PubMed Health⌕ Search

Biomedical subjects

A Bader

Publications and source records attributed to A Bader.

At least 19 recordsLinked to original sources
PubMed1997

Interleukin-6 is necessary, but not sufficient, for induction of the humanC-reactive protein gene in vivo.

We have investigated the involvement of interleukin-6 (IL-6) in the induction of the gene encoding the acute-phase protein human C-reactive protein (hCRP). In transgenic mice the hCRP gene can be induced by lipopolysaccharide (LPS), but not by IL-6. In contrast, hCRP was inducible by IL-6 in primary human hepatocytes and in primary hepatocytes isolated from transgenic mice. To further evaluate the role of IL-6, we introduced the hCRP transgene into animals lacking endogenous IL-6 (IL-6-negative mice). Here, hCRP was not inducible by LPS, but was induced by a combination of LPS and IL-6. These results clearly demonstrate that IL-6 is necessary, but not sufficient, for the induction of hCRP expression. These animal models will allow further dissection of the cytokine network responsible for the regulation of the major human acute-phase reactant CRP.

Animals↗
PubMed1997

Isolation of the putP gene of Corynebacterium glutamicum and characterization of a low-affinity uptake system for compatible solutes.

Corynebacterium glutamicum accumulates the compatible solutes proline, glycine betaine, and ectoine under conditions of high osmolality. Uptake of proline is mediated by both a high-affinity and a low-affinity secondary transport system. The low-affinity uptake system also accepts glycine betaine and ectoine as substrates. In the present study, the gene encoding the high-affinity proline uptake system PutP was isolated by heterologous complementation of Escherichia coli mutant strain WG389, which lacks the transport systems BetT, PutP, ProP, and ProU and is unable to synthesize proline and glycine betaine. This gene (putP) encodes a protein of 524 amino acids that shares identity with the proline transport systems PutP of E. coli, Staphylococcus aureus, Salmonella typhimurium, Haemophilus influenzae, and Klebsiella pneumoniae. Functional studies of PutP synthesized in E. coli mutant strain MKH13, which also lacks the transport systems for compatible solutes and is unable to synthesize glycine betaine, revealed that this carrier system is not regulated by the external osmolality on the level of activity. Km values of 7.6 mM for proline and 1.3 mM for sodium as cotransported ion were determined. Deletion of the putP gene allowed the functional characterization of another proline uptake system with low affinity.

Amino Acid Sequence↗
PubMed1997

Application of a stable cell culture assay for the functional assessment of novel MR contrast agents.

The purpose of this study was to apply a new cell culture assay that preserves hepatocyte orientation and differentiation for screening of MR contrast agents with hepatocyte specificity. Cultured hepatocytes were sandwiched between two layers of collagen, preserving both hepatocyte function and morphology over a prolonged period of time. Plain and rhodaminated monocrystalline iron-oxide particles (MION and MION-rh) and asialoglycoprotein receptor-specific rhodaminated asialofetuin coupled to MION (MION-ASF-rh) were prepared. Dose-dependent competition experiments of these agents were performed with D(+)-galactose to determine the specificity of galactose-mediated cell uptake. To assess the impact of cell integrity on cell uptake dose-dependent functional experiments with two hepatotoxins (ethanol and CCl4) were performed. Normal cell cultures showed significantly higher fluorescent-light emission after incubation with hepatocyte-directed ASF-MION-rh than after incubation with MION-rh. Competition experiments of ASF-MION with galactose showed a dose-dependent decrease in calibrated fluorescent-light emission. Cell cultures treated with hepatotoxins demonstrated a dose-dependent reduction in calibrated fluorescent-light emission following incubation with ASF-MION-rh. The validated assay system allows assessment not only of hepatocyte specificity, but also of hepatocyte damage. Because the assay can be applied to cells from any species (rat, pig, human), it may represent an ideal test system prior to clinical trials of new hepatocyte-directed MR contrast agents.

Animals↗
PubMed1996

Isolation of monoclonal antibodies from cell containing hybridoma broth using a protein A coated adsorbent in expanded beds.

