Effects of staurosporine, U-73122, wortmannin, 4-hydroxynonenal and sodium azide upon the release of secreted beta-amyloid precursor protein from human platelets in response to thrombin stimulation.

In the present study, the release of secreted beta-amyloid precursor protein (AbetaPPs) in response to thrombin stimulation in platelets has been investigated. Incubation of platelets with thrombin produced a concentration-dependent release of AbetaPPs with a concomitant reduction in the AbetaPP remaining in the lysates. The response to thrombin was not affected by pretreatment for 15 min with the phospholipase C inhibitor U-73122, with the protein kinase C inhibitor staurosporine, or with hydrogen peroxide (which at the concentrations used affects the phosphoinositide signalling system in human platelets). In contrast, pretreatment with wortmannin and sodium azide reduced the responses to thrombin. These data would suggest that thrombin may cause the release of AbetaPPs from human platelets via an activation of a phospholipase C-independent pathway. Thrombin-stimulated AbetaPPs release was also reduced by 4-hydroxynonenal. This finding, if it is a phenomenon also found for CNS cells, could be of relevance to the pathogenesis of Alzheimer's disease, given that an accumulation of 4-hydroxynonenal is found in this disease.

Human platelet calcium mobilisation in response to beta-amyloid (25-35): buffer dependency and unchanged response in Alzheimer's disease.

In the present study, the effects of beta-amyloid (25-35) (Abeta (25-35)) upon calcium signalling by the human platelet has been investigated. When assays were conducted using HEPES buffers, Abeta (25-35), but not the inactive peptide Abeta (35-25), produced a robust increase in intracellular calcium that remained after removal of extracellular calcium but was abolished by the phospholipase C inhibitor U-73122. There was no significant difference between the calcium response to Abeta (25-35) in platelets from patients with Alzheimer's disease and from age-matched controls. In contrast to the robust effects on calcium mobilisation in HEPES buffers, very little calcium response to Abeta (25-35) was seen when Krebs (pH 7.8) buffer was used.

Effects of sodium azide on the secretion of soluble amyloid-beta precursor protein and the accumulation of beta-amyloid(1-40) in cultured chick neurons.

Sodium azide has been reported in the literature to reduce the release of secreted amyloid beta-precursor protein (AbetaPPs) and to produce a large increase in the cellular level of an 11.5 kDa C-terminal AbetaPP derivative containing the beta-amyloid (Abeta) sequence. Here we report that 1 mM of sodium azide, reduced the constitutive AbetaPPs secretion from cultured embryonic chick neurons after 12 h of incubation. After 24 h of incubation there was a modest increase in lactate dehydrogenase (LDH) release and no change in MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction, suggesting that the reduced AbetaPPs secretion was not due to the cell toxic effects of NaN(3). However, NaN(3) reduced the accumulation of Abeta(1-40) in the cell lysates and decreased the acetylcholine esterase activity both in cell culture media and in cell lysates. It is concluded that the effect of NaN(3) upon AbetaPP metabolism in the chick cultured neurons may be a rather non-specific effect.

Rapid inhibition by sodium azide of the phosphoinositide-mediated calcium response to serotonin stimulation in human platelets: preservation in Alzheimer's disease.

The effect of sodium azide (NaN(3)) upon platelet Ca(2+) signalling has been investigated. A 60 s preincubation with 1 mM NaN(3) reduced the Ca(2+) response to 1 microM serotonin without a corresponding reduction in the responses to 52 mU/ml thrombin or 70 microM beta-amyloid(25-35) (A beta(25-35)). The effect of NaN(3) upon the response to serotonin, which was not blocked by either glutathione ethyl ester (GTEE) or dithiothreitol (DTT), was similar in platelets obtained from patients with Alzheimer's disease and from age- and gender-matched controls. After a preincubation time of 5 min was used, the Ca(2+) response to thrombin was greatly reduced by 1 mM NaN(3), but not by 50 microM 4-hydroxynonenal (HNE, 50 microM). Platelet levels of HNE and malondialdehyde were not significantly affected by up to 30 min of incubation with NaN(3) at room temperature. It is concluded that the rapid effect of NaN(3) upon the Ca(2+) response to serotonin in human platelets is not mediated by an inhibition of cytochrome c oxidase, and is due to an action proximal to phosphoinositide-specific phospholipase C.

Investigation into the rapid effects of 17 beta-estradiol and neuroactive steroids upon beta-amyloid(25-35)-induced activation of phosphoinositide-specific phospholipase C in human platelets.

