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Object Embryonic stem (ES) cell–derived astrocytes have several theoretical and practical advantages as gene therapy vectors in the treatment of malignant gliomas. The aim of this study was to test the proapoptotic effects of ES cell–derived astrocytes expressing transgenic tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) in human malignant glioma cells. Methods Mouse ES cells containing a doxycycline-inducible transgene were engineered with human TRAIL (hTRAIL) and then directed to differentiate into astrocytes. The ES cell-derived–TRAIL-expressing astrocytes were cocultured with human malignant glioma cells. Reverse transcriptase polymerase chain reaction, immunocytochemistry, terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling, and flow cytometry were used to quantify results. In vitro coculture of ES cell–derived astrocytes expressing hTRAIL with A172 human malignant glioma cells after doxycycline induction caused a significant decrease in cell viability from 85 ± 2% at baseline to 8 ± 2% posttreatment (p < 0.001). Labeling with apoptotic markers showed that cell death occurred by means of apoptosis. A significant increase in apoptotic rate (88 ± 3%) from baseline (4 ± 2%) was found in A172 cells after doxycycline induction (p < 0.005). This effect was superior to the apoptotic rate seen after treatment with recombinant TRAIL (57 ± 2%). A decrease in cell viability and an increase in the apoptotic rate were not found in TRAIL-expressing–ES cell-derived astrocytes after induction with doxycycline or in A172 cells exposed to doxycycline alone. Conclusions Engineering of transgenic hTRAIL by using ES cell–derived astrocytes induced apoptosis in human malignant glioma cells while sparing nontumor astrocytes. The apoptotic effects of transgenic hTRAIL are greater than those of recombinant hTRAIL. Analysis of these results suggests that hTRAIL-expressing–ES cell-derived astrocytes should be considered in the development of new in vivo strategies to treat malignant human gliomas.

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.

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.

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.

Object For gene therapy strategies currently in clinical trials, viral vectors are used to deliver transgenes directly to normal and tumor cells within the central nervous system (CNS). The use of viral vectors is limited by several factors. The aim of this study was to assess whether embryonic stem cell (ESC)–derived astrocytes expressing a doxycycline-inducible transgene can be used as a vector for gene therapy. Methods The authors generated a pure population of ESC-derived astrocytes carrying a transgene, tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), inserted in the chromosome under the control of a highly regulated doxycycline-inducible expression system. Fully differentiated ESC-derived astrocytes were stereotactically transplanted in the mouse brain, and then cell migration and transgene expression were studied. Results The ESC-derived astrocytes started to migrate from the transplant site 48 hours after the procedure. They were found to have migrated throughout the brain tissue by 6 weeks. Transplanted ESC-derived astrocytes expressed the TRAIL transgene after doxycycline induction throughout the duration of the experiment. Teratoma formation was not observed in long-term experiments (12 weeks). Conclusions These data show that ESC-derived astrocytes can be used as delivery vectors for CNS tumors. This technique might have a major impact on the treatment of patients with malignant gliomas and a wide spectrum of other neurological diseases.

Object. Embryonic stem cell (ESC)‐derived astrocytes have many theoretical and practical advantages as vectors for delivery of gene therapy to the central nervous system (CNS). The aim of this study was to generate highly pure populations of ESC-derived astrocytes expressing drug-inducible transgenes, while minimizing contamination by undifferentiated ESCs Methods. Embryonic stem cells carrying a doxycycline-inducible green fluorescent protein (GFP) transgene were induced to differentiate into astrocytes by using feeder cell‐free conditions that are completely defined. More than 95% of these cells expressed the astrocyte markers glial fibrillary acidic protein and GLT-1 glutamate transporter, and the morphological characteristics of these cells were typical of astrocytes. The expression of additional astrocyte markers was detected using reverse transcription‐polymerase chain reaction. Undifferentiated ESCs comprised fewer than 0.1% of the cells after 10 days in this culture. Positive and negative selection techniques based on fluorescence-activated cell sorting were successfully used to decrease further the numbers of undifferentiated ESCs. Fully differentiated astrocytes expressed a GFP transgene under the tight control of a doxycycline-responsive promoter, and maintained their astrocytic phenotype 24 hours after transplantation into the mouse brain. Conclusions. This study shows that transgenic ESCs can be induced to differentiate into highly pure populations of astrocytes. The astrocytes continue to express the transgene under the tight control of a drug-inducible promoter and are suitable for transplantation into the mouse brain. The number of potentially hazardous ESCs can be minimized using cell-sorting techniques. This strategy may be used to generate cellular vectors for delivering gene therapy to the CNS.

