Increased number of high sensitive platelets in hypercholesterolemia, cardiovascular diseases, and after incubation with cholesterol.

The number of low density platelets was found to be increased in patients with hypercholesterolemia, as compared with the number in controls. The percentage increase of the low density platelet subpopulation was even more pronounced in patients with hypercholesterolemia when compared with that in patients suffering from myocardial infarction or angina. In vitro studies with control platelets incubated with cholesterol rich liposomes showed also an increase in the subpopulation of low density platelets. After incubation of control platelets with cholesterol rich liposomes, a higher membrane anisotropy and a higher cholesterol to phospholipid (C/P) molar ratio of the plasma membrane were found. Furthermore, cholesterol-enriched platelets were more sensitive upon thrombin stimulation. The results suggest that a shift of platelet subpopulations to a higher number of low density platelets could be caused by either the level of plasma cholesterol or an in-vitro incubation with cholesterol rich liposomes.

Analysis of GTP-binding proteins, phosphoproteins, and cytosolic calcium in functional heterogeneous human blood platelet subpopulations.

The biochemical basis for the functional heterogeneity of human blood platelets was investigated in terms of protein phosphorylation, cytoplasmic calcium ([Ca2+]i), the ratio of 46 and 50 kDa vasodilator-stimulated protein (VASP), and GTP-binding proteins (G-proteins). Platelets were fractionated by density. Comparing resting low-density platelets (LDP) to high-density platelets (HDP) revealed higher phosphorylation of proteins in the 47, 31, and 24 kDa ranges. A higher phosphorylation of the 20 kDa protein in LDP compared to HDP was related to an enhanced [Ca2+]i, an increased ADP-ribosylation of the inhibitory G-protein (G(i alpha1-3)) and rhoA, and a decreased ADP-ribosylation of the stimulatory G-protein (G(s alpha)). The differences in the ribosylation patterns of the subpopulations were not influenced by thrombin stimulation or exposure to prostaglandin E1 (PGE1). An 18 kDa phosphoprotein was more highly phosphorylated in resting HDP than in LDP. Thrombin exposure caused dephosphorylation of the 18 kDa phosphoprotein in the HDP, but generally increased phosphorylation of both HDP and LDP in the 47, 31, 24, and 20 kDa bands. Preincubation with prostaglandin E1 or sodium nitroprusside diminished the subsequent thrombin-induced increase in phosphorylation, particularly in HDP. In unstimulated HDP, the 50 kDa VASP phospho form was enhanced, whereas in unstimulated LDP the 46 kDa VASP dephospho form was increased. Our findings suggest that the functional heterogeneity of platelets is partly derived from differences in signal transduction mechanisms reflected in varying phosphoprotein patterns and G-protein properties of platelet stimulatory and inhibitory pathways.

Kinetic latex agglutinometry I. A rapid, quantitative immunologic assay for fibrinogen.

A rapid, convenient immunologic assay technic for fibrinogen is reported. Basically a latex agglutination, the assay technic, called kinetic latex agglutinometry, quantitates the increase in light transmission that occurs in a stirred suspension of anti-fibrinogen-coated latex beads after the addition of a plasma or whole blood sample containing fibrinogen. In this system the time "delta t" required for a given transmission change to occur is inversely proportional in a log-log relationship to the quantity of fibrinogen in the plasma sample. The reaction is linear over a fibrinogen concentration of greater than 100-fold. The sensitivity can be adjusted over a wide range, and the assay can quantitate as little as 10 ng fibrinogen. The assay can be used with either plasma or whole blood. When compared to the thrombin clotting time method of Clauss, the correlation coefficient is 0.99 for the plasma assay and 0.95 for the whole blood assay. As a one-step assay employing stable reagents and requiring approximately two minutes per assay for normal plasma, the method is ideally suited for use in the clinical laboratory.

Alterations of blood platelet MAO-B activity and LSD-binding in humans after sleep deprivation and recovery sleep.

Sleep deprivation (SD) is an effective, however short-lived, method of treatment of depression. Preliminary findings suggest that the antidepressive effect of sleep deprivation is mediated by serotoninergic (5-HT) mechanisms. We therefore assessed serotoninergic activity before and after total SD (TSD) as well as after the following night sleep by investigating platelet LSD-binding, MAO B-activity, and 5-HT-content as well as plasma norepinephnne (NE) in 10 healthy men (age: 27.4 +/- 2.8 years). Blood samples were drawn on three consecutive days at 0700, 1300 and 1900 h, respectively. After TSD, a significant increase of LSD-binding KD and Bmax as well as of MAO-B KM and plasma NE could be observed, which, however, vanished after consecutive night sleep. Our findings favour an increased serotoninergic transmission after TSD and thus support the hypothesis, that sleep deprivation exerts its antidepressant effects by pro-serotoninergic mechanisms.

