[Acetaminophen - A modern classic under suspicion]. / Paracetamol - ein moderner Klassiker unter falschem Verdacht.

Paracetamol is one of the most popular and widely used drugs for the treatment of pain and fever and provides safe and effective relief of these symptoms since decades. The mechanism of action is very complex and involves the inhibition of the peroxidase portion of the cyclooxygenase enzyme together with the modulation of the serotoninergic and cannabinoid system. Paracetamol is a safe drug, if used in accordance with the regulations and has demonstrated a superior side effect profile to many widely used NSAIDs.

Angiogenic capacity of endothelial cells in islets of Langerhans.

Transplantation of pancreatic islets reconstitutes glucose homeostasis in diabetes mellitus. Before transplantation, islets are disrupted from the surrounding blood vessels by the isolation procedure, with the grafted tissue being subject to ischemic damage. The survival of transplanted islets is assumed to depend on effective revascularization. Perfusion studies suggest that newly formed microvessels supplying the graft with nutrients are exclusively rebuilt by the host. It is generally not known whether isolated islets contain endothelial cells (EC), which potentially participate in the revascularization process. Therefore, we tried to detect immature EC in isolated islets by transformation with polyoma middle T antigen. Endothelioma cells were generated, implicating the presence of de-differentiated EC within isolated islets. When embedded in a fibrin gel, the islets developed cellular cords consisting of EC, whereas FGF-2 and VEGF stimulated the formation of cord-like structures. Furthermore, we studied the presence of donor EC in islet grafts by using transgenic mice with an EC lineage-specific promotor-LacZ reporter construct (Tie-2LacZ). Following islet transplantation, Tie-2LacZ-positive EC of both donor and recipient were identified in the vicinity of or within the graft up to 3 wk after transplantation. In conclusion, EC and/or their progenitors with angiogenic capacity reside within isolated islets of different species, and their proliferative potential can be stimulated by various inducers. These graft-related endothelia persist after islet transplantation and are integrated within newly formed microvessels.

Specific localization of the catalytic subunits of protein kinase CK2 at the centrosomes.

The protein kinase CK2 holoenzyme is composed of two regulatory beta subunits and two catalytic alpha or alpha' subunits. Although experimental evidence for involvement of the enzyme in the regulation of cell proliferation is accumulating, the exact mechanism of its action is still unclear. The subcellular localization of the enzyme may be a key to its function. We have recently shown that the CK2 holoenzyme is tightly associated with the Golgi complex and the endoplasmic reticulum. Centrosomes, which organize spindle formation during the cell cycle and microtubule cytoskeleton formation and, thereby, the location and orientation of different organelles in the cell, are in close vicinity to the Golgi complex. Because several kinases and phosphatases have been described to regulate the functions of the centrosome, we analysed the association of CK2 with these organelles. Using biochemical cell fractionation and coimmunoprecipitation, we never found the holoenzyme but only the catalytic alpha subunits associated with the centrosome. These data were confirmed by immunoelectron microscopy. Thus, the present data point to a particular role of the catalytic alpha and alpha' subunit of protein kinase CK2, which may be different from their roles in the holoenzyme.

A new model for in vitro clot formation that considers the mode of the fibrin(ogen) contacts to platelets and the arrangement of the platelet cytoskeleton.

The constitution of platelet-fibrin(ogen) contacts, the separation of the platelets initially aggregated, and the rearrangement of the platelet cytoskeleton during clot formation (0.5 to 60 minutes after thrombin stimulation) were investigated using ultrastructural and immunocytochemical techniques. After aggregation, fibrin polymerizing within focal contacts and from degranulating secretory granules contributed to the fibers. The initially formed focal contacts with fibrin obviously persisted during clot formation. The physiological branching of the fibers enabled separation of platelets. The contact associated cytoskeleton formed a constricting and fiber internalizing sphere, but later stress fiber like bundles. As retraction progressed, the cytoskeleton changed to stress fiber connecting focal contacts with fibers. A model of clot formation in vitro is presented that reflects both the contributions of platelets (fibrin fiber internalization and retraction) and of fibers (branching) enabling the retraction.

Localization of protein kinase A and vitronectin in resting platelets and their translocation onto fibrin fibers during clot formation.

