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c mice to Dactylis glomerata pollen by low doses of a purified pollen component
225
INHIBITION OF THE IgE RESPONSE IN BALB/C MICE TO DACTYLIS GLoMEIATA POLLEN BY LOW DOSES OF A PURIFIED POLLEN COMPONENT. Gabriel Peltre, Salah Mecheri and Bernard David, INST. PASTEUR FRANCE. --PARIS,
227
Platelet-Dependant Contraction of Human Bronchus by Platelet-Activating Factor. R.R. Schellenberg, B. Walker, U.B.C. Pul. Res. Lab., Vancouver, B.C. and F. Snyder, Oakridge Univ., Oakridge, Te. This study examined the effects of chemically synthesized (l-hexadecyl-2-acetyl-sb-glycero-3-phosphocholin e) platelet-activating factor (PAF) upon isolated human platelets and human bronchial smooth muscle in vitro. Platelets were isolated from platelet-rich plasma (PRP)by gel filtration over a Sepharose 2B column using Ca++ free, 0.25% human serum albumin Krebs-Henseleit solution. Aggregation studies were done in a Biodata PAP 3 aggregometer at smooth muscle spirals were 37oc. Bronchial obtained from surgical specimens and placed in siliconized organ baths with constant stirring at 37OC, bubbled with 95%02/5% Cop. Gel-filtered platelets (GFP) were more responsive to PAF than PRP. 100% aggregation occurred at 7x10m8M PAF for GFP vs 104M for PRP. Threshold aggregation occurred at 7xlO-llM PAF for GFP vs 10m8M for PRP. Fibrinogen 1.68 mg/ml was added to GFP for all studies. PAF alone caused no contraction of human bronchi. Following addition of GFP l.8x108/ml to the organ bath, PAF 10m6M produced contractions of 26-60s of the maximal carbachol response. The characteristics of contraction differed from that seen with serotonin or leukotrienes. These results suggest PAF-induced human bronchial smooth muscle contraction is due to a yet undefined platelet mediator.
226
MICROVASCULAR RESPONSE TO PLATELET ACTIVATING FACTOR (PAF-ACETHER). Jakob Bjiirk and Got-an Smedegdrd, Uppsala, Sweden Platelet activating factor (PAF-acether) is one of several recently identified phospholipid mediators. PAF-acether exhibit a wide spectrum of biological activities, which renders this ohosoholioid a oossible role in allerqic and inflammatory reactions. We have studied-the effects of PAF-acether on the microcirculation in the hamster cheek pouch using intravital microscopy. PAF-acether added to the superfusion buffer (5 nM final concentration), resulted in a transit arteriolar contriction and increased plasma exudation, as measured by fluoresceinlabelled dextran extravasation. Leukocytes were not affected. Higher doses of PAF-acether (20 nM) induced a more pronounced vasoconstriction and plasma exudation. in addition, large quantities of leukocytes were seen adhering to the vessel walls in postcapillary and larger venules subsequently followed by emigration into the extravascular space. Depletion of neutrophils with antineutrophil serum attenuated the vascular leakage induced with 20 nM but not with 5 nM of PAF-acether. Platelets appeared to be unaffected in all the experiments. These in viva results show that PAF-acether induces vascular smooth muscle constriction and plasma exudation. Higher doses of PAF-acether seem chemotactic to neutrophils, and the more pronounced vascular leakage induced could be due to neutrophil involvement.
By our nitrocellulose immunoprint technic, we have studied the heterogeneity of the allergens(recognized by human seric IgE antibodies) present in a water soluble crude extract of ~actylis gZomerata pollen. We observed a pollen fraction, with an isoelectric point of 4.6, which was not frequently detected as an allergen. This purified protein, named DIP (Dactylis Inhibiting Protein), when injected in Bai iiirce, even at doses up to 100 ug, was unable to elicit any antibody to the dactylis pollen extract as tested by ELISA assay. We induced an IgE response to DaetyZis gZ&rata pollen by injected twice 10 uq of this pollen crude extrciract with aluminum phosphate ahjuvant. This culating IgE antibody level measured by PCA in rats became totally negative one week after injection of 0.1 ug of DIP per mouse. The IgG, IgA and IgM antibodies to Dactylis pollen extract remained unchanged as measured by ELISA assay. We obtained a similar inhibition when the IgE antibodies were raised by sensitizing Balb/c mice with a purified allergen molecule instead of the crude extract. Finaly, a pretreatment by three injections of 0.1 ug DIP.at weeklv intervals before the sensitizinq challenqes of-the mice by the DuctyZis gZon&ta crude" extract prevented the production of any positive PCA reaction by the sera of these mice.