The effects of protamine (6 mg kg-1) injected after heparin (5 mg kg-1) have been studied in five groups of five New Zealand white rabbits. Group I was treated with the sequence heparin-protamine and group II with protamine alone. The animals of groups III and IV received respectively intravenous indomethacin (3 mg kg-1) and BN 52021 (3 mg kg-1), a paf receptor antagonist before the sequence heparin-protamine. Group V was pre-treated with indomethacin and BN 52021 before heparin reversal with protamine. In group I, immediate thrombocytopenia (44.1 +/- 4.6% of baseline level, P less than 0.05) and leucopenia (55.5 +/- 2.3% of baseline level, P less than 0.05) were observed 30 s after protamine reversal of heparin, paralleled with an increase in blood paf levels (27.6 +/- 27.6 vs. 148.2 +/- 48.9 pg ml-1, P less than 0.05). In group II, protamine alone induced no change in platelet count nor in blood paf levels (55 +/- 10 vs. 52.5 +/- 20 pg ml-1, P greater than 0.05). Pre-treatment with indomethacin alone (group III) did not protect the animals against the haematological changes induced by the heparin-protamine complexes. Pre-treatment with the paf receptor antagonist, alone or in association with indomethacin, delayed the occurrence of thrombocytopenia 3 min after protamine administration but the leucopenia was the same as in group I. This study demonstrated that paf is implicated in the immediate thrombocytopenia occurring after protamine reversal of heparin in rabbit.

The renal glomerular and tubular effects of the platelet-activating factor (Paf-acether) were studied, by clearance techniques, in acutely thyroparathyroidectomized Brattleboro rats infused with Paf-acether at the rate of 1.25, 2.5 or 5 ng/min/100 g bw. Paf-acether infusion was accompanied by decreases of urinary flow rate, calcium, and magnesium urinary excretion, whereas decreases of mean arterial pressure and glomerular filtration rate did not exceed 20% of control values for the highest perfusion rate of Paf-acether. These changes in tubular function were partially (calcium excretion) or totally (urinary flow rate and magnesium excretion) reversed after Paf-acether infusion was discontinued. Sodium and potassium excretion did not vary significantly during Paf-acether infusion, but increased dramatically after discontinuation of Paf-acether infusion. Infusion of lyso-Paf-acether or ethoxy-Paf-acether, two biologically less active structural analogues of Paf-acether, did not elicit any change in the variables studied. These data suggest that Paf-acether, when locally released by the kidney in pathological conditions, might affect both glomerular and tubular functions, possibly through different mechanisms.

Platelets-isolated or in conjunction with leukocytes-interact with vessel walls in many experimental and human diseases. Several mediators are held responsible for platelet activation and interaction with leukocytes, among which PAF-acether (platelet-activating factor) is a prime candidate. This phospholipid mediator is released by most inflammatory cells, including neutrophils, by isolated organs such as kidney and heart, is a potent platelet and neutrophil agonist, and exerts major vasoactive properties. Its biosynthesis involves a two-step enzymatic process yielding the active molecule from the membrane alkyl-ether choline-containing phospholipids. The first step implicates a phospholipase A2 that hydrolyzes a long-chain fatty acid (which can be arachidonic acid) from membrane phospholipids, leaving the intermediate compound lyso PAF-acether, a PAF-acether precursor that is acetylated by an acetyltransferase in a second step. It can also result from deacetylation of PAF-acether by an acetylhydrolase. PAF-acether release might explain the intervention of platelets in diseases such as glomerulonephritis and allergic vasculitis, in which the involvement of neutrophils and platelets is frequently noted. The end result of these complex sets of cell-to-cell interactions is the release of most known inflammatory mediators, influencing vascular permeability, cell infiltration, and smooth muscle contraction. Nevertheless, direct evidence for the implication of these rather well-defined cellular and molecular interactions in human pathologic states remains to be obtained.