Physiological stimulation of platelets with thrombin brings about the release of protein kinase A (PKA) into the plasma. In human blood, this kinase singles out and phosphorylates vitronectin (Vn), a multifunctional regulatory protein, which was proposed to play an important role in the control of fibrinolysis. Here we present immuno-cytochemical evidence to show: (i) that intact platelets possess on their surface an ecto-PKA which can preferentially phosphorylate Vn; (ii) that in the resting platelet, both the catalytic and the regulatory subunits of PKA are present on the platelet surface, in the surface-connected canalicular system, and within the alpha-granules of the platelets; (iii) that the process initiated upon platelet activation, which leads to the formation of fibrin fibers and consequently forms the fibrin net, is accompanied by a translocation of PKA, of Vn, and of PAI-1 onto the fibrin fibers. We propose that the localization and the translocation of these proteins in the fibrin net, together with our finding that PKA phosphorylation of Vn reduces its grip of PAI-1, can unleash PAI-1 in its free form. The free PAI-1 can then assume its latent (non inhibitory) conformation, allow plasminogen activators to trigger the formation of active plasmin, and to initiate fibrinolysis.

Global improvement in intellectual and neuropsychological functioning after removal of a suprasellar cystic craniopharyngioma.

This is a case report of a patient who presented with cognitive and behavioral decline and underwent surgery for removal of a suprasellar craniopharyngioma. Previous studies have reported memory impairments in the presence of craniopharyngioma, but information is lacking regarding the impact of craniopharyngioma on other brain functions as well as on intellectual abilities. In this study, comprehensive neuropsychological testing, including intellectual testing, was conducted 9 to 14 days before surgery and 16 to 22 days after surgery. Results before surgery demonstrated average intellectual abilities, which were decreased from premorbid estimates, and diffuse neuropsychological impairments. Postoperatively, significant improvement in both intellectual abilities and neuropsychological test scores was observed. These results suggest that craniopharyngiomas can be associated with impairments in intellectual abilities and in brain functions aside from memory. The results are contrasted with those of previous reports, and implications for future research are discussed.

The role of plasminogen activator inhibitor-1 as inhibitor of platelet and megakaryoblastic cell adhesion.

In the present study the ability of plasminogen activator inhibitor type-1 (PAI-1) to interfere with platelet and megakaryoblastic cell adhesion was investigated. Both cell types exhibited integrin-dependent adhesion in a static system, mediated by alphaIIb beta3 on platelets and alpha v-integrins on different megakaryoblastic cell lines, even though they also expressed alphaIIb beta3. In a concentration-dependent manner, active, but not latent or complexed, PAI-1 abrogated cell adhesion onto vitronectin but not onto fibrinogen or other matrix substrata. Urokinase as well as thrombin neutralized the anti-adhesive effect of active PAI-1. The direct binding of vitronectin, but not of other matrix proteins, to integrin alphaIIb beta3 was blocked by active PAI-1 in a purified system. Since activated platelets release active and latent PAI-1 as well as structurally and functionally distinct forms of vitronectin, the described interactions appear to be physiologically significant. Co-distribution of vitronectin and PAI-1 at sites of fibrin polymers within platelet thrombi was demonstrated by transmission electron microscopy, suggesting an extracellular functional relationship of both release products with regard to cell adhesion. Our data emphasize the regulatory role of active PAI-1 in platelet adhesion to provisional matrix proteins as found during wound healing independent of its anti-proteolytic activity. Furthermore, megakaryocyte maturation may depend on the intact vitronectin-integrin adhesion system that is influenced by PAI-1, thereby proposing a regulatory role for the inhibitor in cellular differentiation.

Intravenous corticotrophin vs. hydrocortisone in the treatment of hospitalized patients with Crohn's disease: a randomized double-blind study and follow-up.