226 COMPARISON
OF THE PHARMACOLOGIC PROPERTIES OF LEUKOTRIENE E (LTE) AND PLATELET ACTIVATING FACTOR (PAF) fN VIVO. M. O'Donnell, Ph.D., and A.F. Welton, Ph.D. Nutley, A. Medford, B.S., New Jersev The present study was undertaken to compare the bronchoconstrictive activities in guinea pigs of two possible mediators of anaphylaxis, leukotriene E4 (LTE) and platelet activating factor (PAF), l-o-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine. Exogenous administration of both LTE and PAF produced dose dependent bronchoconstric's 7 ug/kg i.v. and 1.2 ug/kg i.v., when guinea pigs were ~~~~e%&!ly). H owever, exposed to aerosols of LTE and PAF (30 pg/ml for 5 min), only LTE produced bronchoconstriction. Various pharmacologic agents were evaluated for their ability to protect against LTE (25 pg/kg i.v.) and PAF (10 ug/kg i.v.) induced-equibronchospasm in guinea pigs. Indanethacin (IC mg/kg i-v.) and FPL 55712 (IC 10 mg#?g=iO;4) inhibited LTE but not PAF*ind%?ei bronchocon: strictions. The reported' lipoxygenase inhibitors, diethylcarbamazine, ETYA, NDGA, and 2-amino-4chloro-phenol did not inhibit PAF-induced bronchoconstrictions. Taken together, these results suggest that PAF-induced bronchoconstriction is probably not mediated via cyclooxygenase or lipoxysenase products. The bronchodilator, theophylline, was 10 times more potent against LTE than PAF bronchoconstrictions (IC 's respe%ively). 2 mg/kg i.v. and 21 mg/kg i.v., Conversely, verapamil (IC = 0.015 mg/kg i.v.) 20 PAF rather oreferentiallv antaoonize than LTE-induced b;onchoEonstrictions. Thus, it aooears that LTE and PAF induce bronchoconstrictibn in guinea pigs by different mechanisms.
145
INHIBITION OF THE IgE RESPONSE IN BALB/C MICE TO DACTYLIS GLoMEIATA POLLEN BY LOW DOSES OF A PURIFIED POLLEN COMPONENT. Gabriel Peltre, Salah Mecheri and Bernard David, INST. PASTEUR FRANCE. --PARIS,
227
Platelet-Dependant Contraction of Human Bronchus by Platelet-Activating Factor. R.R. Schellenberg, B. Walker, U.B.C. Pul. Res. Lab., Vancouver, B.C. and F. Snyder, Oakridge Univ., Oakridge, Te. This study examined the effects of chemically synthesized (l-hexadecyl-2-acetyl-sb-glycero-3-phosphocholin e) platelet-activating factor (PAF) upon isolated human platelets and human bronchial smooth muscle in vitro. Platelets were isolated from platelet-rich plasma (PRP)by gel filtration over a Sepharose 2B column using Ca++ free, 0.25% human serum albumin Krebs-Henseleit solution. Aggregation studies were done in a Biodata PAP 3 aggregometer at smooth muscle spirals were 37oc. Bronchial obtained from surgical specimens and placed in siliconized organ baths with constant stirring at 37OC, bubbled with 95%02/5% Cop. Gel-filtered platelets (GFP) were more responsive to PAF than PRP. 100% aggregation occurred at 7x10m8M PAF for GFP vs 104M for PRP. Threshold aggregation occurred at 7xlO-llM PAF for GFP vs 10m8M for PRP. Fibrinogen 1.68 mg/ml was added to GFP for all studies. PAF alone caused no contraction of human bronchi. Following addition of GFP l.8x108/ml to the organ bath, PAF 10m6M produced contractions of 26-60s of the maximal carbachol response. The characteristics of contraction differed from that seen with serotonin or leukotrienes. These results suggest PAF-induced human bronchial smooth muscle contraction is due to a yet undefined platelet mediator.
