Biomedical subjects

T H Müller

Publications and source records attributed to T H Müller.

At least 37 records · Page 2Linked to original sources

PubMed1997

Antagonism of the GPIIb/IIIa receptor with the nonpeptidic molecule BIBU52: inhibition of platelet aggregation in vitro and antithrombotic efficacy in vivo.

The glycoprotein (GP) IIb/IIIa (the alphallb beta3 integrin) found on platelets binds fibrinogen or von Willebrand factor when the platelet is activated, thereby mediating the aggregation of platelets. Blockade of the GPIIb/IIIa should prevent platelet aggregation independent of the substance or substances responsible for activating the platelets. This comprehensive inhibition of platelet aggregation is thought to be an effective therapeutic approach to various clinical thromboembolic syndromes. This study investigated the platelet inhibition provided by blocking GPIIb/IIIa by using a new nonpeptidic molecule, BIBU52, in both in vitro and in vivo models. BIBU52 competes with [125I]fibrinogen for binding sites on human platelets in a Ca2+ and pH-dependent manner with a 50% inhibitory concentration (IC50) of 35 +/- 12 nM. BIBU52 inhibited the aggregation of human platelets in platelet-rich plasma induced by collagen (1-2 microg/ml), adenosine diphosphate (ADP; 2.5 microM), and a thrombin receptor-activating peptide (TRAP; SFLLRNPNDKYEPF-NH2; 25 microM) with IC50 values of 82, 83, and 200 nM, respectively. The inhibition of platelet aggregation by BIBU52 was found to be highly species dependent. BIBU52 inhibited aggregation in plasma from rhesus and marmoset monkeys with an IC50 of 150 nM but was totally ineffective in rat plasma. The selectivity of BIBU52 for inhibiting GPIIb/IIIa in comparison with other adhesion molecules was investigated in a human endothelial cell adhesion assay. The adhesion of human endothelial cells to matrices of vitronectin, fibronectin, collagen I, or laminin was not affected by concentrations as high as 100 microM BIBU52; thus BIBU52 demonstrates a high selectivity for the human GPIIb/IIIa. The antithrombotic effect of BIBU52 in vivo was investigated in three animal models of recurrent arterial thrombus formation. In the guinea pig aorta, BIBU52 inhibited thrombus formation dose dependently, with lack of thrombus formation for 1 h after a bolus dose of 1.0 mg/kg i.v.. Both acetylsalicylic acid and dazoxiben were less effective in this model. In pigs with recurrent thrombus formation in the carotid artery, 1.0 mg/kg i.v. also inhibited thrombus formation. Heparin was not effective in the pig, and acetylsalicylic acid was only partially effective. In the pig, the dose of 1.0 mg/kg i.v. BIBU52 also was associated with a 70% inhibition of collagen-induced platelet aggregation ex vivo but with only a transient prolongation of sublingual bleeding time to a maximum of 2.5-fold and without other hemodynamic effects. In the marmoset monkey, a dose of 10 microg/kg i.v. could abolish recurrent arterial thrombosis. Hemodynamic effects of BIBU52 in anesthetized pigs were not detected in doses < or = 10 mg/kg. These data demonstrate that BIBU52 is a potent and selective antagonist of the human GPIIb/IIIa receptor and capable of substantial inhibition of platelet aggregation in vitro and ex vivo as well as inhibition of arterial thrombus formation in vivo in animal models of thrombosis.

Animals↗

PubMed1997

Synaptic connectivity in cultured hypothalamic neuronal networks.

We have developed a novel approach to analyze the synaptic connectivity of spontaneously active networks of hypothalamic neurons in culture. Synaptic connections were identified by recording simultaneously from pairs of neurons using the whole cell configuration of the patch-clamp technique and testing for evoked postsynaptic current responses to electrical stimulation of one of the neurons. Excitatory and inhibitory responses were distinguished on the basis of their voltage and time dependence. The distribution of latencies between presynaptic stimulation and postsynaptic response showed multiple peaks at regular intervals, suggesting that responses via both monosynaptic and polysynaptic paths were recorded. The probability that an excitatory event is transmitted to another excitatory neuron and results in an above-threshold stimulation was found to be only one in three to four. This low value indicates that in addition to evoked synaptic responses other sources of excitatory drive must contribute to the spontaneous activity observed in these networks. The various types of synaptic connections (excitatory and inhibitory, monosynaptic, and polysynaptic) were counted, and the observations analyzed using a probabilistic model of the network structure. This analysis provides estimates for the ratio of inhibitory to excitatory neurons in the network (1:1.5) and for the ratio of postsynaptic cells receiving input from a single GABAergic or glutamatergic neuron (3:1). The total number of inhibitory synaptic connections was twice that of excitatory connections. Cell pairs mutually connected by an excitatory and an inhibitory synapse occurred significantly more often than predicted by a random process. These results suggests that the formation of neuronal networks in vitro is controlled by cellular mechanisms that favor inhibitory connections in general and specifically enhance the formation of reciprocal connections between pairs of excitatory and inhibitory neurons. These mechanisms may contribute to network formation and function in vivo.