In the present study, the rapid effects of five steroids (17 beta-estradiol, progesterone, allopregnanolone, 3 alpha-hydroxy-5 alpha-pregnan-20-one and 3 alpha-hydroxy-5 beta-pregnan-20-one) and the plant steroid trans-resveratrol upon the calcium response to beta-amyloid(25-35) peptide (A beta(25-35)) in human platelets was measured. A beta(25-35) produced a robust increase in intracellular calcium due to a direct activation of phosphoinositide-specific phospholipase C. None of the steroids significantly affected the response to A beta(25-35). In contrast, trans-resveratrol appeared to increase the response to A beta(25-35) at a concentration that decreased the response to thrombin, although the possibility that these changes are artifactual could not be ruled out. It is concluded that although steroids affect human platelet Ca2+ homeostasis, this is not a rapid event, unless very high concentrations are used.

Further studies of the effects of diamide and hydrogen peroxide on calcium signaling in the human platelet.

Intracellular calcium levels were measured in Fura-2/AM loaded human platelets. Diamide (30 microM) reduced the Ca2+ response to serotonin (1 microM), but not to 4-bromo-A23187 (1 microM). The small reduction in the maximum fluorescence produced by diamide was not mirrored by a change in the sensitivity of Fura-2 pentapotassium salt to Ca2+. Changes in the spectral properties of Fura-2 thus cannot wholly explain the reduced serotonin response following diamide treatment. Hydrogen peroxide (500 microM) produced a steady increase in the observed fluorescence ratio that was partially inhibited by the phospholipase C inhibitor U-73122 (1.5 microM). In the absence of platelets, H2O2 reduced the Ca(2+)-sensitivity of Fura-2 pentapotassium salt, although it is not clear the extent to which this effect contributes to the reduction in the Ca2+ response to serotonin seen after H2O2. It is concluded that some caution may be warranted in the interpretation of the effects of H2O2 upon receptor-mediated Ca2+ signaling as measured using Fura-2.

Characterization of the N-methyl-D-aspartate (NMDA) receptor in the embryonic chick brain.

Relatively little is known about the properties of the NMDA receptor in avian species. In the present study, NMDA receptor pharmacology and function has been studied in the embryonic chick brain. The competitive antagonist ligand [3H]CGP 39653 bound to telencephali membranes from 17-day-old chick embryos with KD and Bmax values of 6.6 nM and 3.3 pmol/mg protein, respectively. The binding was inhibited by CGS 19755, L-glutamate, R-CPP and D-AP5 with pI50 values of 7.57, 7.49, 7.28 and 7.08, respectively. L-glycine, S-AMPA, kainate and MK-801 only weakly affected the binding. The inhibitory potency of NMDA (pI50 value 6.98), however, was greater than seen in other species, suggesting a species difference in receptor structure. Such a possibility was investigated by use of two NMDA-R1 antibodies, AB 1516 and 69921 A, which recognize the C-terminal region and an intracellular domain of the rat receptor, respectively. Immunoreactive signals (approximately 116 kDa) were found for a rat brain homogenate, whereas only 69921A gave an immunoreactive signal in the chick. The functional status of the NMDA receptors was investigated in serum-free cultures of neurons from 8-day-old chick embryos. Glutamate and NMDA were found concentration-dependently, and in a manner that could be antagonized by CGS 19755 and MK-801, to produce cell death. Thus, the present study indicates that chick embryonic brain expresses functional NMDA receptors that are pharmacologically and possibly structurally different from their rodent counterparts.

Presenilin-1 in neuroblastoma cells.

Presenilin-1 expression was investigated in Neuro-2a cells using the alphaPS1Loop antibody. The bulk of the immunoreactivity was found as the approximately 18kDa C-terminal fragment. Hydrogen peroxide treatment of the Neuro-2a cells for 24 hours resulted in a concentration-dependent cytotoxicity with a critical concentration of hydrogen peroxide of approximately 20 mum. Treatment of the cells for 24hr with 10, 20 or 30 mum hydrogen peroxide did not produce obvious effects upon presenilin-1 C-terminal fragment immunoreactivity. It is suggested that the loss of presenilin-1-mediated neuroprotection that has been proposed to occur in Alzheimer's disease is not secondary to hydrogen peroxide-mediated cell stress.

The secretion of soluble amyloid beta precursor protein (A beta PPs) by chick neurons in serum-free primary culture is not regulated by protein kinase C.

