Activation of human neutrophils by electronically transmitted phorbol-myristate acetate.

We report the transfer of the activity of 4-phorbol-12-beta-myristate-13-acetate (PMA) by electronic means. Neutrophils were placed at 37 degrees C on one coil attached to an oscillator, while PMA was placed on another coil at room temperature. The oscillator was then turned on for 15 min, after which cells were usually further incubated for up to 45 min at 37 degrees C before measurement of reactive oxygen metabolites (ROMs) production. In 20 blind experiments, PMA thus 'transmitted' induced ROM production. ROM were not induced when: (1) PMA vehicle or 4-alpha-phorbol 12,13-didecanoate (an inactive PMA analogue) were transmitted; (2) the oscillator was switched off; (3) superoxide dismutase or protein kinase C inhibitors were added to cells before transmission. These results suggest that PMA molecules emit signals that can be transferred to neutrophils by artificial physical means in a manner that seems specific to the source molecules.

Lay theories of anorexia nervosa: a discourse analytic study.

Previous studies on lay theories of anorexia nervosa have examined the 'accuracy' of lay knowledge, and the identification of factors by family and friends that would encourage early interventions (Huon, Brown, & Morris, 1988, 7, 239-252; Murray, Touyz, & Beumont, 1990, 9, 87-93). In contrast to these approaches, we examine lay theories of anorexia nervosa using a critical psychology perspective. We argue that the use of a discourse analysis methodology enables the examination of the construction of lay theories through dominant concepts and ideas. Ten semi-structured interviews with five women and five men aged between 15 and 25 years were carried out. Participants were asked questions about three main aspects of anorexia nervosa: aetiology, treatment and relationship to gender. Each interview was analysed in terms of the structure, function and variability of discourse. Three discourses: sociocultural, individual and femininity, are discussed in relation to the interview questions. We conclude that, in this study, lay theories of anorexia nervosa were structured through key discourses that maintained a separation between sociocultural aspects of anorexia nervosa and individual psychology. This separation exists in dominant psychomedical conceptualizations of anorexia nervosa, reinforcing the concept that it is a form of psychopathology.

Presence of paf-acether in human blood after thin-layer chromatography, but not after high-performance liquid chromatography purification.

After HPLC purification of human blood extracts, paf-acether (paf) was found exclusively as a lipoprotein-bound compound (lipopaf), whereas free-paf was absent. When the same samples (or lipopaf recovered from HPLC) were purified by TLC, both free-paf and lipopaf were detected. The free-paf detected in blood samples could thus result from lipopaf dissociation during TLC purification.

Tissue levels of histamine, PAF-acether and lysopaf-acether in carrageenan-induced granuloma in rats.

The tissue concentrations of several inflammatory mediators were determined from day 0 to day 60 in granuloma induced in rats (n = 105) by injection of carrageenan in the fascia of the latissimus dorsi muscle. Noncollagen proteins (NCP) and the number of polymorphonuclear leukocytes (PMN) and mast cells were also assessed. In comparison with the tissue at time 0, we noted in the inflamed tissue (at 4 h) an increase in total proteins (4.0 +/- 3.0 vs. 84 +/- 12.0%, mean +/- SEM) and PMN (0.0 +/- 0.0 vs. 43.3 +/- 13.4%), and a fall in histamine concentration (from 30.0 +/- 9.0 to 9.0 +/- 4.0 ng/ml). A partial disappearance of mast cells and an increase of PAF-acether (PAF) levels (1.0 +/- 1.0 vs. 30.0 +/- 22.0 ng/ml) was noted at 16 h, whereas lysopaf remained unchanged (3.7 +/- 4.0 vs. 3.5 +/- 1.0 ng/ml). During evolution towards chronic inflammation (day 10-60), NCP decreased, PMN disappeared and mast cells reappeared; the histamine level rose to 11.0 +/- 2.0 mg/ml, thus not reaching back baseline values. Lysopaf rose to 7.1 +/- 12.2 ng/ml and PAF levels increased further to reach 240.0 +/- 153.0 ng/ml at day 10. This study suggests that PAF may contribute to the acute phase of an inflammatory state such as the carrageenan-induced granuloma and that it is also present during the chronic process.

Decrease of ciliary beat frequency by platelet activating factor: protective effect of ketotifen.