Animals↗

PubMed1997

Pharmacodynamic models to evaluate antithrombotics in clinical pharmacology.

The need to prevent thromboembolic events effectively and safely has stimulated an intense search for novel antithrombotics. Parameters derived from in vitro tests with patients' blood are essential for therapeutic monitoring of anticoagulants. Clinical pharmacologic evaluation of novel antithrombotic therapies based on such parameters can easily fail, however, by neglecting pivotal pathophysiologic determinants of thrombus formation. When vascular injury occurs, blood cells, plasma proteins, and the vessel wall intimately cooperate for an adequate local repair. Much remains to be learned about the local and transient interaction of these components. In most tests of platelet function and coagulation proteins, blood samples from treated individuals are stimulated in vitro to assess inhibitory effects. Limitations of such a test strategy for dose-finding studies with antithrombotics may be overcome by measuring activation markers specifically generated on the surface of blood cells or in plasma at the site of thrombosis in patients.

Animals↗

PubMed1997

Inhibition of platelet aggregation as a surrogate marker.

Using acetylsalicylic acid-dipyridamole, a combined thromboxane receptor antagonist-thromboxane synthase inhibitor, and a fibrinogen receptor antagonist as examples, this article discusses the predictive value of several methods that may be employed in the evaluation of antiaggregatory effects and their suitability as surrogate markers for the planning of patient studies. Platelet aggregation in various ex vivo tests and the effects of drugs on these tests were investigated using platelet aggregation in platelet-rich plasma and in whole blood, and in thrombus formation on a thrombogenic surface. Drug-induced inhibition of platelet aggregation is based on the modulation of metabolic processes or interactions at the membrane-receptor level. These effects must be assessed by methods adapted specifically to each mode of action, for example, mediator synthesis inhibition (cyclooxygenase), thromboxane receptor antagonism-thromboxane synthase inhibition, or fibrinogen receptor blockade. Thus a combination of both general and specific methods adapted to the respective mode of action of the test substance can serve as surrogate markers of drug efficacy for the efficient planning of clinical trials.

Animals↗

PubMed1997

Leuko-reduction using an integral Sepacell filter early (4 h, 24 h) or late (48 h, 120 h) after plateletpheresis does not affect platelet function.

Leuco-reduction of aliquots of single-donor platelet concentrates with an integral Sepacell PLS-5A filter was performed 4, 24, and 48 h after apheresis including a control after 120 h (not recommended by the manufacturer) to evaluate the effect of both early and late filtration on the concentrates in a paired study. Highly efficient (> 2 log10) leuko-reduction was consistently observed for filtration any time after apheresis. Various proaggregatory stimuli were tested to determine the stimulus concentration (EC50 values) required for half-maximal aggregation of platelet samples (200 platelets/nl). Neither glycoprotein IIb/IIIa-dependent (thrombin-, collagen-, and APD-induced) nor glycoprotein Ib-mediated (ristocetin-induced) platelet function were affected by filtration 4, 24, 48, or 120 h after plateletpheresis. For single-donor plateletpheresis using a closed system with an integral Sepacell filter, our in vitro results suggest that the timing of leuko-reduction does not affect the platelet-dependent hemostatic qualities of the product.

Equipment Design↗

PubMed1997

Platelet aggregation in response to collagen and thrombin reliably detects the ingestion of low-dose aspirin.

