Immune complex induced pancreatitis: effect of BN 52021, a selective antagonist of platelet-activating factor

A model of acute pancreatitis was developed by induction of an immune complex mediated hypersensitivity reaction in rats. This acute inflammatory reaction was characterized by intense interstitial edema, neutrophil infiltration and margination, and congestion of small vessels whereas serum amylase levels remained unchanged. Microscopic examination of the pancreatic tissue revealed the presence of immune complex deposition around blood vessels and ducts. Vascular permeability, as measured by Evan's blue extravasation increased by 6 fold. In addition, circulating platelets dropped to 50% of normal levels. Injection of platelet-activating factor (PAF) in the peritoneal cavity of rats also produced an increase in vascular permeability in the pancreas. A selective PAF-antagonist, BN 52021 reduced by approximately 50% the increase in vascular permeability produced by immune complex in the pancreas as well as that elicited by intraperitoneal injection of PAF. These results suggest that PAF plays a role in the pathological manifestations of immune complex-mediated pancreatitis.     

In vitro contractile effect of platelet-activating factor on guinea-pig myometrium

Platelet-activating factor (PAF) evoked myometrial contractions in two different patterns, depending on whether spontaneous activity was present. In spontaneously active myometrial strips (58%), both PAF and oxytocin enhanced the amplitude of myometrial contractions. In quiescent myometrial strips, PAF induced contractions characterized by a prompt development of tension, a plateau, and a final, rapid relaxation. In 54% of these strips, PAF-induced contraction was followed by rhythmic activity. PAF contractile response was dependent upon the concentration (0.1-100 nM); the minimal effective concentration of PAF was 0.1 nM and the EC50 was 1 nM. The response to oxytocin (0.01-10 mU/ml), assumed as reference stimulus, was characterized by a prompt development of tension, which was followed by a sustained, slow contraction and relaxation. PAF response was almost completely dependent on cyclooxygenase and partially on lipoxygenase pathways, as inferred from studies with indomethacin and FPL 55712, respectively. A receptor mediated mechanism of PAF action was suggested by specific desensitization of the myometrium to a second challenge with an equimolar concentration of PAF (but not with oxytocin) and the blocking effect of CV 3988, a specific PAF receptor antagonist.     

In vitro contractile effect of platelet-activating factor on guinea-pig myometrium

Platelet-activating factor (PAF) evoked myometrial contractions in two different patterns, depending on whether spontaneous activity was present. In spontaneously active myometrial strips (58%), both PAF and oxytocin enhanced the amplitude of myometrial contractions. In quiescent myometrial strips, PAF induced contractions characterized by a prompt development of tension, a plateau, and a final, rapid relaxation. In 54% of these strips, PAF-induced contraction was followed by rhythmic activity. PAF contractile response was dependent upon the concentration (0.1–100 nM); the minimal effective concentration of PAF was 0.1 nM and the EC₅₀ was 1 nM. The response to oxytocin (0.01–10 mU/ml), assumed as reference stimulus, was characterized by a prompt development of tension, which was followed by a sustained, slow contraction and relaxation. PAF response was almost completely dependent on cyclooxygenase and partially on lipoxygenase pathways, as inferred from studies with indomethacin and FPL 55712, respectively. A receptor mediated mechanism of PAF action was suggested by specific desentization of the myometrium to a second challenge with an equimolar concentration of PAF (but not with oxytocin) and the blocking effect of CV 3988, a specific PAF receptor antagonist.     

Effects of platelet-activating factor and platelet-activating factor: acetylhydrolase on in vitro post-thaw boar sperm parameters

