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Biomedical subjects

A Bader

Publications and source records attributed to A Bader.

At least 91 records · Page 5Linked to original sources
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↗
PubMed1995

A novel bioreactor design for in vitro reconstruction of in vivo liver characteristics.

We have constructed a bioreactor aimed at imitating the three-dimensional micro- and macroenvironment of the liver. In vivo hepatocytes are arranged in plates of cell monolayers and are specifically attached with both sinusoidal surfaces to the space of Disse which contains extracellular matrix. Nonparenchymal cells are located on the other side of the space of Disse toward the sinusoid. For supporting monolayer hepatocytes with bipolar attachment to the extracellular matrix, we used a double gel culture technique that sandwiches hepatocytes between two layers of collagen. In double gel cultures, albumin production increases during an adaptive period to the in vitro environment. In contrast to conventional single gel hepatocytes, double gel hepatocytes maintain expression of sinusoidal microvilli and a polyhedric cell shape in culture as seen by transmission electron microscopy. Albumin production in the bioreactor was stable. The organotypical bioreactor concept is an example of organ mimicry and may provide the basis for the organ-otypical development of a full-sized hybrid artificial liver.

Albumins↗
PubMed1995

Reconstruction of liver tissue in vitro: geometry of characteristic flat bed, hollow fiber, and spouted bed bioreactors with reference to the in vivo liver.

Bioreactors currently being developed for hybrid artificial livers vary greatly with respect to their microenvironment. The specific architecture modifies the relationship parenchymal and nonparenchymal cells have with the exchange surfaces of the bioreactor. Most designs are either based on hollow fiber, spouted bed, or flat bed devices. This diversity is contrasted by the uniform and unique organization of the in vivo liver. The liver cells are arranged as plates and both sinusoidal surfaces of the hepatocytes are enclosed within the matrix of the space of Disse. In this study we intended to define the in vivo liver tissue characteristics in a manner useful for an organotypical approach to hepatic tissue engineering. Transmission electron microscopy of an in vivo liver was utilized to describe these ratios. The ratios defined in this study are based on the constant hepatocellular expression of two sinusoidal surfaces. A relationship is established between the expression of the sinusoidal surfaces and their use as attachment and exchange surfaces inside a bioreactor. The presence of biliary surfaces and nonparenchymal cell surfaces is compared. The functional relevance of an in vivo like extracellular matrix geometry for oxidative biotransformation of primary hepatocytes in vitro was studied using the two model drugs cyclosporin and rapamycin. The generation of the hydroxylated cyclosporin metabolites AM 9 and AM 1 and four rapamycin metabolites was analyzed by high performance liquid chromatography (HPLC). It is shown that the cell-specific biotransformation rates at 1 week in culture in matrix overlayed hepatocytes was 5-10 times that of hepatocytes without matrix overlay. Bilaminar membrane (BLM) bioreactors were used to reconstruct extracellular matrix geometry, three-dimensional cell plates, and sinusoidal analogs in between cell plates.

Animals↗
PubMed1995

Regulation of lineage-specific transcription of the sucrase-isomaltase gene in transgenic mice and cell lines.

Sucrase-isomaltase (SI), a gene expressed exclusively in absorptive enterocytes, was used to examine the molecular mechanisms that regulate cell-specific gene expression in the intestinal epithelium. Transgenic mice were made with a construct containing nucleotides -8,500 to +54 of the mouse SI gene linked to a human growth hormone reporter gene. In adult transgenic animals, high-level transgene expression was limited to the small intestine, with low levels of ectopic expression in the colon. In contrast to the endogenous gene that is expressed only in enterocytes, the transgene was expressed in all four cell lineages, including enterocytes, enteroendocrine, goblet, and Paneth cells. To examine this process of lineage-specific expression further we studied Caco-2 and COLO DM cell lines, which model enterocytes and enteroendocrine cells, respectively. Reminiscent of results in transgenic animals, only Caco-2 cells transcribed the endogenous SI gene, whereas both Caco-2 and COLO DM cells supported transcription from chimeric SI reporter gene constructs. Taken together, these data suggest that each intestinal cell lineage has the cellular machinery to transcribe the SI gene. Moreover, these findings imply that transcription is normally repressed in nonenterocytic cells, possibly via a transcriptional silencer residing outside of the region of the SI gene examined in these studies.

Animals↗
PubMed1995

Metabolism of the immunosuppressant tacrolimus in the small intestine: cytochrome P450, drug interactions, and interindividual variability.