Plasma membrane properties in heterogeneous human blood platelet subfractions modulate the cellular response at the second messenger level.

Three subfractions of human blood platelets differing in density and function exhibited also differences when EPR (electron paramagnetic resonance) spin label measurements and fluorescence polarization were performed. The membrane anisotropy in the plasma membranes was higher in low density platelets as compared with that of platelets of intermediate and higher densities. This higher plasma membrane anisotropy correlated with a significantly higher cholesterol-phospholipid (C:P) molar ratio in the plasma membranes of low density platelets. As compared with the other platelet subfractions the low density platelets exhibited the smallest cAMP increase after activation with the aggregation inhibitor prostaglandin (PGE1), and the highest percentage of inositol phosphate accumulation after thrombin stimulation. The results suggest a high correlation between functional parameters and the plasma membrane architecture of human blood platelets.

Involvement of guanylate cyclase and phosphodiesterases in the functional heterogeneity of human blood platelet subpopulations.

Cyclic nucleotides are involved in the regulation of platelet activation, shape change, and aggregation. In this study we have investigated the role of guanylate cyclase and phosphodiesterase in three functional heterogeneous human blood platelet subpopulations separated according to density. In low-density platelets aggregation was enhanced and inhibited less when cyclic GMP was increased by sodium nitroprusside, compared to high-density platelets. Low-density platelets possessed a lower basal level of cyclic GMP and exhibited a small increase in cyclic GMP after stimulation with sodium nitroprusside. Cyclic GMP-dependent phosphodiesterase activity was similar in high, low, and intermediate-density platelets. In contrast, the activity of the cyclic AMP-dependent phosphodiesterase was higher in low-density compared to high and intermediate-density platelets. These results suggest that regulation of cyclic GMP and cyclic AMP levels plays an important role in the functional heterogeneity of human blood platelets.

Platelet aggregation during abdominal surgery in an experimental pig model: the effects of presurgical antibiotic protocols and volume replacement with hydroxyethyl starch.

The effect of presurgical antibiotic protocols in combination with hemodilution on platelet aggregation was studied. Thirty pigs were randomly assigned to three groups. Group 1 received amoxicillin/clavulanic acid, group 2 metronidazole+cefuroxime, and group 3, as a control, sodium chloride. They underwent laparotomy, massive blood loss, and volume replacement with hydroxyethyl starch 200, followed by an anaphylactoid reaction. Platelet aggregation was measured by the turbidometric method. Neither antibiotic protocols had any effect on platelet aggregation as compared with the control group. In all three groups, aggregation to ADP and collagen was significantly reduced after volume replacement with hydroxyethyl starch. In contrast, the sensitivity to the aggregating effects of collagen was increased as assessed by a higher frequency of responses to low concentrations of collagen and a shortened latency of the aggregation response after collagen addition. Further in vitro studies revealed that dilution of plasma with hydroxyethyl starch specifically induced the changes seen after in vivo volume replacement. The results suggest that the plasma substitute hydroxyethyl starch 200 increases the sensitivity to low doses of collagen, an effect never described before and considered of clinical relevance.

Lowering of body temperature affects human platelet functions and norepinephrine release.

The effect of lowering body temperature on plasma epinephrine, norepinephrine, and platelet density distribution and volume was studied in a placebo-controlled double-blind study. Lowering of body core temperature was induced by either exposure to a cold environment at a temperature of 5 degrees C (CT) or by a single dose of the 5-HT1A agonist ipsapirone (IPS). A third group exposed to an ambient temperature of 28 degrees C was given placebo (PLAC). All of the three groups were investigated in a climate chamber. In the CT group the density distribution of blood platelet subpopulations was shifted to an increase in less dense platelets that were more sensitive towards aggregation-inducing agents. The mean platelet volume in this subpopulation was decreased. Epinephrine was not affected, whereas the increase of norepinephrine was correlated with an increase of platelets that were more sensitive to aggregation-inducing agents in the CT group but not in the PLAC and IPS groups.

Alterations of host defence system after sleep deprivation are followed by impaired mood and psychosocial functioning.