A novel expanded bed adsorbent carrying a recombinant protein A ligand was used for the isolation of monoclonal antibodies from cell containing hybridoma fermentation broth. The untreated effluent from a continuous hybridoma cultivation was applied to the stable expanded adsorbent (Streamline rProteinA), which proved to have a high capacity for the MAb studied (14 mg MAb per ml of adsorbent). A clarified and highly concentrated (up to 50 fold) eluate of high purity was obtained. A scale up of the MAb purification is demonstrated from lab scale (250 mg MAb per purification cycle) to a small pilot scale (2 g MAb per cycle). Low product concentration in the broth in combination with the high capacity of the adsorbent caused long sample application cycles (10-11 h). Experimental problems arising from these long cycle times are discussed with regard to a large scale application of the method.

Antibodies, Monoclonal↗
PubMed1996

3-D coculture of hepatic sinusoidal cells with primary hepatocytes-design of an organotypical model.

Models for cocultures of parenchymal (PC) and nonparenchymal cells (NPC) of the liver relied on mixing the cells in a two-dimensional configuration or on establishing spheroidal aggregates. In vivo hepatic nonparenchymal cells, such as endothelial cells and Kupffer cells, are separated from parenchymal cells by extracellular matrix (ECM). Due to their location outside of the space of Disse they can form a barrier toward the sinusoid. Hepatocytes are attached to ECM of the space of Disse via two opposing sinusoidal surfaces. No three-dimensional coculture model reflecting this specific microenvironment of the liver cell plates in vivo has been available to date. We designed a three-dimensional model by positioning NPC on top of PC enclosed as a monolayer within a collagen sandwich. A gas-permeable membrane support can be used to allow the supply of oxygen to the resulting cell plate also from underneath the cell layers. Morphological analysis was performed by inverse and cross-sectional studies by light microscopy, scanning, and transmission electron microscopy of the coculture model. Cuboidal hepatocytes formed confluent layers below the NPC layer. They regularly expressed bile canaliculi at intercellular contact zones. Both sinusoidal surfaces expressed microprojections. Characteristic NPC including endothelial cells, Kupffer cells, and Ito cells completely covered the second matrix layer within a week. Kupffer cells were located on top of endothelial cells. Ito cells were intermingled and could be identified by their intracytoplasmic lipid droplets. LPS stimulation of cocultures resulted in a depression of albumin secretion. Phase I and phase II metabolites of the cytochrome P-450 1A1 substrate ethoxyresorufin were generated independently from the presence of cocultured NPC. This study describes the development of a novel three-dimensional coculture model, which intends to mimic more closely the microenvironment of the hepatic sinusoid by respecting the specific plate structure of the liver parenchyma. The model could serve as a complex tool to study potential collaborations between PC and NPC of the liver.

Albumins↗
PubMed1996

Tacrolimus (FK 506) biotransformation in primary rat hepatocytes depends on extracellular matrix geometry.

Established in vitro models for studies of hepatic drug biotransformation include the use of primary hepatocytes. In normal liver the space of Disse provides the possibility of bilateral attachment to extracellular matrix for each hepatocyte. This configuration is disrupted by the cell isolation procedure of normal liver tissue, which delivers suspensions of round shaped cells. In standard culture configurations this unphysiologic cell shape terminates in a morphological dedifferentiation and inability to biotransform drugs. This study analyses the relevance of extracellular matrix geometry in hepatocyte monolayer configurations for expression and activity of cytochrome P450 3A. This enzyme is involved in the biotransformation of a large number of pharmaceuticals including the immunosuppressants tacrolimus and sirolimus. Morphological analysis of primary rat hepatocytes cultured with and without overlay of collagen type I was performed by transmission and scanning electron microscopy. Expression and activity of cytochrome P450 3A was studied by Western blot and the use of two model drugs specific for this enzyme. To this purpose the immunosuppressive drugs tacrolimus and sirolimus were used. Metabolites were analyzed by HPLC and HPLC/MS. Two sided attachment to extracellular matrix induces profound changes of the hepatocellular morphology in vitro resulting in the reconstitution of a polyhedric cell shape. This phenomenon is paralleled by an enhanced expression of cytochrome P450 3A and corresponding metabolic activity. As shown for tacrolimus biotransformation, the model may be useful to study complex metabolic patterns. In addition this model may facilitate studies of the kinetics of hepatocellular drug biotransformation in a setting with prolonged stability.

Albumins↗
PubMed1996

Identification of drugs inhibiting the in vitro metabolism of tacrolimus by human liver microsomes.