The small intestinal metabolism of tacrolimus, which is used as an immunosuppressant in transplantation medicine, was investigated in this study. Tacrolimus was metabolized in vitro by isolated human, pig, and rat small intestinal microsomes. The metabolites generated were identified by HPLC/MS. Tacrolimus and its metabolites were quantified using HPLC or HPLC/MS. The cytochrome P450 (CYP) enzymes responsible for tacrolimus metabolism in small intestine were identified using specific CYP antibodies and inhibitors. For characterization of the interindividual variability, microsomes were isolated from small intestinal samples of patients who had undergone resection for various reasons. In an in vitro model using pig small intestinal microsomes, 32 drugs were analyzed for their interactions with tacrolimus metabolism. After incubation with human, rat, and pig small intestinal microsomes, the metabolites 13-O-demethyl and 13,15-O-demethyl tacrolimus were identified. The metabolism of tacrolimus by human small intestine was inhibited by anti-CYP3A, troleandomycin, and erythromycin, indicating that, as in the liver, CYP3A enzymes are the major enzymes for tacrolimus metabolism in the human small intestine. Metabolism of tacrolimus by small intestinal microsomes isolated from 14 different patients varied between 24 and 110 pmol/13-O-demethyl tacrolimus/min/mg microsomal protein, with a mean +/- SD of 54.2 +/- 29.2 pmol/min/mg. Of 32 drugs tested, 15 were found to inhibit small intestinal tacrolimus metabolism: bromocryptine, corticosterone, cyclosporine, dexamethasone, ergotamine, erythromycin, ethinyl estradiol, josamycin, ketoconazole, nifedipine, omeprazole, progesterone, rapamycin, troleandomycin, and verapamil. All of these drugs inhibited tacrolimus metabolism by human liver microsomes as well. It is concluded that tacrolimus is metabolized by cytochrome CYP3A enzymes in the small intestine. The rate of the CYP3A enzymatic activities varies about 5 times from patient to patient, and drugs that interfere with the in vitro metabolism of tacrolimus in the liver also inhibit its small intestinal metabolism.

Animals↗
PubMed1994

Use of organotypical cultures of primary hepatocytes to analyse drug biotransformation in man and animals.

1. In conventional single-gel culture systems for primary hepatocytes, rapid loss of drug metabolizing capacities is a common feature and parallels general loss of function. An organotypical (double gel) culture technique for primary hepatocytes is established by enclosing the cells within two layers of extra cellular matrix. This serves to imitate the in vivo microenvironment within the space of Dissé. Using rat hepatocytes, this technique has been shown previously to maintain protein synthetic functions in vitro and to allow more efficient P450A-dependent biotransformation of drugs than a standard single-gel culture system. 2. The aim was to test the capacity of this organotypical culture model for primary rat and human hepatocytes to generate drug metabolites in a typical species-dependent pattern. 3. Urapidil, an antihypertensive drug, was used as a test compound, since it is metabolized in vivo in a species-dependent manner in rat and man. 4. Primary rat and human hepatocytes were cultured within two layers of collagen and exposed to 2.25 micrograms/ml urapidil for periods of 1-24 h at 3 days in culture. Urapidil metabolites were measured using hplc. 5. Metabolite M1 (hydroxylated product) was produced preferentially in human hepatocyte cultures, and metabolites M2/M3 (O-demethylated, N-demethylated product) were preferentially generated in rat cultures. This corresponded to the in vivo pattern found in man and rat, respectively. 6. Since in vitro urapidil metabolism by human and rat hepatocytes cultured in a double-gel system reflects that in vivo, it is suggested that information from such a system may be useful to predict the metabolic pathway of novel xenobiotics and to direct further toxicological evaluation.

Adult↗
PubMed1993

Retroviral infection and expression of cationic amino acid transporters in rodent hepatocytes.

The susceptibility of rodent hepatocytes to infection by mouse type C retroviruses was examined in vivo and in vitro and compared with the expression of two membrane proteins that function as transporters for the cationic amino acids CAT-1 and CAT-2. CAT-1 expression in rodents determines susceptibility to ecotropic retrovirus infection by serving as the virus receptor. Recently, it has been suggested that CAT-2 may be a receptor for amphotropic murine leukemia virus. In the present study, CAT-1 expression was observed in Hepa1, a cell line derived from a murine hepatoma, and in rat hepatocytes propagated on collagen monolayers in vitro but not in intact or regenerating rat liver in vivo. The expression of CAT-1 correlated with susceptibility to infection by an ecotropic retrovirus encoding beta-galactosidase. CAT-2 expression was observed in hepatocytes in vitro and in vivo, consistent with reports of infection of regenerating and cultured hepatocytes by amphotropic retroviruses. However, introduction of murine CAT-2 into nonpermissive Chinese hamster cells was not sufficient to confer susceptibility to amphotropic retrovirus infection, using a protocol that could easily demonstrate CAT-1-dependent infection by an ecotropic virus. Our data establish CAT-1 as a major determinant of ecotropic retrovirus infection in rodent hepatocytes and suggest that CAT-2 is not a receptor for viruses in the amphotropic subgroup.