In healthy humans, sleep deprivation (SD) has consistently been demonstrated to impair different parameters of the host defence system and of psychosocial functioning. However, the individual timing of these alterations and their possible association have remained unknown so far. We therefore investigated functional measures of the individual host defence system as well as of subjective well-being and psychosocial performance in 10 healthy male adults before and after SD, as well as after recovery sleep. In detail, we examined the number of leukocytes, granulocytes, monocytes, lymphocytes, B cells, T cells, T helper and cytotoxic T cells, natural killer (NK) cells as well as the interleukin-1 beta (IL-1 beta) release from platelets after serotonin (5-HT) stimulation. Mood and psychosocial performance (excitement, energy, ability to work and timidity) were measured by visual analogue scales. Taken together, SD induced a deterioration of both mood and ability to work, which was most prominent in the evening after SD, while the maximal alterations of the host defence system could be found twelve hours earlier, i.e., already in the morning following SD. Our findings therefore suggest an SD-induced alteration of these psychoimmune response patterns in healthy humans preceding deterioration of mood and psychosocial functioning.

Daily variations of functional parameters and density distribution in human blood platelets.

Blood platelets play a critical role in the onset of myocardial infarction, which has been shown to have a circadian rhythmicity with a peak incidence in the morning. In an attempt to correlate platelet parameters with the outcome of cardiovascular diseases, we studied the daily (24-h) variation of the following platelet parameters: distribution pattern of functional heterogeneous platelet subpopulations; serotonin uptake; ketanserin binding; aggregation upon thrombin, serotonin, and ADP stimulation; and platelet count. Furthermore, we analyzed the tryptophan and serotonin concentrations in the blood samples. The percentage of less dense platelets, which represent the subpopulation with the highest preactivation, showed a rhythmicity period of 24 h and an acrophase at 21:18 h. The time course of intermediate and high density platelets was inverse to that of low density platelets. The serotonin uptake exhibited also a rhythmicity with a 24-h period. The acrophase was at 13:50 h. The aggregation curves were inverse to the ketanserin binding curves. The serotonin concentration exhibited a 12-h rhythmicity. The results obtained suggest that (a) changes in platelet activity are reflected by several parameters of platelet function that underlie daily variations; (b) the aggregation curves show a peak in the morning, with an additional peak in the afternoon; and (c) changes in the distribution pattern occur independently from variations in platelet functions like aggregation and serotonin binding.

Short-duration oral terbinafine for the treatment of tinea pedis in HIV-positive patients.

Management of tinea pedis in patients who have the human immunodeficiency virus (HIV) is problematic; in those patients, dermatophytoses may be more difficult to treat than in the general population. This prospective, open-label, multicenter, randomized study evaluated the efficacy and safety of a short course of oral terbinafine for tinea pedis in patients who are HIV positive. Twenty-seven patients were randomized to receive oral terbinafine 250 mg once daily for 2 or 4 weeks; 17 patients with positive initial cultures and follow-up cultures were evaluable for efficacy at week 8. Mycological cure (defined as negative potassium hydroxide [KOH] microscopy and culture results) occurred in 47% (8) of patients; and modified mycological cure (defined as negative follow-up cultures) occurred in 65% (11) of patients. All 27 patients were evaluated for safety. Clinical cure (defined as minimal residual signs and symptoms) occurred in 82% (14) of patients. Oral terbinafine was well tolerated, indicating that regimens of 2 or 4 weeks are safe and effective for the treatment of tinea pedis in patients who are HIV positive.

Changes in lipids and membrane anisotropy in human spermatozoa during epididymal maturation.

Previously it was demonstrated that immature and immotile human spermatozoa from the caput epididymides developed a good progressive motility after in-vitro stimulation with phosphatidylcholine (PC). In order to define the role of PC and membrane anisotropy in epididymal maturation and to determine the exact lipid composition of human spermatozoa during epididymal maturation, spermatozoa from seven epididymides from patients who underwent orchiectomy because of prostatic cancer were investigated. Lipids were determined by high-performance thin-layer chromatography and gas chromatography. Membrane anisotropy was measured by fluorescence polarization. The ratio between PC and phosphatidylserine (PS) plus phosphatidyl ethanolamine (PE) plus sphingomyelin (SM) was significantly higher in spermatozoa from the cauda compared to those from the caput and corpus. This was due to an increase of PC and a decrease of the concentration of PS plus PE plus SM. With regard to fatty acids, those with saturated chains predominated in caput spermatozoa while the highest concentration of unsaturated long-chain fatty acids was in cauda spermatozoa. A lower membrane anisotropy of cauda spermatozoa compared with caput or corpus spermatozoa was found. In conclusion, during epididymal maturation human spermatozoa integrate lipids, particularly PC, which is strongly associated with the induction of progressive motility. A change in the pattern of fatty acids and a decrease in the cholesterol/phospholipid molar ratio cause a decrease in membrane anisotropy in cauda spermatozoa.