The ciliary beat frequency (CBF) of the tracheal epithelial cells controls in part the respiratory tract mucociliary transport efficiency. We investigated the effects on CBF of PAF-acether (PAF) and its metabolite/precursor lyso-PAF. Guinea-pig tracheal rings were incubated for 3 to 6h with 1 microM PAF (C16, C18, C16/C18: 80/20%), lyso-PAF C16 or lyso-phosphatidylcholine (LPC). CBF changes were assessed by microphotooscillography (mean number of measures per ring = 14). We also examined the effect on PAF-induced CBF changes of the PAF receptor-antagonist WEB 2086, the anti-asthmatic/anti-anaphylactic drug ketotifen and the anti-histamine H1 pyribenzamine. CBF of control rings exposed to vehicle only from 0 to 6h showed no significant statistical variations (hertz, mean +/- SEM): 10.8 +/- 0.1 (n of measures = 890). By contrast, 1 microM C16, C18, and C16/C18 PAF significantly inhibited CBF after 3 to 6h incubation. C16 and C16/C18 PAF were more potent than C18 PAF (8.8 +/- 0.2, n = 112, 8.7 +/- 0.2, n = 64, and 9.6 +/- 0.1, n = 537 respectively; ANOVA analysis, p < 0.001 from control). At the same concentration, lyso-PAF also inhibited CBF, 9.5 +/- 0.1 (n = 197, p < 0.001) but not LPC, 10.5 +/- 0.2 (n = 127). WEB 2086 inhibited lyso-PAF and C16/18 PAF-induced CBF decrease. Preincubation (20 min) with ketotifen but not with pyribenzamine (1 microM) also suppressed the CBF inhibitory effect of PAF and lyso-PAF. Incubation of [3H]PAF with tracheal rings from 10 min to 6h resulted in its partial metabolism (25%) into [3H]lyso-PAF and a compound with a short retention time (10 min). [3H]lyso-PAF incubated for 3h with tracheal rings was partially metabolized (10%) into [3H]PAF and a compound with a short retention time. The PAF-induced decrease of CBF is congruent with its influence on pulmonary clearance, possibly via a specific receptor, since WEB 2086 abolished the effect of PAF. The inhibition of the PAF-induced CBF decrease by ketotifen may contribute to the therapeutic properties of this antiallergic drug.

Biochemical and cellular effects of heparin-protamine injection in rabbits are partially inhibited by a PAF-acether receptor antagonist.

The origin of the thrombocytopenia and leucopenia induced by protamine-heparin complexes is unknown. We studied the biochemical and cellular effects of protamine (6 mg x kg-1, i.v.) injected after heparin (5 mg x kg-1, i.v.) in New Zealand rabbits. After protamine injection (0.5 min) increases in blood platelet-activating factor (PAF-acether, PAF) (27.6 +/- 27.6 to 148.2 +/- 48.9 pg x ml-1, P < 0.05), thrombocytopenia (403 +/- 64 to 166 +/- 13 cells x 10(-3) x mm-3, P < 0.05) and leucopenia (7650 +/- 930 to 4300 +/- 668 cells x mm-3, P < 0.05) were noted. Plasma thromboxane B2 increased at 1 min (125.6 +/- 24.4 to 879.7 +/- 141.0 pg x ml-1, P < 0.01). Protamine alone induced no change. Indomethacin (3 mg x kg-1, i.v.) did not counteract the effects of heparin-protamine. Pretreatment with the PAF receptor antagonist BN 52021 [9H1, 7a-(epoxymethano)-1 H,6aH-cyclopenta[c]furo[2,3-b]furo-[3',2',3,4]cyclopenta[1,2-d]fur an-5,9, 12(4H)trione,3-tert-butylhexahydro-4,7b,11 hydroxy-8 methyl] alone (3 mg x kg-1, i.v.) delayed thrombocytopenia and reduced plasma thromboxane B2 concentration but did not modify leucopenia. Thus thrombocytopenia and thromboxane B2 release triggered by heparin-protamine may be potentiated by the release of PAF.

Regulation of platelet-activating factor production in gastric epithelial cells.