Adrenocorticotrophic hormone (ACTH) and corticosteroids have no maintenance values for inflammatory bowel disease but serve to reduce the severity of disease. The effectiveness of intravenous corticotrophin versus hydrocortisone in ulcerative colitis has been determined including whether previous steroid therapy influenced the better response to one rather than the other, but no such studies have ever been done in Crohn's disease. Eighty-eight patients hospitalized with moderate-to-severe Crohn's disease (Present-Korelitz [P-K] Index -3 to -2 and the International Organisation for the Study of Inflammatory Bowel Disease-Crohn's & Colitis Foundation of America [IOIBD-CCFA] Index, mean 14, range 5-23) were treated in a prospective, randomized, double-blind clinical trial to receive either continuous intravenous infusion of 120 U/day of ACTH (44 patients) or hydrocortisone 300 mg/day (44 patients). Patients were also subdivided into those who received oral steroids during the 30 days prior to intravenous therapy and those who had not. Response was followed on a daily basis and tabulated at 3, 5, and 10 days. Patients were followed from 1-3 years to determine the later status. After 10 days of intravenous therapy 36 of 44 patients (82%) who received ACTH and 41 of 44 patients (93%) who received hydrocortisone fully responded (P-K index +3 and IOIBD-CCFA Index mean of 3). At the end of the study, response to intravenous ACTH and hydrocortisone was not statistically different whether or not patients received oral steroids during the 30 days prior to admission, although the response to IV ACTH tended to be faster at 3 days in those who had received previous steroid therapy. Intravenous ACTH and hydrocortisone are equally effective in achieving therapeutic goals in patients with Crohn's disease who have not achieved results with oral medications. Moreover the response rate was high (mean 88%), serving to buy time for establishment of successful maintenance programs of treatment with oral 5-ASA and immunosuppressive drugs for 69% of patients at 1-3 years.

A new model for quantitative in vivo microscopic analysis of thrombus formation and vascular recanalisation: the ear of the hairless (hr/hr) mouse.

The alteration of rheological blood properties as well as deterioration of vascular perfusion conditions and cell-cell interactions are major determinants of thrombus formation. Herein, we present an experimental model which allows for quantitative in vivo microscopic analysis of these determinants during both thrombus formation and vascular recanalisation. The model does not require surgical preparation procedures, and enables for repeated analysis of identical microvessels over time periods of days or months, respectively. After i.v. administration of FITC-dextran thrombus formation was induced photochemically by light exposure to individual arterioles and venules of the ear of ten anaesthetised hairless mice. In venules, epi-illumination induced rapid thrombus formation with first platelet deposition after 0.59 +/- 0.04 min and complete vessel occlusion within 7.48 +/- 1.31 min. After a 24-h time period, 75% of the thrombosed venules were found recanalised. Marked leukocyte-endothelial cell interaction in those venules indicated persistent endothelial cell activation and/or injury, even after an observation period of 7 days. In arterioles, epi-illumination provoked vasomotion, while thrombus formation was significantly (p <0.05) delayed with first platelet deposition after 2.32 +/- 0.22 min and complete vessel occlusion within 20.07 +/- 3.84 min. Strikingly, only one of the investigated arterioles was found recanalised after 24 h, which, however, did not show leukocyte-endothelial cell interaction. Heparin (300 U/kg, i.v.) effectively counteracted the process of thrombus formation in this model, including both first platelet deposition and vessel occlusion. We conclude that the model of the ear of the hairless mouse allows for distinct in vivo analysis of arteriolar and venular thrombus formation/recanalisation, and, thus, represents an interesting tool for the study of novel antithrombotic and thrombolytic strategies, respectively.

Memory-enhancing effects of benzodiazepines in mice.

In the present study, post-trial effects of clonazepam and diazepam on inhibitory avoidance learning under two different experimental conditions (i.e., 0.25 or 0.75 mA footshock) have been investigated. Both clonazepam (0.5 mg/kg) and diazepam (2 and 8 mg/kg) enhanced retention when administered immediately after the 0.25 mA footshock applied during training of the inhibitory avoidance task. In contrast, clonazepam and diazepam proved ineffective after the 0.75 mA footshock. The results suggest a post-training memory-enhancing effect of clonazepam and diazepam depending on the experimental conditions.

The formation of compound granules from different types of secretory organelles in human platelets (dense granules and alpha-granules). A cryofixation/-substitution study using serial sections.

The secretory pathway of dense granule contents in cryofixed and cryosubstituted human platelets was investigated by electron microscopy of serial sections. The spherical dense granules in resting platelets were separated by cytoplasm. No contact between these granules and the plasmalemma or the membranes of alpha-granules or other membranes could be observed. Stimulated platelets (thrombin, 0.1-0.5 IU/ml for 20-30 sec) contained dense granules with long protrusions. Tight contacts between the membranes of these granules with the plasmalemma and with membranes of alpha-granules were detected (apposition). Fusion events took place at these sites. After fusion of the dense granules with the plasmalemma the organelles were swollen and contained remnants of their dense matrix. After fusion between dense and alpha-granules compound granules were formed which included remnants of matrices from both types of organelles. It is concluded that the secretory pathway is similar for both types of organelles.