226
MICROVASCULAR RESPONSE TO PLATELET ACTIVATING FACTOR (PAF-ACETHER). Jakob Bjiirk and Got-an Smedegdrd, Uppsala, Sweden Platelet activating factor (PAF-acether) is one of several recently identified phospholipid mediators. PAF-acether exhibit a wide spectrum of biological activities, which renders this ohosoholioid a oossible role in allerqic and inflammatory reactions. We have studied-the effects of PAF-acether on the microcirculation in the hamster cheek pouch using intravital microscopy. PAF-acether added to the superfusion buffer (5 nM final concentration), resulted in a transit arteriolar contriction and increased plasma exudation, as measured by fluoresceinlabelled dextran extravasation. Leukocytes were not affected. Higher doses of PAF-acether (20 nM) induced a more pronounced vasoconstriction and plasma exudation. in addition, large quantities of leukocytes were seen adhering to the vessel walls in postcapillary and larger venules subsequently followed by emigration into the extravascular space. Depletion of neutrophils with antineutrophil serum attenuated the vascular leakage induced with 20 nM but not with 5 nM of PAF-acether. Platelets appeared to be unaffected in all the experiments. These in viva results show that PAF-acether induces vascular smooth muscle constriction and plasma exudation. Higher doses of PAF-acether seem chemotactic to neutrophils, and the more pronounced vascular leakage induced could be due to neutrophil involvement.
By our nitrocellulose immunoprint technic, we have studied the heterogeneity of the allergens(recognized by human seric IgE antibodies) present in a water soluble crude extract of ~actylis gZomerata pollen. We observed a pollen fraction, with an isoelectric point of 4.6, which was not frequently detected as an allergen. This purified protein, named DIP (Dactylis Inhibiting Protein), when injected in Bai iiirce, even at doses up to 100 ug, was unable to elicit any antibody to the dactylis pollen extract as tested by ELISA assay. We induced an IgE response to DaetyZis gZ&rata pollen by injected twice 10 uq of this pollen crude extrciract with aluminum phosphate ahjuvant. This culating IgE antibody level measured by PCA in rats became totally negative one week after injection of 0.1 ug of DIP per mouse. The IgG, IgA and IgM antibodies to Dactylis pollen extract remained unchanged as measured by ELISA assay. We obtained a similar inhibition when the IgE antibodies were raised by sensitizing Balb/c mice with a purified allergen molecule instead of the crude extract. Finaly, a pretreatment by three injections of 0.1 ug DIP.at weeklv intervals before the sensitizinq challenqes of-the mice by the DuctyZis gZon&ta crude" extract prevented the production of any positive PCA reaction by the sera of these mice.
226 COMPARISON
OF THE PHARMACOLOGIC PROPERTIES OF LEUKOTRIENE E (LTE) AND PLATELET ACTIVATING FACTOR (PAF) fN VIVO. M. O'Donnell, Ph.D., and A.F. Welton, Ph.D. Nutley, A. Medford, B.S., New Jersev The present study was undertaken to compare the bronchoconstrictive activities in guinea pigs of two possible mediators of anaphylaxis, leukotriene E4 (LTE) and platelet activating factor (PAF), l-o-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine. Exogenous administration of both LTE and PAF produced dose dependent bronchoconstric's 7 ug/kg i.v. and 1.2 ug/kg i.v., when guinea pigs were ~~~~e%&!ly). H owever, exposed to aerosols of LTE and PAF (30 pg/ml for 5 min), only LTE produced bronchoconstriction. Various pharmacologic agents were evaluated for their ability to protect against LTE (25 pg/kg i.v.) and PAF (10 ug/kg i.v.) induced-equibronchospasm in guinea pigs. Indanethacin (IC mg/kg i-v.) and FPL 55712 (IC 10 mg#?g=iO;4) inhibited LTE but not PAF*ind%?ei bronchocon: strictions. The reported' lipoxygenase inhibitors, diethylcarbamazine, ETYA, NDGA, and 2-amino-4chloro-phenol did not inhibit PAF-induced bronchoconstrictions. Taken together, these results suggest that PAF-induced bronchoconstriction is probably not mediated via cyclooxygenase or lipoxysenase products. The bronchodilator, theophylline, was 10 times more potent against LTE than PAF bronchoconstrictions (IC 's respe%ively). 2 mg/kg i.v. and 21 mg/kg i.v., Conversely, verapamil (IC = 0.015 mg/kg i.v.) 20 PAF rather oreferentiallv antaoonize than LTE-induced b;onchoEonstrictions. Thus, it aooears that LTE and PAF induce bronchoconstrictibn in guinea pigs by different mechanisms.
145