The authors have previously reported that platelet-activating factor (PAF), a phospholipid mediator with potent proinflammatory activities, is produced in the gastric mucosa and stimulates gastric acid secretion in humans and animals. In the present study they used the human gastric tumour cells HGT1 (clone 6) to examine whether PAF production is regulated by neuromediators. PAF was extracted by ethanol and assayed by the washed platelet aggregation test. HGT1 cells produced PAF spontaneously (110 +/- 20 pg 10(6) cells). The addition of vasoactive intestinal peptide (VIP; 10(-9) to 10(-7) mol L(-1)) or of histamine (10(-5) to 10(-3) mol L(-1)) increased PAF production by three- to fivefold, while the addition of carbachol (10(-7) to 10(-4) mol L(-1)) increased PAF production up to sevenfold. PAF production was also increased up to 10- to 13-fold, in a dose- and time-dependent manner, by the addition of calcium and two- to threefold by the addition of phorbol myristate acetate (PMA; 10(-7) to 10(-5) mol L(-1)). However, the addition of dibutyryl cyclic AMP (dBcAMP; 10(-6) to 10(-4) mol L(-1) was without any effect. This is the first report showing PAF production by gastric epithelial cells in response to histamine, VIP and carbachol. Furthermore, the findings are consistent with a central role of calcium in this production. The results of this study, together with those of previous studies from the authors' laboratory, support the hypothesis that PAF is a physiological mediator of gastric acid secretion.

Liver and plasma concentrations in paf-acether and its precursors after partial hepatectomy.

Liver and plasma concentrations in paf-acether (paf) and related phosphocholines, i.e., lysopaf and the ether lipid 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (AAGPC) were studied in rats following two-third hepatectomy. We report a rapid increase in hepatic content of the 3 phospholipids at early steps of the regeneration process, when hepatocytes are switching from G0 to G1 (time 2-6 h). Later on, throughout G1 and at the G1-S transition, these concentrations decreased progressively. They were back to sham-operated or intact control levels at 50 h. In the plasma of hepatectomized animals, no comparable changes were detected. However, an increase in both circulating paf and lipoprotein-bound paf concentrations was measured during the regenerating response. This report is, to our knowledge, the first one on paf level variations following 2/3 hepatectomy. In rats, partial resection of the liver was shown to initiate rapid and complex cascades of biochemical changes involving growth factors, neurotransmitters and interleukins among others. Our data are in good agreement with reported increases in both total phospholipid content and synthesis of phosphatidylcholine, a paf precursor, in the regenerating liver. At present, the possible functional significance of high paf concentrations measured over the 'priming' stage of the induced proliferative wave is suggested as a working hypothesis. However, on the one hand, the observed paf response is noteworthy in view of its cytokine-related action, i.e., stimulation of IL-6 production by different cell types (endothelial, macrophagic). On the other hand, it could represent an in vivo confirmation of previously reported in vitro paf effects inducing c-fos and c-jun expression, two members of the so-called 'cellular immediate-early gene' family.

Intraluminal excretion of PAF, lysoPAF, and acetylhydrolase in patients with ulcerative colitis.

PAF-acether (PAF) is a phospholipid synthesized by numerous inflammatory cells. PAF can produce several pathological changes in various organs, especially in the colon. In animals PAF causes colonic ulceration and inflammation, which are similar to the anatomic lesions seen in human ulcerative colitis. The aim of this study was to measure in vivo colonic production of PAF in active ulcerative colitis using a modified colonic perfusion method. Ten patients with active ulcerative colitis and six control patients were investigated. A colonic segment was continuously perfused with a buffer and the liquid was recovered 20 cm distally, after a 45-min period of equilibration, at 20-min intervals. PAF, lysoPAF, and acetylhydrolase were measured in the colonic samples. PAF and lysoPAF outputs were significantly higher in patients with active ulcerative colitis compared to controls patients. There was a significant correlation between colonic PAF output and, respectively, macroscopic mucosal lesions and myeloperoxidase colonic output. We thus conclude: (1) the colonic perfusion method allows in vivo study of the metabolism of PAF during ulcerative colitis and could also be used to study the efficiency of PAF antagonists in UC; and (2) colonic production of PAF is increased during ulcerative colitis and correlated to local injury and inflammation. Whether or not PAF plays a role in the pathogenesis of ulcerative colitis remains open for further investigations.

Inhibition of PAF-acether effects on isolated guinea pig hearts by zinc ions (Zn2+).

PAF-acether is a phospholipid synthesized by most animal tissues and exerting a strong decrease on the heart's contractile force and coronary flow. PAF-acether (10(-9) and 10(-10)M) was administered to isolated guinea pig hearts perfused via the Langendorff apparatus with Chenoweth solution. Zinc (1.5 microM) is known to benefit heart function thus, Zn2+ (1.5, 7.5, and 30 microM) was added in the perfusing solution before or after PAF-acether administration. Contractile force, coronary flow, and heart rate were recorded by means of a Narco MK-IV Physiograph throughout all modes of perfusion. Calcium inhibitor (Verapamil 10(-10)M) and Pb+2 Co2+ (1.5 x 10(-6)M) were used subsequently in the perfusing solutions in order to elucidate some of the Zn and PAF interactions observed. All hearts were analyzed for their Zn and Ca content by means of an Atomic Absorption Spectrophotometry (AAS). Our data suggest that low concentrations of zinc (1.5 microM) can strongly inhibit PAF-induced decrease of contractile force and coronary flow. Zinc-inhibiting effects on PAF's negative inotropic action (myocytic level) is not exerted through Zn-Ca antagonism. Nevertheless, a Zn-Ca antagonism in the arteriolar level cannot be excluded. Zinc inhibits PAF selectively only if it is administered before PAF injection and this strongly suggests a receptor interaction between the metal and the phospholipid at the heart level.

