSG/GSH ratio (3.8+/-0.5 vs. 4.1+/-0.6%) and by decreased SOD (15.63+/-1.78 vs. 14.58+/-1.51 10(3)U/mmol Hb) and GSH-Px (485.1+/-107.1 vs. 434.9+/-101.7 U/mmol Hb). Despite the decline of the oxygen radical source during rest, the oxidative stress in the blood did not decrease in all runners.

Histone H1-mediated transfection: serum inhibition can be overcome by Ca2+ ions.

PURPOSE: One of the drawbacks of polycationic and cationic liposomal gene transfer is its sensitivity to serum. Gene therapy requires the transfectant-DNA complex to be resistant to serum as well as blood. Since Ca2+ has proved to be an efficient cofactor of polycationic gene transfer, we decided to investigate its effects on transfection in the presence of serum. METHODS: We studied transgene expression of luciferase gene (pCMV Luc) on ECV 304 human endothelial cells using H1 histone and DOSPER as transfectants in the presence of 0-100% fetal calf serum. RESULTS: H1-and DOSPER-mediated transfection was found to be inhibited by serum above the concentration of 10%. If 2 mM Ca2+ or 2 mM Ca2+/0.1 mM chloroquine was included in the culture medium which replace the transfection mixture and was left on the cells for 24 hours postincubation, the inhibiting effect of even 100% serum was overcome. CONCLUSIONS: A high serum level does not interfere with binding and uptake of H1- and DOSPER-DNA complexes, but inhibits subsequent steps such as endosomal escape. Ca2+ in the form of nascent calcium phosphate microprecipitates and other lysosomolytical agents facilitate endosomal/lysosomal release by their fusigenic and membranolytic activity.

Immunocytochemical visualization of transfected DNA in cultured cells.

Nonviral transfection is one of the modern methods for the incorporation of foreign genes into cells. This process involves uptake of foreign genetic material by the cell and further trafficking through the cytoplasm to the nucleus. Elucidation of cytoplasmic pathways of transfection complexes can be useful to improve already existing gene delivery systems or to establish new systems. To monitor transfection complexes in the cell during transfection, we elaborated a method for the visualization of transfection complexes by introducing digoxigenin-labelled nucleotides into foreign DNA followed by detection of digoxigenin label with the use of antibodies directed against digoxigenin. This procedure allowed the visualization of DNA in transfection complexes and to monitor these complexes in cells during transfection.

Calcium ions as efficient cofactor of polycation-mediated gene transfer.

We investigated the effect of calcium on the transfection of non-viral DNA transfer systems. Cationic proteins such as the nuclear protein H1, the polycation polylysine and a number of commercial transfection agents exhibited high transfection rates in the presence of Ca2+. Without Ca2+ H1 and HMG1 were inactive in transfection of the human permanent endothelial cell line ECV 304 while cationic liposomes such as Lipofectin and Lipofectamine did not show any Ca2+ dependence. More detailed experiments showed that Ca2+ was replaceable by the lysosomotropic agent chloroquine. Furthermore, it was possible to separate the transfection-enhancing role of Ca2+ from the actual transfection process by adding Ca2+ to the cells after the transfection period and still to obtain a significant transgene expression. This makes it possible to distinguish between cellular uptake of H1 (or mediator)-DNA complexes and endocytotic release. We also replaced soluble Ca2+ by Ca-phosphate precipitates not containing DNA and obtained similar transfection results. This allowed us to suggest that the addition of free Ca2+ to the transfection medium resulted in nascent Ca-phosphate microprecipitates. The known fusogenic and membranolytic activity of such microprecipitates could facilitate the transport through and the release of the transfecting complexes from the endosomal/lysosomal compartment.

Ligand-histone H1 conjugates: increased solubility of DNA complexes, but no enhanced transfection activity.