Cryopreservation of boar sperm compromises fertility after thawing by reducing sperm longevity and inducing acrosome reaction-like changes. In an attempt to improve the post-thaw motility and acrosome integrity of boar sperm, semen was frozen using a modified Westendorf method in which the medium was supplemented with either platelet-activating factor (PAF) or a recombinant platelet-activating factor:acetylhydrolase (PAF:AH; Pafase) before or after freezing. Platelet-activating factor is a phospholipid that is present in boar semen and PAF:AH is the naturally occurring enzyme that converts PAF to biologically inactive Lyso-PAF. Addition of PAF to the cryopreservation medium improved post-thaw motility immediately after thawing and after 3h incubation at 37 degrees C (60.0+/-0.0% and 25.0+/-2.9%; mean+/-S.E.M.) compared to the control sperm (41.7+/-1.7% and 10.0+/-2.9%; P<0.05). Acrosome integrity was higher immediately after thawing and after 3 and 6h incubation at 37 degrees C when sperm were frozen in the presence of Pafase (55.7+/-3.2%, 45.7+/-3.7% and 23.0+/-3.1%), compared to the control sperm (42.7+/-1.5%, 25.7+/-5.7% and 12.3+/-2.7%) and sperm frozen in the presence of PAF (33.0+/-3.7%, 26.3+/-2.2% and 11.7+/-0.3%; P<0.05). Addition of PAF to sperm after thawing improved motility immediately post-thaw (41.6+/-2.6%), compared with addition of Pafase (23.3+/-2.2%) or the control sperm with no supplementation of the medium (26.7+/-2.2%; P<0.05). However, this beneficial effect was lost by 3h post-thaw. Supplementation of boar semen cryopreservation medium with PAF and Pafase appeared to have beneficial effects on the in vitro quality of the sperm post-thaw.     

Role of platelet-activating factor (PAF) in the ovoimplantation in the rat: effect of the specific PAF-acether antagonist, BN 52021

The role of PAF (platelet-activating factor) in early pregnancy has been recently postulated. Indeed, platelet count is markedly reduced immediately prior to and returns to normal following ovoimplantation. Using a pharmacological approach, we therefore further investigated the possible involvement of PAF in ovoimplantation. BN 52021 (10 nmol), a PAF antagonist, was administered after fecondation in the lumen of the left uterine horn. A group of animals was injected with the vehicle alone. The animals were sacrificed 1 week latter and the number of implanted embryos in the treated horn and in the untreated contralateral one was assessed. When injected on day 4 of pregnancy, the PAF antagonist, BN 52021, inhibited in a dose-dependent fashion ovoimplantation suggesting a role for this lipid mediator in this process. Total inhibition of ovoimplantation was observed in the horns treated with indomethacin, NDGA or BW 755 C. In addition, a significant inhibition of ovoimplantation was also produced by the lipoxygenase inhibitor, EP 10045 (10nmol). Since both PAF and leukotrienes trigger the generation of prostaglandins in various tissues, our results indicate that these two lipid mediators may be implicated in early stages of the inflammatory reaction accompanying ovoimplantation and may contribute to the local generation of cyclooxygenase metabolites.     

Partial characterization of the embryo-derived platelet-activating factor in mice

The platelet-activating factor (PAF) produced by mouse embryos showed similar kinetics of action and dose-response curve, in a bioassay, as did 1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphocholine (PAF-acether). The activity of the embryo-derived PAF was not affected by inhibitors of the ADP (pyruvate kinase with phosphoenol pyruvate) or cyclo-oxygenase (indomethacin) pathways of platelet activation. Chlorpromazine, an inhibitor of the PAF-acether pathway of platelet activation, caused a significant inhibition of the effects of embryo-derived PAF. Phospholipases A2, C and D significantly inhibited the activity while lipase had no effect, suggesting a phospholipid structure. All the embryo-derived PAF was found in the chloroform fraction after chloroform:methanol (2:1 v/v) extraction, as was PAF-acether. Both factors migrated at a similar rate (Rf 0.10-0.12) on silica thin-layer chromatography (chloroform:methanol:water; 65:35:4 by vol.). The embryo-derived PAF therefore displays chemical, biochemical and physiological properties similar to those of PAF-acether.     