Studies on the surface properties of human lymphocytes by photon correlation spectroscopy technique.

Electrokinetic behaviour of human lymphocytes was studied by photon correlation spectroscopy technique on laser IR-spectrometer. The electrophoretic mobilities (EPMs) were measured for peripheral blood lymphocytes (PBL) and CEM-C12 T cell line in 1:1 electrolyte at 22 degrees C. Plots of mobility vs ionic strength in the range 0.001-0.1 M were compared with theoretical curves calculated from (i) the Smoluchowsky formula, (ii) the simplified form of the Dukhin-Deryaguin equation which takes into account the fact that the mobility decreases due to the relaxation effect and (iii) the equation suggested by Donath and Pastushenko, which takes into account the influence of cell glycoprotein layer (GPL) on the EPM values. It has been found that the first two equations describe the experimental data with the assumption that surface charge density (sigma) decreases and width of the hydrodynamically immobile layer (L) increases with decreasing ionic strength; the relaxation effect turns out to be insignificant for the cell charges and sizes under consideration. In agreement with these findings, the third equation is approximately consistent with experimental data on the condition that GPL is allowed to expand with decreasing ionic strength, with simultaneous decrease of its full charge density (sigma(f)). The results are compared with relevant evidence for erythrocytes. The possible applications of the inferences arrived at in electrophoretic studies of cell behaviour are also discussed.

Human umbilical vein endothelial cells: specific binding of platelet-activating factor and cytosolic calcium flux.

An interaction of the platelet-activating factor (Paf) with endothelial cells was investigated using human umbilical vein endothelial cells. Confluent endothelial cells bound [3H]Paf in the presence of 0.25% fatty acid-free serum albumin after culture in media containing either heat-inactivated foetal calf serum or serum substitute. The Scatchard analysis of the saturated specific [3H]Paf binding showed a Bmax of 2.5 fmol indicating 2800 binding sites per endothelial cell. [3H]Paf binding was partially reversible at 20 degrees and 4 degrees and endothelial cells partially metabolized [3H]Paf at 20 degrees but not at 4 degrees. [3H]Paf binding and Paf-mediated increase of cytosolic free calcium were inhibited by specific Paf receptor antagonists which do not interfere with Paf metabolism. Immortalized umbilical vein endothelial cells bound [3H]Paf specifically after culture in the presence of insulin (20 hr, 0.4 U/mL) with non-specific binding in the absence of insulin. The results show that specific Paf binding mediated calcium flux in human endothelial cells.

Voltage-dependent ion channels on human basophils: do they exist?

The presence of voltage-dependent ion channels (particularly Ca2+ channels) on the surface of 'non excitable' cells such as human basophils is a matter of debate. Indeed, in basophils, Ca2+ entry or mobilization is not sufficient by itself to trigger secretion, although enhanced cytosolic Ca2+ concentration increases it. In order to address this question, we used a two-signal model and we report here experiments which suggest the presence of voltage-dependent structures directly or indirectly linked to membrane Ca2+ pathways. Indeed, it is known that, in the presence of PMA at threshold concentration (1st signal), elevation of cytosolic Ca2+ (2nd signal) induces histamine release. We observed that a depolarizing external solution (high K+) induced a Ca(2+)-dependent release of histamine from PMA-treated human basophils. High K+ alone did not induce histamine release. Although the voltage-sensitive component and the physiological relevance of this mechanism remain to be defined, these results suggest that this voltage-dependent Ca2+ influx in the human basophil could contribute to the up-regulation of histamine release.

Treatment of carrageenan induced arthritis by the platelet activating factor antagonist BN 50730.