[Influence of measurement time and reacton medium on kinetic immunturbidimetric protein determination (author's transl)]. / Der Einfluss von Messzeit und Reaktionsmilieu bei kinetischen immunturbidimetrischen Proteinbestimmungen.

For fixed-time rate analysis, the turbidity curve, which is the reference system of immunturbidimetric protein determination, depends on the time of first measurement, on the subsequent measurement time interval and on the composition of the reaction medium. The IgG/anti-IgG system has been chosen as an example to demonstrate how the reaction parameters can be optimized. In this context particular consideration has been given to some of the analyzers commonly used in larger laboratories (ABA-100, CentrifiChem, Cobas Bio, GEMSAEC, Gilford 3500).

Biopsychological changes after bungee jumping: beta-endorphin immunoreactivity as a mediator of euphoria?

A study on 12 novice bungee jumpers was performed to investigate the influence of acute psychological stress on levels of cortisol in saliva, beta-endorphin immunoreactivity as well as the number of leukocytes in peripheral blood. In addition, heart rate and blood pressure as well as ratings on emotional states were recorded. Furthermore, correlations between ratings on mood and biochemical stress markers were computed. As expected, subjective ratings on anxiety were increased prior to the jump and were markedly reduced after the jump. Salivary cortisol was also increased after the jump and decreased to baseline within the next hour. In contrast, ratings on euphoria increased markedly after performing the jump and remained highly elevated for the next 30 min. An increase of more than 200% in beta-endorphin immunoreactivity after the jump was observed. In contrast to levels of cortisol, the concentration of beta-endorphin recorded immediately after the jump was significantly correlated with ratings on euphoria obtained at subsequent measurements indicating a relationship between beta-endorphins and euphoria. Additional increase of the number of blood leukocytes and of heart rate and blood pressure indicate that various systems of the organism are markedly affected by the exceptional eustress of bungee jumping.

Correlation of membrane anisotropy with function in subpopulations of human blood platelets.

Human blood platelets were fractionated on a discontinuous Percoll gradient into high density (HD), intermediate (ID), and low density (LD) platelets. The subpopulations were characterized with regard to [14C]serotonin uptake and release, cAMP content, aggregation, and membrane anisotropy, which is inversely related to membrane fluidity. Membrane anisotropy, which was high in LD platelets, was found to decrease with increasing density (LD greater than ID greater than HD). LD platelets showed significant lower cAMP levels and [14C]serotonin uptake than the total platelet population (TPP) and ID and HD platelets. Upon ADP and serotonin stimulation the cAMP content was reduced in all platelet populations with the exception of HD platelets in which cAMP was unchanged. Upon thrombin stimulation the cAMP content was reduced only in TPP and LD platelet population and it was increased in HD platelet population. Thrombin activation changed the anisotropy only in LD platelets. Thrombin at a concentration of 0.001 U/ml reduced whereas 0.01 and 0.05 U thrombin/ml increased the membrane anisotropy significantly. As compared with TPP and the other subpopulations, LD platelets were most sensitive upon ADP and thrombin stimulated [14C]serotonin release as well as upon ADP, serotonin and thrombin induced aggregation. The findings suggest that the differing functional abilities of the platelet subpopulations are correlated to the various membrane anisotropies observed in these fractions.

The antagonism of ipsapirone induced biobehavioral responses by +/- pindolol in high and low impulsives.

The present study was conducted to investigate whether +/- pindolol antagonizes ipsapirone induced biobehavioral changes in a personality dependent way. Our previous work demonstrated that high impulsives show higher immune cell responses than low impulsive subjects upon treatment with ipsapirone. A total number of 80 healthy male volunteers received placebo (N = 20) or 10 mg ipsapirone (N = 20), 30 mg +/- pindolol (N = 20), or a combination of 30 mg +/- pindolol and 10 mg ipsapirone (N = 20). Each group consisted of 10 low and 10 high impulsive subjects. Since 5-HT related drugs induce thermoregulatory responses, the study took place in a climate chamber with a constant ambient temperature. Blood samples (for measurement of CD4+ cell counts) were drawn from an indwelling catheter invisibly for the subjects. The results clearly demonstrate that the ipsapirone induced decreases in body temperature and number of peripheral CD4+ cells are more pronounced in high impulsives. +/- Pindolol antagonizes thermoregulatory and CD4+ cell responses. The results are discussed with respect to mechanisms of alteration in 5-HT function related to impulsivity.