The metabolism of amyloid precursor protein (A beta PP) in chick neurons cultured in serum-free medium is described. A beta PP immunoreactivity, detected with the 22C11 antibody, was seen at approximately 135 and approximately 120 kDa. A beta PPs (approximately 120 kDa) was released from the cells and could be detected in the culture medium without the need of a purification step. The content of A beta PPs increased with time after medium change, but was not affected by either carbachol (100 microM), glutamate (50 microM), veratrine (20 microM), oleic acid (200 microM), A23187 (5 microM), phorbol 12, 13-dibutyrate (PDBu, 1 microM), staurosporine (1 microM), Gö 6976 (1 microM) or okadaic acid (50 nM) although the combination of PDBu and okadaic acid reduced the secretion. Addition of the muscarinic receptor agonist carbachol to the neurons increased the rate of phosphoinositide breakdown. In Western blot experiments using antibodies to the alpha, beta II and epsilon isoforms of protein kinase C and conditions whereby robust signals could be seen with rat brain lysates, no immunoreactive bands that could be inhibited by appropriate positive control peptides were seen. It is concluded A beta PPs production by chick neurons in culture is mainly constitutive in nature.

Hapten inhibition of dextran anaphylaxis. Nine years of post-marketing surveillance of dextran 1.

All spontaneous reports to the manufacturer and to WHO's database INTDIS regarding adverse reactions to clinical dextran after preinjection of dextran 1 and to dextran 1 alone 1983-1991 were collected. During 1983-1991 a total of 4.8 million doses of Promit were sold in fourteen countries. The incidence of severe DIAR (grades III-V) to clinical dextran after the prophylactic use of hapten inhibition was approximately one case per 200,000 doses of dextran 1. In Sweden, where reporting of severe adverse drug reactions is mandatory, the incidence was one case per 70,000. This indicates a 35-fold reduction in the incidence of severe DIAR compared with the use of clinical dextran without dextran 1. Only two fatal reactions were reported; the incidence was therefore one case per 2.4 million doses, indicating a 77-fold reduction. Both these occurred in patients with extremely high titers of DRA. Side effects to dextran 1, mostly mild, were reported in one case per 100,000 doses. These side effects were not antibody mediated. It is concluded that the introduction of hapten inhibition with dextran 1 has greatly reduced the risk for serious side effects to dextran, making dextran one of the safest colloids in use.

Serum concentrations of hyaluronan and proteoglycan in joint disease. Lack of association.

Circulating hyaluronan originating from the synovial membrane and circulating proteoglycan released from cartilage were determined by specific assays in patients with osteoarthritis (OA), rheumatoid arthritis (RA), reactive arthritis or juvenile chronic arthritis (JCA). Elevated hyaluronan concentrations were found in OA and RA, suggesting proliferation of the synovial membrane in both diseases. The proteoglycan concentrations were highest in OA and polyarticular JCA indicating increased turnover of cartilage matrix. The concentrations of the macromolecules did not correlate except in the group with JCA. Serum concentrations of hyaluronan and proteoglycan thus differ between disease groups and may reflect different aspects of the arthritic process.

Does vaccination of splenectomized patients against pneumococci induce dextran-reactive antibodies?

Polyvalent pneumococcal vaccine containing polysaccharides from serotypes known to be cross-reactive with dextran was used for vaccination of 72 patients either shortly before or on the average 6.25 years after splenectomy. The titer of dextran-reactive antibodies was determined by passive hemagglutination before as well as 6 weeks and 6 months after vaccination. Already prior to vaccination the patients had moderately elevated titers of these antibodies and the titer was somewhat reduced during the study period. Small intergroup differences were observed but the titers of patients who had received cytostatic and/or radiation therapy did not appear to react differently to vaccination. It is concluded that this type of vaccine does not induce dextran-reactive antibodies when given to asplenic individuals.

Histopathological lung changes in immune complex mediated anaphylactic shock in humans elicited by dextran.

Severe dextran-induced anaphylactic reaction (DIAR) is being recognized as a form of immediate IgG mediated immune complex reaction. Support for this pathogenesis is found in the correlation between the titer of dextran-reactive antibodies of IgG class and the severity of the reaction. Autopsy records were reviewed in 27 certified cases of fatal DIAR. The most frequent macroscopic findings were dilatation of the right side of the heart and acute pulmonary stasis. Autopsy lung specimens were collected from 17 of these patients. In 15 of the 17 lung specimens pulmonary microemboli were found. The microemboli had the appearance of hyaline eosinophilic globules, and the lung vasculature also contained leukocytes, platelets and disintegrated erythrocytes. These findings show similarity to the findings in a monkey model of known IgG mediated anaphylaxis, and give further support to the proposed pathogenesis of severe DIAR.