OBJECTIVE: To evaluate the role of platelet activating factor (PAF) in the early stage of arthritis. METHODS: Arthritis was induced in rabbits by weekly intra-articular injections of carrageenan. A PAF receptor antagonist, BN 50730, was used as a preventive or curative agent. RESULTS: BN 50730 was able partially to prevent the development of arthritis, and was also active on established arthritis. The joint arthritis scores of BN treated animals were significantly lower than those of the non-treated animals. The blood concentrations of PAF, PAF bound to lipoproteins (lipo-PAF), and its precursor, lyso-PAF, were not correlated with clinical variations. CONCLUSIONS: The present data demonstrate a therapeutic action of a PAF antagonist in experimental arthritis and suggest a critical role for PAF during the early stage of arthritis.

Correlations between PAF-acether and tumor necrosis factor in rheumatoid arthritis. Influence of parenteral corticosteroids.

The aim of this study was to evaluate the presence of PAF-acether (PAF), its specific degrading enzyme acetylhydrolase, and tumor necrosis factor (TNF) concentrations in blood and synovial fluid (SF) from patients with active RA. The variations of the mediators were also evaluated after corticosteroid perfusions in 7 patients. Lipo-PAF (PAF complexed to lipoproteins) was the main form of PAF detected both in blood and in SF, whereas unbound PAF was uncommon. Acetylhydrolase activity was also present in SF, with a strong correlation between serum and SF levels. TNF was detected in most of the samples, and TNF and acetylhydrolase levels were strongly correlated both in blood and in SF. Despite dramatic clinical improvement, corticosteroid treatment was not accompanied by a significant reduction of the concentration of blood mediators, suggesting that these molecules should not be considered as markers of disease activity.

Modulation of stress proteins by Cd2+ in a human T cell line.

We previously showed in a human T cell line (CEM-C12 cells) that Cd2+ induced gene expression of stress proteins, metallothionein-IIA and heat shock protein 70 in a time- and dose-dependent manner. In the present study, CEM-C12 cells were pretreated for 24 h with 1 microM Cd2+ and then challenged with toxic concentrations of this metal. We found that maximal expression of the metallothionein-IIA and heat shock protein 70 genes was increased and this maximal level occurred at higher Cd2+ toxic concentrations. Actinomycin D chase experiments indicated that Cd2+ pretreatment did not modify metallothionein-IIA mRNA stability. The modulatory effect of Cd2+ pretreatment was dose-dependent from 100 pM to 1 microM. Such pretreatment also enhanced resistance to Cd2+ toxicity. Finally, verapamil, a calcium/potassium channel blocker displaced the dose-response curve for Cd2+ toxicity as well as metallothionein-IIA and heat shock protein 70 gene expression to higher Cd2+ concentrations.

Cadmium induces apoptosis in a human T cell line.

Cadmium, a potent toxic metal, poses a serious environmental threat but the mechanisms of its toxicity remain unclear. In the present study, we investigated the nature of cadmium-induced cell death in the human T cell line CEM-C12. Cadmium was time- and dose-dependently toxic for CEM-C12 cells, cell death being preceded by chromatin condensation and DNA fragmentation. Quantification of the latter indicated an increase above 4 microM cadmium, with maximal fragmentation at 8 to 10 microM. By contrast, when CEM-C12 cells were exposed to higher cadmium concentrations (50 microM), cell death increased without concomitant chromatin condensation or DNA fragmentation. Thus, cadmium at low and high concentration kills CEM-C12 cells by apoptosis and necrosis, respectively. Addition of cycloheximide reduced the apoptotic effect of cadmium, suggesting that cadmium-induced apoptosis is an process depending on protein synthesis. Verapamil, a calcium/potassium channel blocker, markedly increased the viability of CEM-C12 cells treated by low cadmium concentrations and prevented DNA fragmentation. The apoptotic effect of cadmium suggests a possible mechanism for lymphocyte damage occurring after in vivo exposure to cadmium.

Anti-IgE induces the opening of non selective cation channels on human basophils.

Basophils play a major role in allergic reactions-particularly in late phase reactions-by releasing histamine and other mediators of inflammation. Although transmembrane ion fluxes are thought to play an important role in the modulation of histamine release, little is known about ion pathways through the basophil membrane. We thus studied human basophils from normal subjects (n = 25 cells) with the patch-clamp method. We observed that IgE-dependent activation of human basophils led to the opening of non selective cation channels with a 20pS conductance. This was obtained when the patch pipette was applied onto the cell surface and sealed onto it in order to measure transmembrane currents on a small surface of intact basophils (cell-attached configuration). Non selective channels with the same 20pS conductance were also observed when a membrane patch was detached from basophil and its inner side placed in a Ca(2+)-containing medium (inside-out configuration). These data are a first contribution of the patch-clamp method in the understanding of ion movements in human basophils.