The exposure of blood donors to aspirin is not reliably excluded by pharmacokinetic measurements due to the irreversible effects of aspirin which persist even after elimination of aspirin and its metabolites from plasma. Tests of platelet functions do overcome this deficit, but are usually limited by substantial inter-individual variability of parameters of platelet function. We have evaluated platelet aggregation ex vivo in 20 aspirin-treated (100 mg single oral dose/day) patients in comparison with a control group of 20 aspirin-free donors. The results demonstrate a significant reduction in the collagen(2.5 micrograms/ml)-induced platelet aggregation by aspirin treatment, whereas thrombin(50 mumol/l TRAP-6)-induced platelet aggregation was not affected at all. Assessment of collagen-induced platelet aggregation relative to platelet responses of the same subject elicited either by thrombin or by a combination of collagen and thrombin does substantially improve the reliability of functional assays of aspirin.

Administration, Oral↗

PubMed1997

Routine rhesus-D genotyping in amniotic fluid: analysis of exon 10 versus intron 4 by polymerase chain reaction.

Both the exon 10 and the intron between exons 4 and 5 of the Rh-D gene were analyzed in DNA from amniotic cells by polymerase chain reaction (PCR) in order to determine the fetal Rh-D status. Residual (0.3-2 ml) samples of amniotic fluid obtained by diagnostic amniocentesis in Rh-D-negative women were analyzed. It is important to standardize the sampling and preanalytical storage and to optimize DNA extraction procedures as well as the detection of PCR products for maximal sensitivity. Genotyping was successful in approximately 90% (229 of 256) of amniotic fluid samples tested. Consistent Rh-D results were obtained by both methods in all 229 typed samples with a single exception. Our experience demonstrates that routine genotyping of the Rh-D blood group in small volumes of amniotic fluid (0.5-2 ml) is feasible. Its validity is currently tested by comparison of the prenatal genotype with the serotype of the newborn.

Amniocentesis↗

PubMed1996

Calcium channel types contributing to excitatory and inhibitory synaptic transmission between individual hypothalamic neurons.

The contribution of L-, N-, P- and Q-type Ca2+ channels to excitatory and inhibitory synaptic transmission and to whole-cell Ba2+ currents through Ca2+ channels (Ba2+ currents) was investigated in rat hypothalamic neurons grown in dissociated cell culture. Excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs) were evoked by stimulating individual neurons under whole-cell patch-clamp conditions. The different types of high-voltage-activated (HVA) Ca2+ channels were identified using nifedipine, omega-Conus geographus toxin VIA (omega-CTx GVIA), omega-Agelenopsis aperta toxin IVA (omega-Aga IVA), and omega-Conus magus toxin VIIC (omega-CTx MVIIC). N-, but not P- or Q-type Ca2+ channels contributed to excitatory as well as inhibitory synaptic transmission together with Ca2+ channels resistant to the aforementioned Ca2+ channel blockers (resistant Ca2+ channels). Reduction of postsynaptic current (PSC) amplitudes by N-type Ca2+ channel blockers was significantly stronger for IPSCs than for EPSCs. In most neurons whole-cell Ba2+ currents were carried by L-type Ca2+ channels and by at least two other Ca2+ channel types, one of which is probably of the Q-type and the others are resistant Ca2+ channels. These results indicate a different contribution of the various Ca2+ channel types to excitatory and inhibitory synaptic transmission and to whole-cell currents in these neurons and suggest different functional roles for the distinct Ca2+ channel types.

Animals↗

PubMed1996

Rhesus D genotyping in amniotic fluid.

Rhesus D genotyping by PCR of amniotic fluid offers a very valuable tool for prenatal care of Rhesus D-alloimmunized women. Misleading results have been reported for the method of Bennett et al. analysing the 3'-terminal end of the RhD gene. We have observed also problems with Rhesus D genotyping of DVI-variant samples in the assay of Simsek et al. utilizing size differences of intron 4 between the Rhesus D and the Rhesus CcEe gene. We, thus, prefer to amplify two different regions including the 3'-terminal region of the Rhesus D gene to further minimize the very low risk of false-negative results. This rapid Rhesus D genotyping usually does not require more than 2 ml of amniotic fluid.

Amniocentesis↗

PubMed1995

Signaling by E-selectin and ICAM-1 induces endothelial tissue factor production via autocrine secretion of platelet-activating factor and tumor necrosis factor alpha.