The metabolite of molsidomine, 3-morpholino-syndnonimine (SIN-1) is a vasodilator and an inhibitor of platelet activation induced by adenosine-5'-diphosphate (ADP), arachidonic acid (AA) and thrombin. We present the results of SIN-1 on platelet aggregation induced by paf-acether and on the biosynthesis of this mediator by washed rabbit platelets. SIN-1 inhibits submaximal platelet aggregation induced by 50 pM paf-acether. This inhibition is dose-dependent (SIN-1 IC50: 37 +/- 10 nM, n = 3). SIN-1 also inhibits submaximal aggregations induced by ADP (IC50: 13 +/- 11 nM) and AA (IC50: 26 +/- 12 nM) (n = 3-5). Finally, SIN-1 inhibits dose-dependently the formation of lyso paf-acether (the precursor of paf-acether) and of thromboxane A2, and AA freeing by thrombin-stimulated platelets. Thus SIN-1 has a wide spectrum activity on platelets. He is both capable of inhibiting platelet aggregation induced by all used agonists and to inhibit the formation of paf-acether and thromboxane A2, two powerful mediators of platelet aggregation.

Activated peritoneal macrophages (M phi) from mice injected with Bacilli Calmette-Guérin, trehalose dimycolate, a defined immunostimulant derived from Mycobacterium tuberculosis, or streptococci C74 (St), synthesized two to three times less paf-acether (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in response to a zymosan challenge than did (0.7 +/- 0.2 nmol/mg protein) resident-M phi (R-M phi). To assess at which step the paf-acether biosynthetic pathway was impaired, the content in phospholipid paf-acether precursors was evaluated. The alkyl-acyl-glycerophosphocholine content was comparable in R-M phi (50.4 +/- 18.6 nmol/mg protein) and activated-M phi (40.0 +/- 6.5 to 63.9 +/- 7.7 nmol/mg protein), as well as the lyso paf-acether content (0.85 +/- 0.18 nmol/mg protein for R-M phi vs 0.63 +/- 0.16 to 1.23 +/- 0.21 nmol/mg protein for activated macrophages). The nonlimiting rate of the phospholipid substrate was strengthened by experiments showing that incubation of the various populations with lyso paf-acether did not yield increased amounts of paf-acether. Similarly, incubation of R-M phi or St-M phi with sodium acetate increased paf-acether production to the same extent in both populations, ruling out the acetate substrate deficiency as the cause of the impaired production. The level of acetyltransferase activity, the enzyme that transfers an acetate moiety of acetyl coenzyme A (acetyl-CoA) onto lyso paf-acether, was very low in thioglycolate (TG)-elicited M phi but high in R-M phi and activated ones. In all cases, it was increased by two to three times upon zymosan challenge. This suggested that increased paf-acether catabolism and not impaired anabolism could be responsible for the marked reduced formation noted in activated macrophages. The addition of acetyl-CoA (200 microM) to the various macrophage monolayers restored paf-acether formation by activated cells to R-M phi values but with delayed kinetics as compared with paf-acether formation induced by zymosan. The enhancing effect of acetyl-CoA on paf-acether production was inhibited upon oleyl-CoA addition, suggesting that acetyl-CoA may increase paf-acether production by preventing the reacylation of lyso paf-acether resulting from paf-acether degradation. In conclusion, the paf-acether output in some inflammatory macrophages may be regulated by the level of acetyltransferase activity, because it is observed in TG-M phi. However, we present the first evidence for another mechanism of regulation, most probably related to the deacylation/reacylation of paf-acether precursors and metabolites.

Platelet aggregation is triggered by at least three distinct pathways. The first one is mediated by adenosine diphosphate, the second one by arachidonic acid metabolites and the third is defined somehow by exclusion of the first two. In fact rabbit platelets synthesize, during their activation, another substance with potent aggregating activity, namely platelet-activating factor or PAF-acether. Indirect pieces of evidence are presented, which suggest that PAF-acether is the mediator of the third pathway of platelet aggregation.