We introduced galactose and a short RGD sequence as ligands into H1 histone to target the asialoglycoprotein receptor or integrins on cells expressing these receptors. The efficiency of the gene transfer mediated by galactosylated H1 histone was strongly affected by the transfection conditions. Galactosylation of H1 led to an increase of the basic H1-mediated gene transfer activity only, when H1 itself did not develop its optimal transfection activity. Under other conditions any specific gene transfer mediated by the asialoglycoprotein receptor was covered by the high transfection efficiency of H1 itself. Similar results of a marginal increase in the transfection efficiency were obtained by conjugates of a short RGD sequence and H1. This unexpected failure in the receptor specificity of both conjugates could be due to the unspecific cell-binding capacity of the H1 moiety and to increasing solubility of the complexes as shown by gel shift and solubility measurements.

Polycation-mediated transfection: how to overcome undesirable side effects of sticky DNA complexes.

Using polycationic transfection one encounters undesired persistent binding to cells of sticky polycation/DNA complexes. These complexes simulate transfection under conditions where no uptake is expected e.g. at 4 degrees C if the uptake is by endocytosis. To overcome this problem, using H1/DNA complexes, we developed an easy and nontoxic method for removing the sticky complexes not taken up during the transfection phase. The cells are simply washed with isotonic (0.1 M) MgCl2 solution, which enables the complete removal of the complexes by their rapid dissolution.

Acid nuclear extracts as mediators of gene transfer and expression.

In an attempt to demonstrate transfection-active DNA packaging proteins in the cell nucleus, we prepared acid nuclear extracts with perchloric acid and subsequent protein fractions by stepwise acetone precipitation. The original extract and these fractions containing different compositions of nuclear proteins were used as DNA packaging agents. After the formation of complexes between these protein fractions and reporter genes, the addition of these complexes to the cells resulted in high transfection rates. Gel electrophoresis shows that the most active fractions contain histone H1 and HMG17. HMG1 exhibits a smaller activity. This result was confirmed by positive transfection experiments with commercial histone H1. Our results show that the transfection activity of acid nuclear protein fractions and histone H1 is dependent on the presence of calcium.

A new efficient method for transfection of neonatal cardiomyocytes using histone H1 in combination with DOSPER liposomal transfection reagent.

Although cationic lipids are successfully used for gene transfer in vitro, primary cells such as neonatal cardiomyocytes frequently resist efficient transfection. We show here that the polycationic lipid DOSPER in combination with histone H1 was much more efficient in transfection of neonatal cardiomyocytes than DOSPER alone or other cationic lipids. This has been shown for transfection with the reporter plasmids pSV beta-gal and pCMV luc. If viral transfections are not possible, this mild method is an alternative to transfect cardiomyocytes.

The ratio of formation of prostacyclin/thromboxane A2 in HUVEC decreased in each subsequent passage.

In this study, we analyzed the antioxidative potential (SOD-, GSH-Px-activity) and the basal, H2O2- and ATP-stimulated formation of PGI2 and TXA2 in human umbilical vein endothelial cells (HUVEC) of different passages. The subcultivation of cells partly represents the process of aging. Both subcultivation of the cells and the H2O2 incubation did not significantly influence the activity of SOD and GSH-Px. H2O2 (0.1 mM and 1.0 mM) stimulated the generation of PGI2 and TXA2 in the cell passages time dependently. The formation ratio of PGI/TXA2 changed from 640:1 (0.1 mM H2O2) or 430:1 (1.0 mM H2O2, 40 min incubation) at the 1st passage, to 13:1 and 17:1, respectively, at the 4th passage. This resulted from the reduction of the PGI2 synthesis connected with more pronounced TXA2 formation. The same behavior was found in the basal and ATP-stimulated eicosanoid formation. Based on this, the age-dependent activation of the oxygen radical formation could be responsible for the modified eicosanoid metabolism resulting in vascular complications in the elderly.

H1 and HMG17 extracted from calf thymus nuclei are efficient DNA carriers in gene transfer.

In this article we describe the chromatographic separation of acid nuclear protein fractions which have previously been shown to be active in DNA transfection experiments. By combining anionic and cationic ion exchangers, we were able to separate and identify some of the active proteins. In addition to HMG1, already known for its transfection activity, we have identified histone H1 and HMG17 as further transfection-active proteins. The highest transfection activity was associated with H1 and another nonidentified protein showing a somewhat higher electrophoretic mobility than H1. We have also found that the presence of CaCl2 in a low concentration in the cell culture medium is an important requirement for transfection.