Evidence for platelet-activating factor secretion during immune activation in dogs

To elucidate the potential physiological significance of platelet-activating factor (PAF) in acute bronchoconstriction, we studied the effect of Ascaris suum antigen on the tachyphylactic response to PAF in 15 natively allergic mongrel dogs in vivo. Active bronchial tension was measured isometrically, and mediator secretion was measured as the arteriovenous difference (AVd) in plasma concentration across the lungs. Administration of PAF into the bronchial artery caused dose-related contraction in five control dogs (maximal active tension = 11.8 +/- 1.68 g/cm) that paralleled the increase in the AVd for serotonin (4,188 +/- 175 pg/ml) but not histamine (maximal AVd less than 6.0 ng/ml). The response to PAF was highly tachyphylactic. In contrast to PAF, 1:10 concentration of intra-arterial antigen caused substantial release of histamine (AVd = 308 +/- 57.1 ng/ml; P less than 0.001 vs. PAF). Diminished responsiveness (2-log shift in threshold and maximal contraction; P less than 0.001) to PAF was demonstrated in five dogs after 1:10 antigen, compatible with endogenous release of PAF during prior immune challenge in the same animals. Administration of Ascaris antigen caused a leftward shift in the dose-response curve to serotonin and only mild tachyphylaxis to the maximal response to histamine. Our data are compatible with physiological participation of PAF in eliciting bronchial smooth muscle contraction during the acute phase of immune activation caused by A. suum antigen.     

Experimental Trypanosoma cruzi infection in platelet-activating factor receptor-deficient mice

The generation of an inflammatory response driven by Trypanosoma cruzi or its subproducts appears to be essential for tissue injury and disease pathogenesis. However, this inflammatory response is also relevant in the control of T. cruzi replication. The lipid mediator platelet-activating factor (PAF) has been implicated in a number of pathological conditions characterized by tissue inflammation. In the present study, we aimed at evaluating the role of PAF during T. cruzi infection by using mice that were genetically deficient in the PAF receptor. We observed that infected hearts of PAFR(-/-) mice had an increased number of parasite nests, associated with a more intense inflammatory infiltrate. This was associated with greater parasitemia and lethality. When wild-type and PAFR(-/-) mice were compared, there were no marked changes in the kinetics of the expression of MCP-1, RANTES, IFN-gamma and TNF-alpha in heart tissue of infected animals. Moreover, serum concentrations of TNF-alpha, nitrate and parasite-specific IgM were similar in both groups of mice. In vitro, macrophages from PAFR(-/-) animals did not phagocytose trypomastigote forms when activated with PAF, leukotriene B(4) or MCP-1 and produced less nitric oxide when infected and activated with IFN-gamma. These results are consistent with the hypothesis that endogenous synthesis of PAF and activation of PAF receptors control T. cruzi replication in mice in great part via facilitation of the uptake of the parasite and consequent activation of macrophages.     

The dark reactions of rye phytochrome in vivo and in vitro

The dark reactions of Secale cereale L. cv. Balbo phytochrome have been investigated in coleoptile tips and in extensively purified extracts of large molecular weight phytochrome. Destruction, but not reversion, was detected in vivo. The effects of various inhibitors of an in vitro phytochrome-degrading protease did not support a view of proteolytic attack as the basis of in vivo destruction. In vitro, rye phytochrome (about 240,000 molecular weight) reverted extremely rapidly, even at 5 C. The reversion curves were resolved into two first order components. The previously studied 60,000 molecular weight species, obtained by controlled proteolysis of large rye phytochrome, showed a similar two-component pattern, but a much slower over-all reversion rate. This reduction in rate was caused mainly by the reversion of a greater percentage of the small phytochrome as the slow component. Sodium dithionite markedly accelerated the reversion rate of both large and small forms, but oxidants, at concentrations low enough to avoid chromophore destruction, had no effect. Both large and small crude Avena sativa L. phytochrome showed two-component reversion kinetics.     