There is evidence indicating that bioreactive lipid mediators, PAF-acether (platelet-activating factor: 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) and arachidonic acid (AA) metabolites, are formed upon deacylation of ether lipids. M phi obtained from mice treated with the sterile irritant thioglycolate exhibited an impaired formation of PAF-acether, leucotrienes (LT) C4 and prostaglandins (PG). In order to assess whether the impaired formation in lipid mediators is a general feature of M phi found at inflammatory sites, we have compared the capacity of resident (R-M phi) and immunostimulant-activated M phi to release, upon a zymosan challenge, PAF-acether and cyclooxygenase and lipoxygenase products. Activated M phi was obtained from mice injected intraperitoneally with non-viable C74 streptococci (St-M phi), bacilli Calmette-Guerin (BCG-M phi) or trehalose dimycolate (TDM-M phi), a defined immunostimulant isolated from Mycobacterium tuberculosis. All populations were capable of releasing PAF-acether. However, the amount of cell-associated PAF-acether was reduced by 75-90% in activated M phi populations as compared to R-M phi. Although the acetyltransferase level was comparable, acetyl-CoA supplementation restored the formation of PAF-acether by activated M phi to control (R-M phi) level. The amount of 14C-AA metabolites released by St-M phi was much lower compared with TDM-M phi or R-M phi, as was the amount of LTC4 detected as SRS contractile activity after HPLC.(ABSTRACT TRUNCATED AT 250 WORDS)

Paf-acether (platelet-activating factor) is one of the most potent mediator of inflammation released from and acting on most cells that participate in inflammatory diseases. Its molecular structure is 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine. Two metabolic steps are involved in its biosynthesis: the action of a phospholipase A2 on choline-containing membrane alkyl-ether lipids results in the production of lyso paf-acether and acetylation of the lyso compound by an acetyltransferase yields the biologically active molecule. Membrane alkyl-ether lipids can therefore be considered as potential precursors of paf-acether and their composition has been studied in various cell types. In this work, we investigated the presence of paf-acether in E. coli. Our results showed that paf-acether can be obtained from E. coli K12 under a variety of bacterial growth conditions. Paf-acether from E. coli exhibited the same physicochemical and biological characteristics as synthetic paf-acether and that from eucaryotic cells. Therefore, it appears that E. coli itself has the ability of producing paf-acether, a result that could be of some importance with respect to the pathogenesis of Enterobacteria and the use of E. coli in the recombinant DNA technology.

The direct in vitro antigen-induced degranulation of rabbit basophils based on a simplified one-step method for the staining of these cells is reported. Degranulation was assessed by counting the number of basophils before and after exposure to antigen at 37 degrees C in the presence of Ca++. The degranulation was IgE-mediated, as shown by its passive transfer to nonimmunized rabbits by IgE-containing serum; also, the reaction was dependent on the presence of Ca++ ions and normal cell metabolism. The technique allowed the detection of serum factors capable of blocking the degranulation. It provided a means for following IgE sensitization at the cellular level, which is a better index of immediate hypersensitivity than the presence of circulating specific IgE.

Platelet-activating-factor is a new mediator of anaphylaxis released in numerous mammalian species from basophils and mastocytes by antigens, anti-IgE antiserum, ionophore A 23187, C3a, C5a and neutrophil cationic protein. It is probably a lyso-1-phosphatidylcholine, as evidenced by structural studies isomg phospholipases. It may explain the involvement of platelets in immunopathology.