1. Tacrolimus, an immunosuppressive macrolide, is metabolized by enzymes of the cytochrome P450 3A subfamily. In this study, 34 drugs were tested for their interactions with tacrolimus metabolism by human liver microsomes. 2. Fifteen drugs which inhibit the in vitro metabolism of tacrolimus were identified: bromocriptine, corticosterone, dexamethasone, ergotamine, erythromycin, ethinyloestradiol, josamycin, ketoconazole, miconazole, midazolam, nifedipine, omeprazole, tamoxifen, troleandomycin and verapamil.

Bromocriptine↗
PubMed1996

Metabolism of the macrolide immunosuppressant, tacrolimus, by the pig gut mucosa in the Ussing chamber.

1. The macrolide tacrolimus (FK506), used as an immunosuppressant, is a cytochrome P450 (CYP) 3A substrate in the liver. The metabolism of tacrolimus and the transport of its metabolites in the pig gut was studied in the Ussing chamber. Tacrolimus and its metabolites were quantified by h.p.l.c./mass spectrometry. 2. In the Ussing chamber, demethyl, didemethyl, hydroxy and hydroxy-demethyl tacrolimus were generated. Their formation was concentration- and time-dependent. The metabolite pattern was not different from that after incubation of tacrolimus with human small intestinal microsomes. 3. The metabolite formation was highest in the duodenum and declined in the order duodenum > jejunum > ileum > colon > stomach. 4. Since tacrolimus metabolism was inhibited by the specific CYP3A inhibitors, troleandomycin and ketoconazole, we concluded that these enzymes are involved in intestinal metabolism of tacrolimus. 5. Tacrolimus metabolites re-entered the mucosa chamber (> 90%) and passed through the small intestinal preparation into the serosa chamber. 6. It is concluded that tacrolimus is metabolized in the intestine, that the metabolites are able to re-enter the gut lumen and also enter into the portal vein and that small intestinal metabolism and transport is at least in part responsible for the low oral bioavailability of tacrolimus.

Animals↗
PubMed1996

Hepatic tissue culture model for study of host-parasite interactions in alveolar echinococcosis.

An in vitro model for growth and differentiation of the metacestode tissue of the tapeworm Echinococcus multilocularis is described. This model simulates the organotropism of the parasite toward the liver of the intermediate host. In the presence of collagen-embedded primary hepatocytes from rats and humans, which can be kept in culture for 2 to 3 months, the parasitic vesicles grew by exogenous budding and multiplied about 12-fold within 3 weeks. In contrast, without the hepatocytes, the metacestodes rapidly degenerated. Development of protoscolices was seen only in the presence of rat hepatocytes but not in coculture of the metacestodes with hepatocytes of human origin, thus reflecting the in vivo situation during infection of rodents and in alveolar echinococcosis in humans. The experiments indicated that growth of the metacestodes and development of protoscolices depended on soluble low-molecular-weight factors released by the hepatocytes. The in vitro-grown metacestodes did not differ morphologically from the larvae found in infected intermediate hosts, and their infectivity was completely maintained. This report describes the first in vitro model of alveolar echinococcosis and will be the basis for future studies on host-parasite interactions of this important zoonosis.

Animals↗
PubMed1996

Drug interactions and interindividual variability of ciclosporin metabolism in the small intestine.

The undecapeptide ciclosporin is used as immunosuppressant after organ transplantation and for therapy of immune diseases. Low and variable bioavailability of ciclosporin has been attributed to its metabolism in the small intestine. The aim of the present study was to investigate drug interactions and interindividual variability of ciclosporin metabolism in the small intestine. Ciclosporin metabolism was studied in vitro using microsomes isolated from the small intestine of humans and pigs. The metabolites generated were quantified by HPLC and identified by mass spectrometry. Using specific antibodies and inhibitors, we showed that, as in the liver, cytochrome P450 3A (CYP 3A) enzymes are responsible for ciclosporin metabolism in the human small intestine. Of the 28 xenobiotics included in the study, 16 drugs, all well-known CYP 3A inhibitors, inhibited ciclosporin metabolism in the small intestine. In the small intestine of different patients, the rate of metabolism varied by a factor of 10. Ciclosporin was metabolized faster by small intestine microsomes from female (n = 4) than from male (n = 10) patients (p < 0.009).

Adult↗
PubMed1996

[Natural estrogens and the cardiovascular system].