Ultrastructural aspects of platelet adhesion on subendothelial structures.

Platelet adhesion to ligands within the subendothelium induces platelet activation. Ultrastructural and immunocytochemical investigations were carried out to obtain information on the microtopography of the adhesive interactions. Rabbit platelets which react in vivo with endothelial lesions (V. jugularis), and human platelets which react in vitro with collagen were observed. It was shown that the reaction of adhering platelets depends on the micro-topography of the adhesive surface 1. On plane surfaces the platelets spread 2. Collagen fibers induce the formation of focal contacts. They are initiated by binding of ligands to certain transmembrane receptors. The contact mediates the formation of the contractile gel, which acts as a constricting sphere internalizing surface bound ligands, i.e., retraction of collagen networks. Most of the experiments hitherto dealt with platelets that spread. The described findings show that the reaction mentioned above plays a more important role than previously imagined and may be the physiological response of platelets interacting with subendothelial components in vessel lesions.

Textured biomaterials as a model for studying formation of focal contacts and rearrangement of the contractile cytoskeleton in platelets.

Fibres of textured biomaterials (BM) enable platelets to adhere with formation of focal contacts. The contact structure and the reaction of the contact associated contractile cytoskeleton were studied using fibres of different flexibility/mobility: butyl-S-Sepharose (S), Polysulfone (PS) and Polyurethane (PU). Ultrastructural and immunocytochemical investigations were carried out to obtain information on the influence of tension on (1) the structure of the focal contacts; (2) the constricting cytoskeleton known to retract adherent collagen or fibrin fibres and (3) the cable-like bundles of actomyosin as observed in the clot. Fibre network from S spheres and 0.3 mm thick frozen sections of PS or PU were incubated with citrated PRP or with washed platelets at 37 C for 6 to 30 min while stirring for contact or activation with ADP or thrombin. Flexible fibres of the BM were found in deep invaginations of the plasmalemma associated with the constricting cytoskeleton. Focal contacts (mediated by fibrinogen as shown immunocytochemically) with fibres which were fixed in the texture or inflexible (PU) induce cable-like bundles of micofilaments containing myosin. These bundles pass across the cytoplasm and connect the contacts with the fibres or with other platelets, as demonstrated by computer-assisted 3-dimensional reconstruction. The model used indicates that retraction is possible as long as fibres are mobile and that cable-like bundles occur when the locomotion of platelets is blocked by immobile fibres. The interaction of platelets with textured BM reflects the situation during collagen or fibrin condensation. The findings may contribute to an understanding of platelet reactions on textured surfaces in grafts.

Contact-induced neutrophil activation by platelets in human cell suspensions and whole blood.

Platelet-dependent activation of polymorphonuclear neutrophils (PMNL) was investigated with a lumi-aggregometer in heparinized whole blood and platelet-PMNL suspensions. The lumi-aggregometer allowed us to simultaneously monitor increases in impedance or light transmission as consequences of platelet aggregation and luminol-enhanced chemiluminescence (CL) as a measure of the oxidative burst in PMNL. Aggregation and platelet-PMNL contacts were also checked by light and electron microscopy. In whole blood, adenosine diphosphate (ADP) and the thromboxane A2 mimetic U 46619 induced the aggregation (increase in impedance) and the CL, which were both suppressed by EDTA, arginyl-glycyl-aspartyl-serine (RGDS) peptide, and the absence of stirring. In contrast, FMLP caused only CL that was unaffected by EDTA, RGDS peptide, and nonstirring. Similar observations were obtained with mixed suspensions containing washed platelets and PMNL at their physiologic concentrations. ADP, U 46619, and thrombin induced both aggregation (increase in light transmission) and CL, whereas FMLP caused CL but only very weak aggregation. Exogenous fibrinogen strongly enhanced the effects of ADP and U 46619. Iloprost, EDTA, RGDS peptide, red blood cell (RBC) ghosts, and nonstirring inhibited the effects induced by the platelet agonists, but were ineffective on the CL induced by FMLP. Treatment of platelets with aspirin did not affect the CL of PMNL induced by platelets. Microscopic examination, the requirements of stirring, Ca2+, and fibrinogen, and the inhibitory effects of RGDS peptide and RBC ghosts show that stimulated platelets activate PMNL in a contact-dependent manner that depends on fibrinogen binding. This was confirmed by the immunochemical demonstration of fibrinogen (but not of fibronectin) in the contact spaces between activated platelets and PMNL. Because supernatants and lysates of resting or thrombin-stimulated platelets did not induce the CL of PMNL, soluble agonists did not appear to be involved. Nonstimulated washed platelets also caused CL of PMNL that required stirring and Ca2+ and was inhibited by RBC ghosts. No CL occurred in unstimulated stirred whole blood, suggesting that a preactivation of platelets during the preparation may be responsible for the effects of unstimulated washed platelets. The results show that platelets provide a strong stimulus for PMNL that requires intercellular contact. Fibrinogen exposure on the platelet surface seems to be necessary for the activation of PMNL by stimulated platelets.