Animals↗
PubMed1993

Cyclosporine metabolite pattern in blood from patients with acute GVHD after BMT.

The cyclosporine (CYA) metabolite patterns in blood were evaluated in patients with liver dysfunction after allogeneic BMT. Fifty-five BMT patients were included in the study until discharge from hospital. Blood trough levels of CYA and 12 of its metabolites were quantified using HPLC. The patients were assigned to three groups: group I (no acute GVHD, n = 23), group II (acute GVHD of the skin and good liver function, overall acute GVHD: grade I: n = 18, grade II: n = 2) and group III (acute GVHD and liver dysfunction, overall acute GVHD: grade II: n = 2, grade III/IV: n = 8). Analysis of the trough blood concentrations of CYA and its metabolites revealed higher concentrations of metabolite AM19 in group III than in the other groups without reaching statistical significance. During acute GVHD of the liver, the metabolites AM19 (p < 0.01), AM1c9 (p < 0.05) and AM1A (p < 0.05) were significantly elevated compared with patients with normal liver function while CYA and all other metabolites did not differ. The CYA metabolite pattern in patients with acute GVHD and liver involvement was identical with that of liver graft patients during acute graft rejection, while the metabolite patterns of the patients without acute GVHD paralleled that of kidney grafted patients with normal liver function. Acute GVHD of the liver leads to an impaired elimination of CYA with increased blood concentrations of single CYA metabolites.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute Disease↗
PubMed1992

Dephosphorylation of pp19: a common second signal for human T cell activation mediated through different accessory molecules.

Accessory molecules are thought to provide essential regulatory signals for T cell activation. In order to identify specific intracellular events linked to triggering through accessory surface receptors, mAbs against CD2, CD3, CD4, and CD8 were employed to activate resting human T lymphocytes in vitro. Subsequently, intracellular phosphorylation of phosphoprotein (pp) 19, a recently identified substrate of a serine phosphatase involved in CD2 mediated T cell triggering, as well as functional parameters (responsiveness to IL-6, production of IL-2 and IFN-gamma) were determined. As in responses to CD2 mAbs, cross-linking of CD4 and/or CD8 to the TCR-CD3 complex but not CD3 cross-linking alone promoted pp19 dephosphorylation. This early event was in all cases followed by particular late functional responses, i.e. induction of IL-6 responsiveness and secretion of IL-2. In marked contrast, no relationship was found between pp19 dephosphorylation and IFN-gamma production. Taken together, a common intracellular pathway appears to exist in which signals mediated through CD2, CD4, and CD8 merge to promote monokine responsiveness and IL-2 production in human T cells. Dephosphorylation of pp19 thus appears to represent a process which is linked to critical 'second signals' involved in the generation of antigen induced T cell responses.

Antibodies, Monoclonal↗
PubMed1991

A serine phosphatase is involved in CD2-mediated activation of human T lymphocytes and natural killer cells.

We investigated early activation events after T cell triggering via the Ag receptor (TCR/CD3) complex as compared to activation via the CD2 surface molecule. To this end, resting peripheral human T lymphocytes were preincubated with 32P-orthophosphate and subsequently exposed to mitogenic mAb directed at either TCR/CD3 or CD2 for varying time periods. Cells were lysed and postnuclear lysates subjected to two-dimensional-gel electrophoresis (IEF and SDS-PAGE). As early as 10 min after stimulation through CD2, dephosphorylation of a cytosolic 19-kDa protein was observed. In contrast, this protein remained phosphorylated in unstimulated as well as CD3 activated T cells. Phosphoprotein (pp) 19 dephosphorylation was transient because, at later time points (2-4 h) after CD2 triggering, this protein was phosphorylated again. Phosphoaminoacid analysis indicated that pp19 is dephosphorylated on serine residues. Identical results were obtained using a CD2+ but TCR/CD3- human NK cell clone indicating that pp19 dephosphorylation occurs independent of surface expression of a TCR/CD3 complex. These data show that, in addition to protein phosphorylation events, serine dephosphorylation is involved in T cell triggering. More important, a selective signaling mechanism appears to be linked to T cell activation through the CD2 pathway.

Antibodies, Monoclonal↗