Mouse bone marrow-derived mast cells passively sensitized with monoclonal IgE released paf-acether (platelet-activating factor) and beta-hexosaminidase when challenged with the specific antigen. The formation and the release of paf-acether followed an early increase in the activity of the acetyltransferase, the main enzyme in paf-acether biosynthesis. The antigen-induced activation of the acetyltransferase was dependent on physiologic temperature and on the presence of Ca2+. By using microsomal fractions from unchallenged and challenged mast cells, the Vmax values were 3.5 and 12.0 nmol/min/mg of protein, respectively, whereas in both cases a Km value for acetyl-coenzyme A of 172 microM was measured. The stimulation of acetyltransferase could be mimicked in vitro under experimental conditions which favor phosphorylation, i.e. adding ATP and Mg2+ to lysates from unchallenged mast cells. In contrast, ATP and Mg2+ were uneffective on lysates from challenged cells that exhibited high level of acetyltransferase activity, suggesting that phosphorylation of the enzyme already took place at the time of cell stimulation. Moreover, addition of alkaline phosphatase to microsomal fraction obtained from either antigen-challenged mouse bone marrow-derived mast cells or unchallenged cells, resulted in 52% and 43% loss of acetyltransferase activity, respectively. Phorbol myristate acetate treatment of cells doubled the enzyme activity supporting the phosphorylation hypothesis. Thus, we report on the immunologic activation of a key enzyme for paf-acether synthesis and on the mechanism of this activation in a pure mast cell population. A link between bridging of IgE receptors and the activation of an enzyme critical to the formation of a lipid mediator is thereby evidenced.

Intratracheal administration of platelet-activating factor (paf-acether) induced transient bronchoconstriction in baboons. Using an automated isotopic monitoring system, we found that intratracheal administration of paf-acether also elicited transient accumulation of platelets labeled with 111In oxine within the pulmonary vasculature after the increase in maximal peak inspiratory pressure. Bronchoalveolar eosinophilia was evident 1 h after challenge. The increases in peak inspiratory pressure and bronchoalveolar eosinophilia were inhibited by prophylactic administration of the antiasthma drug ketotifen but not by pyribenzamine, suggesting that the effects of ketotifen are unrelated to H-1 receptor antagonism. Platelet accumulation was not affected by ketotifen or pyribenzamine. This study suggests that paf-acether may be a mediator of the eosinophil recruitment in bronchial asthma and that inhibition of this phenomenon by ketotifen may contribute to the therapeutic efficacy of this drug.

Platelet-activating factor (a 1-0-alkyl-2-acetyl glyceryl-3-phosphorylcholine, P.A.F.-acether) causes the aggregation of platelets from various Mammalian species and the release of their granule content. P.A.F.-acether activity has been recovered in vitro from perfused isolated Rat kidneys, stimulated by the ionophore A 23187. The maximum release was reached 10 min. after addition of the ionophore. P.A.F.-acether from kidney exhibited the same physico-chemical and biological characteristics as P.A.F.-acether from leucocytes. These data demonstrate that the kidney secretes a mediator of immediate hypersensitivity (P.A.F.-acether) in the veinous vasculature. Therefore the kidney itself has the ability of inducing intravascular platelet aggregation with subsequent local increase in vasopermeability.

Platelet-activating factor (P.A.F.) is a mediator of anaphylaxis released from human and Rabbit basophils which causes aggregation of platelets and release of their vasoactive amines. We have induced the release of P.A.F. from Rat peritoneal cells (P.C.) with ionophore A 23187. After fractionation of P.C. on 5-15% Ficoll gradients, P.A.F. was obtained from macrophage-rich but not from mastocyte-rich fractions and from adherent cells but not from non adherent cells. These data suggest an important new function for the macrophage: aggregation of platelets and release of their vasoactive amines and others mediators of inflammation.

An anaphylactic mechanism involving IgE-sensitized basophils is necessary for immune complex deposition in rabbits. Basophils under stimulation by the IgE-bound antigen actively release a platelet-activating factor. Increased vascular permeability results from action of vasoactive amines from basophils and from aggregated platelets and possibly direct action of PAF on vascular walls. This allows trapping of circulating complexes along vascular membranes. Once deposited complexes produce tissue injury by neutrophil-complement-dependent mechanisms. However, complex deposition seems pathogenic in itself for the glomerular basement membrane. PAF is a 1,100-dalton MW lipid, with hydrophobic properties and positive charge; it exists in human basophils. Patients suffering from SLE or undergoing anaphylactic shocks exhibit marked reduction of basophil counts and PAF level. We propose that anaphylactic increase of vasopermeability may be a common feature at early stages of many immune diseases associated with severe structural injury.