Comparative effects of carbamazepine, phenytoin, diazepam and clonazepam on inhibitory avoidance learning in mice.

Four antiepileptic drugs were investigated in an inhibitory avoidance task in mice. Following IP administration 30 min prior to training, carbamazepine (32 mg/kg), phenytoin (30-60 mg/kg), diazepam (2-8 mg/kg) and clonazepam (0.125-0.5 mg/kg) impaired retention. When administered 30 min prior to the retention test none of the drugs under investigation affected retention. The drugs did not affect latencies in the hot plate test. This indicates that in the case of pretraining drug administration effects on retention cannot attributed to elevated pain thresholds. Carbamazepine and phenytoin impaired avoidance learning at doses above those which prevent electroshock induced tonic hindlimb convulsions. Diazepam and clonazepam were effective at lower than anticonvulsant doses. The results of the study are relevant to the evaluation of CNS side effects of anti-epileptic drugs in mice.

Transport of anti-glycoprotein IIb/IIIa-antibodies into the alpha-granules of unstimulated human blood platelets.

The redistribution of the antibody-glycoprotein (GP) IIb/IIIa complex was investigated with the immuno-gold labeling technique in order to trace its transport in resting platelets. Washed platelets were incubated in the presence of aspirin and a prostacyclin analogue (iloprost) with three different monoclonal antibodies (Gi3, J15 and P2) against GPIIb/IIIa. The examination of ultrathin serial sections showed that the surface labeling was internalized into the surface connected membrane system (SCS). Labels were found within the alpha-granules after 40 min and the number of labels increased during longer incubation periods (max. 120 min). The transport possibly involved coated membranes. The alpha-granules were neither found to be altered during this process nor were any morphological signs of platelet activation detectable. The anti-GPIIb/IIIa complex remained membrane-associated during the transfer. These observations indicate that the membrane-GPIIb/IIIa complex was stable and transported from the surface into the alpha-granules of resting platelets. Since this transport was not influenced by iloprost or by aspirin it may be interpreted as constitutive endocytosis.

Role of internalization in platelet activation induced by collagen fibers--differential effects of aspirin, cytochalasin D, and prostaglandin E1.

Washed human platelets were stimulated with fibrillar collagen and platelet aggregation was prevented by non-stirring conditions. In these samples, electron microscopy revealed three fractions of platelets: 1) a majority without contacts to the collagen fibers, 2) with focal contacts to collagen, and 3) a small fraction of platelets with internalized collagen. All platelets had undergone shape change, and exhibited an internal contraction and granule release. However, only those with internalized collagen were completely degranulated. The internalized collagen was found to be in close contact to the contractile sphere in the platelet center, as it was demonstrable with computer assisted 3-D reconstruction from serial sections. Aspirin inhibited neither the adhesion to collagen nor its internalization by internal contraction. Also it did not impair the shape change and degranulation in the platelet fractions that internalized collagen. However, aspirin blocked the shape change and internal contraction of the other platelets and inhibited the [3H]serotonin release. Cytochalasin D 0.1 microM also suppressed the internalization of collagen, the shape change, the formation of a contractile gel, the degranulation, and the [3H]serotonin release in all platelets, whereas the number of platelets that adhered to collagen remained unchanged. The same effects were produced by prostaglandin E1. If the platelets were stimulated with the TXA2 mimetic, U46619, cytochalasin D at 0.1 microM had no effect; but at 20 microM it strongly enhanced the degranulation and the [3H]serotonin release, although the platelets remained discoid. It is concluded that collagen triggers a focal activation of an adherent platelet at the site of its initial contact to collagen.(ABSTRACT TRUNCATED AT 250 WORDS)