The significant relationship between hormone substitution therapy during menopause and reduced cardiovascular risk has been demonstrated in many studies. The beneficial effect of natural estrogens on plasma lipids has been questioned, accounting for no more than 25% of the vascular effect. Natural estrogens act on the vascular wall by favoring the penetration of potassium into the cell and stimulating synthesis of prostacycline synthetase and prostaglandin cyclooxygenase. Capacity for dilatation is partially restored and sensitivity to vasoconstrictor substances is reduced by increased synthesis of endothelium derived relaxing factor (EDRF). In addition to the endothelium-dependent mechanism, there is also an endothelium-independent mechanism due to the anti-calcium effect and endothelin antagonism. Thus natural estrogens increase vascular flow and reduce resistance. Besides the direct or indirect vascular effect, estrogens also have an anti-atherogenic effect resulting from a modulation of gene expression due to specific receptors situated on smooth muscle and endothelial cells. Indications for hormone substitution have been modified due to this better understanding of the different actions of natural estrogens. Patients with atheromatosis or hypertension are legitimate candidates.

Blood Flow Velocity↗
PubMed1995

Ultrastructural and functional differentiation of hepatocytes under long-term culture conditions.

BACKGROUND: Studies on hepatocytes grown in different culture systems have shown that these cells rapidly dedifferentiate on a single support with liquid medium on top (single gel technique). However, in systems sandwiching them between two layers of extracellular matrix (double gel technique), the cells are able to regain and maintain typical light microscopical appearance and function. Their ultrastructural morphology is as yet unknown. METHODS: Isolated, adult rat hepatocytes were grown in both systems, and their fine structure (thin section electron microscopy) and the functional ability of albumin production (immunoassay) were studied and compared in both culture systems after 2, 7, and 14 days. RESULTS: The hepatocytes in conventional single gel culture did not completely regain their normal morphology and rapidly underwent progressive dedifferentiation. This was characterized by loss of cell polarization in terms of obliteration of the bile canaliculi-like intercellular expansions, loss of cell membrane differentiations, and reduction of organelles. Cytoskeletal components gradually increased, building up large filamentous zones underneath the plasma membrane. In double gel culture, the hepatocytes reachieved and maintained intact morphology and polarity over at least 14 days. The bile canaliculi were formed, preserved, or even enlarged and were associated with dense peribiliary bodies and Golgi fields. The plasma membrane facing both collagen layers bore numerous cytoplasmic microprojections like the sinusoidal surfaces of the hepatocytes in situ. Cell organelles, glycogen particles, and lipid droplets were always present. CONCLUSIONS: The hepatocyte is a cell type in which ultrastructural and functional differentiation are strongly interdependent. For these cells, the morphological microenvironment (i.e. the bipolar position of the extracellular matrix) may be as important or even more decisive for maintenance of normal cell differentiation than modifications of the composition of the matrix itself or addition of other cell types, as focused in other studies.

Albumins↗
PubMed1995

An organotypical in vitro model of the liver parenchyma for uptake studies of diagnostic MR receptor agents.

Testing of receptor-specific MR contrast agents targeted to the liver is hampered by a shortage of viable in vitro models with in vivo-like hepatocellular morphology. Coated pits are ultrastructural signs of an active receptor mediated endocytosis in hepatocytes. Expression of coated pits by matrix overlaid hepatocytes was studied by transmission electron microscopy. Binding of a rhodaminated asialoglycoprotein receptor agent (MION-ASF-rh) was assessed by fluorescence microscopy. Fluorescence of cells exposed to MION-ASF-rh with D(+)-galactose reduced fluorescent light emission to a level of 58% of MION-ASF-rh-induced fluorescence. After preincubation with the hepatotoxin CCl4 a dose-dependent decrease in fluorescent light emission resulted. Hepatocytes maintained a homogeneous cell surface expression, with microprojections, coated pits, and vesicles on both sinusoidal surfaces. Matrix overlaid primary hepatocytes constitute a viable, morphologically and functionally differentiated model. This model can be used to study receptor binding, uptake, and blockage of diagnostic magnetopharmaceuticals under controlled conditions.

Animals↗
PubMed1995

Clinical effects and maternal and fetal plasma concentrations of epidural ropivacaine versus bupivacaine for cesarean section.