Hapten inhibition and dextran anaphylaxis.

Hapten inhibition with dextran 1 has been used in Sweden since 1982 to prevent severe anaphylactic reactions to dextran. This has led to a reduction in the reports of severe reactions to dextran from 22 per 100,000 units of dextran used between 1975 and 1979, to 1.2 per 100,000 units between 1983 and 1985. The number of fatal reactions decreased from 23 to one. More than 600,000 units of dextran were used during each period.

Adverse reactions to dextran in Sweden 1970-1979.

In a retrospective investigation of the period 1970-1979, 478 reports on cases of dextran-induced anaphylactoid/anaphylactic reactions (DIAR) were found. In 458 of these dextran was judged to be the causative agent. The number of units of dextran 40 and 70 used during this decade was 313 598 and 1 051 668, respectively. During the last five years, when reporting of severe adverse drug reactions was mandatory, the incidence of severe DIAR per unit administered was 0.013% for dextran 40 and 0.025% for dextran 70. The incidence of fatal DIAR was 0.003% and 0.004%, respectively. We found a male: female ratio of 1.5: 1 for all DIAR while for the most severe DIAR this ratio was 3: 1. The mean age of the patients was higher in severe than in mild DIAR. Allergic disease did not predispose for severe DIAR. Diabetes, cardiovascular and pulmonary diseases and chronic inflammation were more frequent among the patients who had suffered the most severe DIAR. 22 reactions were characterized by fever and/or shivering, and in 19 of these cases the reaction occurred after infusion of more than one unit of dextran. Severe DIAR occur after infusion of a few ml of dextran. Fatal DIAR during anaesthesia occurred almost exclusively in patients under spinal or epidural blockade. A favourable outcome of resuscitation in DIAR with cardiac arrest was possibly promoted by the infusion of large amounts of crystalloid fluids. We could not show that therapy with steroids, epinephrine or isoprenaline influenced the outcome. Fatal DIAR occurred even after infusion of only 0.5-1 ml of dextran, and we strongly advise against the use of "biological test-doses".

Prevention of dextran-induced anaphylactic reactions by hapten inhibition. I. A Scandinavian multicenter study on the effects of 10 ml dextran 1, 15% administered before dextran 70 or dextran 40.

In an open prospective study the prevention of dextran-induced anaphylactic reactions (DIAR) by hapten inhibition was investigated in 29 252 patients. Forty-nine hospitals in Sweden, Norway and Finland participated in the study which was running for two years. Ten ml of dextran 1, 15%, (Promiten, Pharmacia AB, Uppsala, Sweden) with a weight average molecular weight of 1 000 dalton was injected intravenously two minutes before infusion of dextran 70 (Macrodex) or dextran 40 (Rheomacrodex). DIAR were graded according to severity from I to V. Six grade III and one grade IV reactions occurred, representing an incidence of severe DIAR of 0.024%. Compared to a large historical control material, preinjection of 10 ml of dextran 1 exerts a partially protective effect, but no statistical proof can be given. Consequently trials have been started with a 20 ml dose of dextran 1. Adverse reactions to the preinjection of 10 ml of dextran 1 were observed in 0.072%. These reactions were of a mild and shortlasting nature and considered to be of minor clinical importance.

Prevention of dextran-induced anaphylactic reactions by hapten inhibition. III. A Scandinavian multicenter study on the effects of 20 ml dextran 1, 15%, administered before dextran 70 or dextran 40.

In an open, prospective multi-center study prevention of dextran-induced anaphylactic reactions (DIAR) was attempted by intravenous injection of 20 ml dextran 1, 15%, Mw 1000 dalton, (Promiten), two minutes before start of infusion of dextran 70 (Macrodex) or dextran 40 (Rheomacrodex). Of 34955 patients investigated, only one developed a severe DIAR of grade III (0.003%). The incidence of severe DIAR after preinjection of 20 ml dextran 1 is significantly lower than that after injection of 10 ml dextran 1 (p = 0.01), and is also markedly lower than that of a large Swedish historical control material (0.037-0.050%). The incidence of mild DIAR is not affected by dextran 1. Adverse reactions to dextran 1 of a generally mild and short-lasting nature were observed in 20 patients. It is recommended that patients who are going to receive dextran 70 or 40 are given a prophylactic i.v. injection of 20 ml dextran 1 before the first unit of clinical dextran to minimize the risk of severe DIAR.