Paf-acether (platelet-activating factor, 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine) is a potent mediator of inflammation and allergy, synthesized by various cell types. It is noteworthy that IgE-sensitized alveolar macrophages are capable of releasing this mediator which, in turn, induces a platelet-dependent, cyclooxygenase-independent acute bronchoconstriction in the guinea pig, monkey, and man. Indirect evidence suggests that this mediator may also play a role in human asthma as an inducer of bronchial hyperreactivity, but clear definition of its importance in any disease must await study of the effects of specific, selective antagonists.

Addition of 1 microM dexamethasone (DM) to bone marrow-derived mast cells (BMMC) induced a time-dependent increase in cell histamine content. The latter reached a plateau of 2.5 micrograms/1 x 10(6) cells after 11 days in culture, compared with 100 ng/1 x 10(6) for untreated BMMC. Steroids, such as beta-estradiol, androsterone, and testosterone (1 microM), did not alter the histamine content of BMMC, whereas progesterone (1 microM) induced a moderate increase. Other glucocorticosteroids also enhanced histamine content, suggesting that the observed increase was specific for glucocorticosteroid. Treatment of BMMC with 1 microM DM for 14 days inhibited the Ag-induced, IgE-mediated release of histamine, beta-hexosaminidase, platelet-activating factor-acether, LTB4, and LTC4 by 65 +/- 3%, 66 +/- 1%, 93 +/- 3%, 66 +/- 2%, and 74 +/- 10%, respectively (mean +/- 1 SD, n = 3). In contrast with untreated cells which produce less than 2 ng/1 x 10(6) cells PGD2 after Ag challenge, DM-treated BMMC generated 16.8 +/- 0.3 ng/1 x 10(6) cells PGD2. Moreover, most of DM-treated BMMC became Alcian blue+/safranin+ and by ultrastructure, exhibited numerous cytoplasmic granules filled with abundant and uniform electron-dense matrix. The present results indicate that DM-treated BMMC exhibit biochemical and functional properties different from immature untreated cells, suggesting that a maturation-like process occurred in vitro during DM treatment.

The role of the basophils in acute serum sickness of rabbits was examined by monitoring daily the absolute number of basophils before, during and after the disease period. After antigen (bovine serum albumin, BSA) elimination, levels of serum IgE and in vitro basophil degranulation in the presence of BSA were determined. The results showed that the onset of glomerular lesions depends upon the simultaneous occurrence of circulating immune complexes greater than 19 S and of an in vivo basophil depletion--probably equivalent to degranulation--reaching 70% of the pre-disease number. Post-disease antigen-dependent in vitro degranulation of the basophils and levels of serum IgE anti BSA did not prove to be good indexes of basophil sensitization. Our data suggest that basophils are instrumental at early stages of the deposition of immune complexes, most probably through their sensitization by membrane-bound IgE antibodies.

The activity of the acetyltransferase capable of transferring the acetyl moiety of acetyl-CoA onto 2-lyso PAF-acether (1-alkyl-sn-glycero-3-phosphocholine) to form PAF-acether was compared in ionophore A23187-stimulated and in non-stimulated rat peritoneal cells. Stimulation resulted in a doubling of the acetyltransferase activity within 30 s. This effect was abolished in the presence of EDTA (1 mM) or EGTA (1 mM) and restored by addition of Ca2+ (10 mM). The specificity of acetyltransferase measured in ionophore-stimulated as well as in untreated cells is the same. In both situations we observed the same Km values for acetyl-CoA, whereas the Vmax values were different. The wide similarities of the two enzyme preparations lead us to conclude that stimulation by the ionophore involves an increase in the number of enzyme molecules rather than a change in the kinetic parameters of the acetyltransferase.