Effect of inhaled platelet-activating factor on circulating neutrophils and platelets in vivo and ex vivo in man

We studied the effect of five successive inhalations of platelet-activating factor (PAF) on airway calibre, circulating neutrophil and platelet counts and the activation of these cells ex vivo in normal subjects. PAF (24 ug) caused a mean maximal fall of the expiratory flow rate at 70% vital capacity from a partial manoeuvre (Vp30) of 46.4 +/- 6.2% (p less than 0.001); there was a tachyphylactic response to further doses of PAF. Circulating neutrophil counts fell by 54.3 +/- 10.6% (p less than 0.005) with immediate recovery and with a rebound neutrophilia by the fifth inhalation. Platelet counts showed no significant changes. Aggregation of platelets in platelet-rich plasma to PAF and ADP in vitro at 15 min after the first, second and fifth PAF inhalations was not significantly altered. Chemiluminescence responses of neutrophils to PAF (0.01, 0.1, 1 and 10 microM) in vitro were reduced at 15 min after the fifth PAF inhalation, but this was only significant at 1 microM PAF. Methacholine inhalation did not cause any changes in responsiveness of neutrophils to PAF ex vivo. We conclude that ex vivo platelet desensitisation cannot be used as an index of endogenous PAF release, but reduced responsiveness of neutrophils ex vivo is not a sensitive indicator.     

Effect of inhaled platelet-activating factor on circulating neutrophils and platelets in vivo and ex vivo in man

We studied the effect of five successive inhalations of platelet-activating factor (PAF) on airway calibre, circulating neutrophil and platelet counts and the activation of these cells in normal subjects. PAF (24 ug) caused a mean maximal fall of the expiratory flow rate at 70% vital capacity from a partial manoeuvre (Vp30) of 46.4 ± 6.2% (p < 0.001); there was a tachyphylactic response to further doses of PAF. Circulating neutrophil counts fell by 54.3 ± 10.6% (p < 0.005) with immediate recovery and with a rebound neutrophilia by the fifth inhalation. Platelet counts showed no significant changes. Aggregation of platelets in platelet-rich plasma to PAF and ADP at 15 min after the first, second and fifth PAF inhalations was not significantly altered. Chemiluminescence responses of neutrophils to PAF (0.01, 0.1, 1 and 10 μM) were reduced at 15 min after the fifth PAF inhalation, but this was only significant at 1 μM PAF. Methacholine inhalation did not cause any changes in responsiveness of neutrophils to PAF . We conclude that platelet desensitisation cannot be used as an index of endogenous PAF release, but reduced responsiveness of neutrophils is not a sensitive indicator.     

Release of eicosanoids in rat peritoneal cavity stimulated with platelet-activating factor (PAF). Effect of the PAF-antagonist BN-52021

Platelet activating factor (PAF; 10 micrograms) was injected in the peritoneal cavity of rats in the absence or presence of the PAF antagonist BN-52021 (5 mg/kg). Thirty min later, the peritoneal cavity was washed with 3 ml of saline, the fluid was collected and the concentrations of selected eicosanoids were measured using novel enzyme immunoassays. PAF increased by 2.9, 2.8 and 1.7 fold the levels of thromboxane B2, prostaglandin E2 and leukotriene B4 respectively in the peritoneal fluid. The stimulatory effects of PAF was reduced by 42, 51, and 86% for thromboxane B2, prostaglandin E2 and leukotriene B4 respectively by the specific PAF antagonist. These results confirm the presence of specific PAF receptors in tissues and/or cells of rat peritoneal cavity and underline the complex interactions between PAF and eicosanoids.     

Effects of the platelet-activating factor antagonist BN 52021 on the hemodynamics of rats with experimental cirrhosis of the liver

Previous studies from this laboratory have shown that rats with experimental cirrhosis of the liver induced by the combined administration of oral phenobarbital and inhaled carbon tetrachloride show an hyperdynamic status with enhanced cardiac output (CO), and decreased mean arterial pressure (MAP) and peripheral vascular resistance (PVR). Cirrhotic rats also showed an increased vascular permeability. All these phenomena are similar to some of the known effects of the systemic infusion of low doses of synthetic platelet-activating factor into the systemic circulation of normal rats. The measurement of the levels of platelet-activating factor in samples of blood demonstrated significantly higher levels in cirrhotic (2.65 +/- 0.39; n = 10) than in control rats (1.50 +/- 0.57 ng/ml; n = 10; p less than 0.05). The hemodynamic changes induced by the intravenous injection of the platelet-activating factor receptor antagonist BN 52021 (5 mg/kg body weight) have been measured in 10 control and 10 cirrhotic male Wistar rats, using a radioactive microsphere technique. BN 52021 induced no significant hemodynamic changes in control animals. However, in cirrhotic animals it induced a significant decrease in CO with increase in PVR. MAP increased slightly but not significantly. From these data it can be deduced that platelet-activating factor plays a role in the hemodynamic derangement shown by cirrhotic rats and that these derangement can be reversed by BN 52021, a highly selective antagonist of the platelet-activating factor receptor.     