Based on previous studies showed adhesion molecule-dependent induction of tissue factor upon endothelium-lymphocyte interactions, we investigated whether E-selectin and ICAM-1 are linked to signaling pathways leading to tissue factor gene expression. Cellular interaction was mimicked by antibody cross-linking of E-selectin and ICAM-1 on the surface of human umbilical vein endothelial cells (HUVECs), resulting in induction of tissue factor mRNA and protein expression. Tissue factor production could be independently abolished by antibodies against TNF-alpha and by WEB 2086, a platelet-activating factor (PAF) receptor antagonist. Because WEB 2086 prevented the production and/or secretion of TNF-alpha by HUVECs, these results provide evidence for E-selectin- and ICAM-1-linked signal pathways leading to tissue factor synthesis in endothelial cells via an autocrine feedback loop involving PAF and TNF-alpha secretion.

Antigen-Antibody Reactions↗

PubMed1995

Lymphocyte adhesion to human endothelial cells induces tissue factor expression via a juxtacrine pathway.

To study the effect of lymphocyte adhesion on the procoagulant activity of endothelial cells, we have stimulated HUVECs with interferon-gamma to upregulate adhesion molecules. Subsequent addition of lymphocytes induced the expression of tissue factor (TF) by HUVECs. Both CD4+ and CD8+ T-cells promoted this TF synthesis via distinct adhesion molecules (CD4+ T-cells: E-selectin and ICAM-1; CD8+ T-cells: MHC-I molecules). In addition, tumor necrosis factor-alpha and -beta (TNF alpha, TNF beta) and platelet-activating factor (PAF) were involved in lymphocyte-mediated TF expression on HUVECs. We demonstrate that PAF plays a pivotal role in this process. Adhesion of lymphocytes to endothelial cell surface molecules induced the release of PAF. PAF, in turn, caused the production of TNF alpha and TNF beta, both of which are potent stimulators of TF expression.

Azepines↗

PubMed1995

[Sleep of sleep research personnel--results of the Munster Congress survey].

Due to different knowledge of sleep and sleep disorders (i.e. focussing) sleep quality of sleep-researchers may be better or worse than the sleep of other scientists. On the Münster Congress for Sleep-research and Sleep medicine in March 1994 we investigated this question using the Pittsburger Sleep Quality Index (PSQI). 46 sleep researchers, 52 practitioners and 103 scientists of other disciplines from the Münster University filled out the questionnaire. Results show no difference in overall sleep quality between the three groups, but only sleep quality in the group of sleep researchers shows a bimodal distribution suggesting a subpopulation with more serious sleep problems. 10 to 15% of the total academic population can be regarded as "poor" sleepers. Especially the group of practitioners, in which we found the highest values of daytime sleepiness, seems to be a risk-population for sleep disorders.

Adult↗

PubMed1994

6,6-Disubstituted Hex-5-enoic acid derivatives as combined thromboxane A2 receptor antagonists and synthetase inhibitors.

A series of omega-disubstituted alkenoic acid derivatives were designed and synthesized as antithrombotic inhibitors of thromboxane A2 synthetase and thromboxane A2 receptor antagonists. Hexenoic acid derivatives with a 3-pyridyl group and a 4-(2-benzenesulfonamidoethyl)phenyl substituent were found to be optimal with regard to the dual mode of action. The most potent compound, (E)-6-(4-(2-(((4-chlorophenyl)sulfonyl)amino)ethyl)phenyl)-6-(3-pyridyl) hex-5-enoic acid (36), inhibits thromboxane A2 synthetase in gel-filtered human platelets with an IC50 value of 4.5 +/- 0.5 nM (n = 4), whereas an inhibitory effect on cyclooxygenase is seen only at a much higher concentration (IC50: 240 microM). Radioligand-binding studies with [3H]SQ 29,548 in washed human platelets revealed that 36 blocks the prostaglandin H2/thromboxane A2 receptor with an IC50 of 19 +/- 5 nM (n = 5) and is therefore 85-fold more potent than another combined thromboxane A2 synthetase inhibitor/receptor antagonist, Ridogrel (4). Compound 36 inhibits the collagen-induced platelet aggregation in human platelet-rich plasma and whole blood with an EC50 of 1 microM (Ridogrel: 16 microM) and 100 nM, respectively, and was selected for further development.

Blood Platelets↗

PubMed1994

Calcium-activated non-selective channels in the nervous system.