Effect of hypochlorite (HOCl)-modified low density lipoproteins and high density lipoproteins on platelet function.

As hypochlorous acid (HOCl) might be a potential candidate for generation of modified (lipo)proteins in vivo , the present study was aimed at investigating the effects of HOCl-modified lipoproteins on platelet function in vitro. Lipoproteins modified with HOCl at concentrations that occur physiologically did not induce spontaneous platelet aggregation. However, low density lipoproteins (LDL; 100 to 500 microg protein/ml) increased platelet aggregation and fluorescence anisotropy in response to ADP (1 and 10 microM) and thrombin (0.1 and 0.5 U/ml) as well as malondialdehyde (MDA) formation as a function of increasing HOCl-concentrations (0.2 to 1.6 mM). HOCl-modified high density lipoprotein subfraction 3 (HDL3) had no effect on platelet function.

Effect of repeated immobilization on serotonin metabolism in different rat brain areas and on serum corticosterone.

The effect of daily repeated 10 min immobilization on the serotoninergic neurotransmission and serum corticosterone levels was studied. Male Lewis rats were immobilized for a 10 min period daily once or on 5 consecutive days. Serotoninergic neurotransmission was followed using differential in vivo pulse voltammetry with carbon fibre electrodes measuring extracellular 5-hydroxyindoleacetic acid (5-HIAA) levels. Recordings were performed in brain areas involved in the control of behaviour, mood, and stress response such as the frontal cortex, the hippocampal CA-3 and dentate gyrus, the striatum, and the raphe nuclei dorsalis (NRD) and medialis (MRN). The first immobilization resulted in an increase of the extracellular 5-HIAA levels in all areas under study, except the striatum where no reaction was observed. The major effect was recorded in the frontal cortex, showing an increase of about 400% as compared to control, which lasted for 3h after the end of the immobilization period. Beginning on day 2 in all areas, except the striatum, a consecutive habituation to the stressor seemed to occur, since the stress-induced increase in the voltammetric signal was found to be reduced after consecutive immobilization. Serum corticosterone levels were measured directly after a single and after 5 daily immobilization periods. After single immobilization the serum corticosterone level was found to be about 270 ng/ml. After the 5th immobilization about 300 ng/ml were detected. These differences were not found to be significant. In summary, our data indicate that the serotonin metabolism shows habituation in nearly all brain areas after repeated immobilization, though the corticosterone level at the end of the immobilization period was comparable after single and repeated immobilization.

Decreased platelet membrane anisotropy in patients with adrenoleukodystrophy treated with erucic acid (22:1)-rich triglycerides.

Low platelet count and bleeding diathesis have been observed in patients with adrenoleukodystrophy (ALD) treated with erucic acid (22:1)-rich triglycerides ("Lorenzo's oil'). To investigate possible alterations of biophysical membrane properties, we measured platelet membrane anisotropy, which is inversely related to membrane fluidity, in 16 patients with and in 3 patients without treatment. In patients on treatment, platelet membrane anisotropy was significantly decreased. Additionally, we found increased platelet concentrations of 22:1 and compromised in vitro platelet aggregation response. The decrease of platelet membrane anisotropy is probably a main cause of bleeding diathesis. Long-term haematological side-effects must be considered in ALD patients treated with Lorenzo's oil.

Effects of interferon-gamma, interleukin-1 beta, and tumor necrosis factor-alpha on the serotonin metabolism in the nucleus raphe dorsalis of the rat.

The effects of the cytokines interferon (IFN)-gamma, interleukin (IL)-1, and tumor necrosis factor (TNF)-alpha on the serotoninergic transmission in the nucleus raphe dorsalis (NRD) were studied after peripheral and central application. The studies were performed in the freely moving rat using differential pulse voltammetry with multicarbon fibre electrodes to study the extracellular levels of the serotonin (5-HT) metabolite 5-hydroxyindoleacetic acid (5-HIAA). The extracellular 5-HIAA levels were not changed in the NRD after peripheral application of rat recombinant IFN-gamma, but elevated by the cytokines IL-1 beta and TNF-alpha. After intracerebroventricular (i.c.v.) application the cytokines IFN-gamma, IL-1 beta and TNF-alpha stimulated the serotoninergic transmission in the NRD. Our data suggest that the effect of peripherally elevated cytokine concentrations on the serotonin metabolism in the NRD of the rat is cytokine-dependent. In this respect the T-cell and NK-cell cytokine IFN-gamma acts clearly different when compared to the mainly macrophage-derived cytokines IL-1 beta and TNF-alpha, and plays a different role in the communication between immune and central nervous system.