The simultaneous evaluation of platelet behaviour in vivo and of the accompanying bronchoconstriction in the guinea pig is described. Arachidonic acid induces bronchoconstriction, accompanied by, but independent from, thrombocytopenia, whereas collagen induces bronchoconstriction also accompanied by, but dependent from, thrombocytopenia. In both cases bronchoconstriction is due to cyclo-oxygenase metabolites of arachidonic acid. Use of potential inhibitors of thromboxane synthetase failed to reveal which of prostaglandin endoperoxides or thromboxane A2 is responsible for aspirin-inhibitive bronchoconstriction and thrombocytopenia. In contrast to PGE1 prostacyclin failed to interfere with bronchoconstriction by serotonin or by arachidonic acid, even though thrombocytopenia by the latter was suppressed. Bronchoconstriction by collagen, in contrast, was inhibited by nanogram doses of prostacyclin, confirming platelet-dependency. The combined bronchoconstriction/thrombocytopenia test in guinea pigs can discriminate sites of action of anti-inflammatory drugs, of agents which block specific platelet and/or bronchial receptors, which stimulate the cyclic AMP system, or generically which interfere with the mechanisms of bronchoconstriction and of thrombocytopenia.

The bronchomotor effect of intratracheal administration of PAF-acether (60 micrograms X kg -1) was investigated in 37 curarized baboons mechanically ventilated with constant volume and frequency. PAF-acether caused an immediate bronchoconstriction as assessed by a marked increase in peak inspiratory pressure with no change in static pulmonary compliance and chest X-rays. There was a concomitant fall in arterial PO2 and a significant increase in ventilated unperfused lung zones. A decrease of circulating platelets and leucocytes was also observed. Local anesthesia with lidocaine and atropine did not prevent PAF-acether-induced bronchoconstriction although both markedly reduced the bronchial response to histamine. Albuterol significantly reduced the bronchial response to PAF-acether. Pretreatment with aspirin (80 mg X kg -1 iv) did not prevent the bronchoconstriction caused by PAF-acether, and intravenous or intratracheal arachidonic acid caused no bronchial response. Thus the role of cyclooxygenase metabolites of arachidonic acid in PAF-acether-induced bronchoconstriction is unlikely. In conclusion, an acute bronchoconstriction probably not triggered by stimulation of irritant receptors of the airways and associated with aggregation of platelet takes place subsequent to intratracheal administration of PAF-acether. These data suggest that PAF-acether might play a role in the pathogenesis of human asthma.

Prostaglandins (PG) and leukotrienes (LT)—arachidonic acid-dependent metabolites—and paf-acether (platelet-activating factor)—an ether phospholipid—are potent mediators of allergic and inflammatory reactions. Their structures, chemical synthesis and biosynthetic pathways have been recently described. These mediators are produced by various cells with proinflammatory activities including the macrophages (a) upon interaction with a specific secretagogue stimulus (phagocytosis of zymosan particles, immune-complexes); (b) in IgE-dependent hypersensitivity reactions; (c) upon interaction with one of these mediators. Formation of these mediators by macrophages depends upon their local environment. Qualitative and/or quantitative variations in their synthesis are observed depending on the tissue they are derived from (alveole or peritoneum) and on the type of inflammation (immunologic specific or not). Their potent biological activities (increase of vascular permeability, smooth muscle contraction, cardiac and vascular effects and/or chemotactism) suggest a role for these mediators in various pathologies.

The ability of Paf-acether to induce increased plasma protein extravasation (IPPE) was assessed in the skin of rats by measuring extravasation of 125I-bovine serum albumin. Paf-acether elicited dose-dependent IPPE with a threshold of 0.04 pmol and a maximum at 4.5 nmol, with an effective concentration (EC50) of 23 pmol. The EC50 of the 1-0-ester Paf-acether analog was 1.5 nmol whereas 2-lyso Paf-acether was ineffective in inducing IPPE. Serotonin and histamine were respectively 100 and 1,00-fold less potent than Paf-acether. High doses of Paf-acether also resulted in a dose related accumulation of III-indium oxine labeled platelets. Ultrastructural and radioisotopic studies showed that Paf-acether-induced IPPE was not dependent upon platelet accumulation. In addition, platelet release products were ruled out in this phenomenon, since a "cocktail" of indomethacin, methysergide and pyrilamine-maleate failed to significantly reduce Paf-acether-induced IPPE. Neutrophils were not involved in Paf-acether -induced IPPE since nitrogen mustard-treated animals presented with IPPE not significantly different from controls. Finally, the specificity of Paf-acether on IPPE formation was demonstrated by its complete prevention by prior intravenous injection of Paf-acether (6 ug/kg), but not of Paf-acether analogs, histamine or serotonin.