BACKGROUND: Ropivacaine is a new amide local anesthetic structurally similar to bupivacaine and mepivacaine. Previous studies showed that ropivacaine has a similar clinical effect as bupivacaine with regard to sensory anesthesia and slightly less motor blockade than bupivacaine. Ropivacaine appears to be less cardiotoxic and arrhythmogenic than bupivacaine. The clinical and pharmacokinetic effects of 0.5% ropivacaine (5 mg/ml) versus 0.5% bupivacaine (5 mg/ml) when used epidurally for elective cesarean section were investigated. METHODS: Using a randomized, double-blind study design, 60 ASA physical status 1 or 2 term parturients presenting for elective cesarean section received either 0.5% bupivacaine (150 mg) or 0.5% ropivacaine (150 mg) epidurally in appropriate fractionated doses over a 10-min period. Onset, duration, and regression of sensory and motor blockade were noted until complete resolution was observed. Quality of intraoperative anesthesia and abdominal wall muscle relaxation were noted. Maternal plasma concentrations of local anesthetic were determined before anesthetic administration and 5, 10, 20, 30, and 60 min and 2, 3, 6, 8, 12, and 24 h after drug injection in 20 subjects. Umbilical cord blood was obtained at time of delivery for acid-base values and determination of the free and total plasma concentration of local anesthetic. Neonates also were examined for neurobehavioral assessments by Scanlon's and Neurologic and Adaptive Capacity Scores at 2 and 24 h after delivery. RESULTS: All patients received satisfactory anesthesia for operation. The onset, duration, and regression of sensory blockade were similar for both groups. Onset of degree 1 and 2 motor blockade was faster, and duration of degree 1 motor block was longer in the group receiving bupivacaine. Hemodynamic sequelae were similar between groups. All neonates had 5-min Apgar scores of 7 or greater and normal acid-base values and neurobehavioral assessments. Pharmacokinetic analysis showed that the Cmax was similar for both drugs (1.3 +/- 0.09 for ropivacaine and 1.1 +/- 0.09 micrograms/ml for bupivacaine). The T1/2 of the terminal decline in plasma concentration was shorter for ropivacaine versus bupivacaine (5.2 +/- 0.60 versus 10.9 +/- 1.08 h, respectively; P < 0.01). The free (i.e., unbound) concentrations of ropivacaine were approximately twice those of bupivacaine in both maternal and neonatal blood at the time of delivery. The ratio of umbilical vein to maternal vein concentration of unbound drug was 0.72 for ropivacaine and 0.69 for bupivacaine. CONCLUSIONS: Ropivacaine, 0.5%, epidurally provided satisfactory and similar sensory anesthesia compared to 0.5% bupivacaine for elective cesarean section. The Cmax was similar for both drugs, although the terminal half-life of ropivacaine was significantly shorter, and the blood concentrations of free ropivacaine were significantly greater than that for bupivacaine. These values were less than concentrations shown to be toxic in animals.

Adolescent↗
PubMed1995

Parallel blood concentrations of second-generation cyclosporine metabolites and bilirubin in liver graft recipients.

Cyclosporine, a cyclic undecapeptide, is currently the major immunosuppressant used after liver transplantation. Since it is unclear whether or not cyclosporine metabolites play a part in toxicity, high concentrations of metabolites should be avoided. The quantification of cyclosporine metabolites requires immunoassays using nonspecific antibodies cross-reacting with metabolites or high-performance liquid chromatography (HPLC) analysis. Since no guidelines are available to date concerning when such additional analysis is required, it was the aim of this study to define biochemical parameters that parallel cyclosporine elimination and indicate whether or not cyclosporine elimination is impaired, requiring quantification of cyclosporine metabolites. One hundred and thirty adult liver graft recipients were included in a prospective study during their first hospital stay. Cyclosporine and 11 metabolites were quantified in blood every second day using radioimmunoassay and HPLC. When the cyclosporine metabolite patterns in trough blood samples of patients with impaired liver function were compared with those of patients with good liver function, concentrations of metabolites AM19 and AM1A were found to be elevated. Serum concentrations of conjugated and total bilirubin were significantly correlated with blood trough concentrations of AM19 and AM1A, while there was no correlation with cyclosporine or its first-generation metabolites. Distribution statistics showed that liver graft patients with impaired cyclosporine elimination had total bilirubin concentrations in serum > 60 mumol/l L. No correlation was found between bile acids and the concentrations of metabolites AM19 and AM1A, suggesting that the ion-coupled transport system is not quantitatively involved in cyclosporine excretion and that bilirubin and cyclosporine metabolites are eliminated by the same transport system through the biliary membrane. It is concluded that bilirubin and cyclosporine metabolite concentrations are strictly parallel and that the total bilirubin concentration in serum may be used as an indicator of impaired cyclosporine elimination.

Administration, Oral↗