结核分枝杆菌核酸疫苗的构建及其小鼠免疫效果观察

将结核杆菌ＥＳＡＴ－６和ＭＰＴ－６４的编码基因分别插入真核表达载体ＪＷ４３０３构建重组质粒,用重组擀凿对４周龄的ＢＡＬＢ／Ｃ和Ｆ１代小鼠进行肌肉和皮内地末次免疫后１０天采血交分离血清,进行ＥＬＩＳＡ检测。结果表明,小鼠能产生较强的体液免疫,特异性ＩｇＧ平均水平分别为参考血清的２．２０倍３．１９倍,且对两种品系小鼠进行的相同途径的免疫,其抗体水平差异不显著,而不同途径的重组质粒免疫,抗体水平略有差异     

The enhancement of the immune response by pain stimulation in mice. I. The enhancement effect on PFC production via sympathetic nervous system in vivo and in vitro

Effects of catecholamines and osmotical and physical stimuli on the induction of anti-sheep red blood cells (SRBC) plaque-forming cells (PFC) were investigated in (C57BL/6 X BALB/c)F1 mice in vivo and in vitro. The anti-SRBC PFC from mice immunized with 5 X 10(7) SRBC was markedly increased by daily s.c. injections of epinephrine. The enhancement of PFC by epinephrine was completely blocked by preadministration with propranolol and hexamethonium, but not with phentolamine. The PFC was increased by osmotic and physical stimuli given once a day for 4 days after immunization with SRBC. The enhancement of PFC by these stimuli was completely blocked by preadministration with propranolol and hexamethonium. The enhancement of PFC by physical stimuli was observed in nonimmunized mice when spleen cells from stimulated mice were cultured with SRBC in vitro. In normal mice, the enhancement of PFC was observed 2 hr after one physical stimulation. However, spleen cells from mice given two physical stimuli did not show the enhancement of PFC after treatment with anti-Thy-1.2 antibody and complement, nor after removal of nonadherent cells. Next, the serum obtained from mice 30 to 60 min after a physical stimulation enhanced PFC of normal mice spleen cells in vitro, but the enhancement was abolished by the addition of propranolol. The enhancement of anti-SRBC PFC by s.c. injection of epinephrine suggested that the autonomic nervous system, especially the sympathetic nervous system, was activated by a local stimulus effect of the injection. This enhancement of anti-SRBC PFC appear to be due to the activation of antigen non-specific helper T lymphocytes by the beta-actin of endogenous catecholamines from the adrenal gland.     

Inhibition of immune complex-induced enteropathy by three different platelet-activating factor receptor antagonists

We previously showed that intravenous injection of rat anti-BSA-BSA complexes (IC) prepared in 5x antigen excess rapidly induced a striate pattern of serosal (to mucosal) hemorrhage and vascular congestion throughout the small intestine of the Sprague-Dawley rat. In this study, we tested the effect of three different platelet-activating factor (PAF) receptor antagonists on the development of lesions. L-652, 731, a synthetic derivative of kadsurenone (at doses of 1.3–2.7 mg/kg), SRI 63–675, a substituted quinolinium compound (6.7–15 mg/kg), and WEB 2086, a thienotriazolodiazepine (5–25 mg/kg) were each capable of completely or partially inhibiting IC-induced enteropathy in the majority of animals tested. Pretreatment with WEB 2086 prevented IC-induced hemoconcentration but not neutropenia. The antagonists did not lower the level of blood complement nor interfere with the fall in complement induced by administration of IC. The ability of PAF receptor antagonists to completely or partially inhibit IC-induced small intestinal lesions suggests that endogenous PAF is a major mediator of IC-induced enteropathy.