In the decade, since the first description of calcium-activated non-selective (CAN) channels in cardiac myocytes, pancreatic acini and neuroblastoma, this type of channel has been shown to have a ubiquitous distribution across a variety of tissues. Recently, their role in the function of cells of the nervous system has become better delineated. Because CAN channels pass depolarizing current, respond to cytoplasmic Ca2+ activity and do not inactivate, they are capable of producing maintained depolarization of neurons. This property endows upon CAN channels an important role in both physiological functions and pathological processes of the nervous system.

Animals↗

PubMed1994

Pharmacology of current and future antithrombotic therapies.

This article reviews the mode of action of pharmacologic agents for anti-thrombotic therapy. It refers to both clinically established drugs and new approaches currently under preclinical or clinical development. Advantages and limitations of the different pharmacologic strategies are discussed with special attention to their effects on local interaction between the blood and the vessel wall.

Animals↗

PubMed1993

Characterization of neurochemical phenotypes in cultured hypothalamic neurons with immunohistochemistry and in situ hybridization.

The expression of neurochemical phenotypes was studied in long-term cultures of dissociated embryonic neurons from rat hypothalamus. With time in culture, these neurons establish a complex dendritic and axonal network, as indicated by staining with antibodies against microtubulin-associated protein (MAP2) and neurofilaments (SMI32 and SMI33) as well as GABA and glutamate decarboxylase mRNA immunoreactivity. Neurons expressing neuropeptide Y (NPY) mRNA and NPY peptide and opioid-like peptides as well as vasopressin were observed. Further, weakly acetylcholinesterase- and NADPH diaphorase (nitric-oxide synthase)-labelled neurons were present. In conclusion, the neurochemical phenotypes reported for hypothalamic neurons in vivo can be observed in these cultures. This indicates that the culture conditions allow morphological and molecular differentiation. These findings support the view that long-term hypothalamic cultures provide a valuable model for studying mechanisms of neurosecretion in hypothalamic networks.

Acetylcholinesterase↗

PubMed1993

The effects of heparin and annexin V on fibrin accretion after injury in the jugular veins of rabbits.

We compared the relative abilities of unfractionated heparin and annexin V to prevent fibrin accretion onto injured jugular veins in vivo. Heparin was used to accelerate the inhibition of thrombin by antithrombin III, and annexin V was used to inhibit the assembly of the prothrombinase complex on phospholipid surfaces, thereby blocking thrombin generation. Rabbit jugular veins were isolated in situ, a 2 cm segment was injured by perfusing it with air, and then blood flow was re-established. Five minutes later, each rabbit was injected with heparin (20 U/kg) or annexin V (0.3 mg/kg) and then with 125I-fibrinogen. The amount of 125I-fibrin accumulation onto each injured vessel wall segment was measured 4 h later. Each injured vessel was completely de-endothelialized as a result of the air perfusion as demonstrated by electron microscopy. 125I-fibrin accretion onto the injured jugular veins was enhanced 2.4-fold as compared to the uninjured veins in sham-operated animals. Heparin treatment did not reduce fibrin accretion, whereas, annexin V treatment decreased fibrin accretion by 60%, p < 0.05. This latter effect was achieved without sustained circulating anticoagulation. Additional experiments confirmed that the inhibitory effect of annexin V on fibrin accretion was associated with a surface specific effect, since more annexin V bound to the injured jugular vein segments as compared to the non-injured jugular veins.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Calcium buffering in bursting Helix pacemaker neurons.

Bursting pacemaker neurons of the snail Helix pomatia were voltage-clamped and Ca currents in response to depolarizing steps were recorded. Simultaneously, changes in intracellular Ca concentrations were measured using the fluorescent dye fura-2 and a highly sensitive digital camera. Ca influx through voltage-gated channels induced a spatially non-uniform increase in intracellular Ca. The Ca signals decayed with a time constant of about 5 s. By increasing the concentration of the indicator dye, its Ca-buffering capacity was enhanced and Ca transients in response to depolarization were diminished. Thereby, the endogenous Ca buffer capacity could be determined and was calculated to be about 480 buffered ions for every free Ca ion. The buffer capacity did not vary significantly with the amount of Ca influx within the range tested, suggesting that the buffer is not saturated at Ca concentrations of up to 1 microM.

Animals↗