Platelet-activating factor (PAF-acether; 1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine) is released from murine peritoneal adherent cells by inflammatory and non-inflammatory stimuli. We have found, in extracts from these cells, an enzyme activity that synthesizes. PAF-acether from synthetic lyso-PAF-acether by transferring the acetyl moiety of acetyl-coenzyme A onto the lyso-PAF-acether molecule. The enzyme is stabilized by 1 mM dithiothreitol, is calcium-dependent, has an apparent Km of 172 microM for acetyl-CoA and is active in a 6-8 pH range. When the acetyl-CoA substrate is replaced by propionyl-CoA, an ether lipid is produced which turns out to be as potent an aggregating agent as PAF-acether. In all cases, the products of the reaction were characterized by their behaviour in platelet-aggregation tests and their high-pressure liquid chromatography (HPLC) elution profiles. The precise definition of this acetyl-transferase is of primary importance for the development of new pharmacological agents capable of moduling a potent platelet aggregating factor.

PAF-acether is a 1-O-alkyl-2 acetyl-sn-glycerol-3-phosphorylcholine which triggers platelet aggregation independently from ADP release and thromboxane A2 formation. PAF-acether-aggregated rabbit platelets did not respond to a second challenge with the same agonist, but still aggregated to arachidonic acid. This desensitization to PAF-acether was a dose-dependent phenomenon. Specific cross-desensitization to PAF-acether was achieved using PAF-acether from four cellular origins, synthetic PAF-acether and its 2-propionyl analogue. Conversely, 2-lyso-PAF-acether and PAF-acether enantiomer were unable to induce specific desensitization. These results suggest that PAF-acether, whatever its origin, exhibits identical active molecular site, and that the presence and the stereospecific position of the 2-acetyl group are critical for the interaction of PAF-acether with platelets.

We have studied the molecular structure of platelet-activating factor" (P.A.F.), a mediator of inflammation obtained from blood leukocytes, macrophages, and platelets themselves. We have semi-synthetized a substance that possesses all the known physicochemical and biological characteristics of P.A.F. from hog leukocytes. This was performed by successive methylation, hydrogenation, and acetylation of lysophosphatidylethanolamine plasmalogen. We therefore propose the following structure for P.A.F.: 1-0-alkyl-2-acetyl-glyceryl-3-phosphorylcholine. This molecular structure is not yet described among the numerous substances capable of inducing platelet aggregation and release.

Isolated glomeruli and tubular and medullary cells obtained from perfused kidneys from Wistar rats were stimulated with ionophore A 23187 (0.5 to 6 microM) or kept overnight at pH 9.5. The amount of platelet-activating factor (Paf-acether) formed was measured in the ethanolic cell extracts using aggregation of washed rabbit platelets. 2-Lyso Paf-acether present in cells was transformed into Paf-acether by chemical acetylation and measured in the same manner as Paf-acether. Microsomes from glomeruli and medullary and tubular cells were prepared, and the acetyltransferase activity was measured. Paf-acether was formed in a dose-dependent fashion by glomeruli and medullary cells, and maximal formation with 3 microM ionophore A 23187 was 1.9 +/- 0.2 and 1.1 +/- 0.2 pmoles/mg of protein, respectively. Paf-acether was not recovered from tubular cells. Three cell types produced large amounts of 2-lyso Paf-acether when incubated at alkaline pH. Only glomeruli generated appreciable quantity of 2-lyso Paf-acether upon ionophore A 23187 stimulation. The acetyltransferase specific activities in ionophore A 23187-stimulated glomeruli and medullary and tubular cells were 3.8 +/- 0.8, 0.3 +/- 0.1, and 0.2 +/- 0.1 nmoles of Paf-acether/10 min/mg of protein, respectively. This study demonstrates the formation of Paf-acether by two distinct populations of kidney cells, pointing out the glomerular cells, besides the already known medullary cells, as capable of forming Paf-acether.