Histologic characterization of late asthmatic response in guinea pigs]

We examined lung tissues in a guinea pig model actively sensitized by inhalation of aerosolized ovalbumin (OA) and found reproducible late bronchial responses (LBR) 7 h and 24 h after OA challenge. Light microscopic examination revealed bronchoconstriction, damage to the epithelium, eosinophil infiltration in both the epithelium and subepithelium, retention of mucous in the pulmonary bronchial lumens and a decrease in the mucous content of Periodic Acid-Schiff (PAS) stain positive cells in the bronchial epithelium in LBR. An increase in the number of PAS-stain positive cells of in the bronchial epithelium was also observed in LBR. Electron microscopic examination revealed eosinophil migration around mast cell at 7 hs after OA challenge. Two different mechanisms of degranulation of eosinophil specific granules were observed in LBR. In one mechanism, the granule core changes after its matrix has changed. In the other mechanism, the granules were exocytosed as a whole, accompanied by the cell membrane and densely clumped chromatin, suggesting burst of eosinophil.     

Involvement of platelet activating factor (PAF) in ovalbumin antigen-induced late asthmatic response and increase of airway hyperresponsiveness in a guinea pig experimental model of asthma

Platelet activating factor, a potent chemical mediator, has been implicated in the pathogenesis of asthma in terms of inflammatory cell recruitment and activation. We have recently demonstrated that repeated antigen (ovalbumin; OA) exposure by inhalation to guinea pigs results in a development of late asthmatic response (LAR) in more than 50% of the animals and significant increase in airway hyperresponsiveness (AH). We have studied the effect of WEB 2086, a specific PAF receptor-antagonist, on this model. Respiratoly resistance (Res) of guinea pigs was measured by a oscillation technique and AH was evaluated by the provocative concentration of aerosols of histamine causing 200% increase of Rrs over the baseline Rrs (PC200 Hist). Four out of 5 actively sensitized and diphenhydramine-pretreated animals developed LAR 3 to 9 hr after allergen (20 mg/ml OA, 10 min inhalation)-induced immediate bronchoconstriction (LAR). Treatment with WEB 2086 (3 mg/kg intravenously) 30 min before and 3 hr after the exposure suppressed LAR clearly without affecting the IAR. Significant increase in AH from 2.80 +/- 0.03 to 2.51 +/- 0.01 and 2.60 +/- 0.08 (p less than 0.05, n = 8) of PC200 Hist (mg/ml, log) was observed 24 hr and 5 day after the OA exposure, respectively. The WEB 2086 treatment also prevented the increase of AH after the OA exposure (PC200 Hist; 2.82 +/- 0.09 before the challenge 2.80 +/- 0.07 and 2.75 +/- 0.09 24 hr and 5 days after, respectively. n = 8). Administration of WEB 2086 did not affect baseline Rrs and PC200 Hist in normal guinea pigs without any antigen challenge. We conclude that WEB 2086 is capable of preventing the development of LAR and increase in AH, and thus PAF may play an important causal role in LAR and increased AH observed in asthma.     

Platelet activating factor (PAF) and lyso-PAF in inflamed human skin

The extent of PAF synthesis and its role in inflammatory skin disorders are not known. Skin exudates were collected from suction blisters or bbrasions on healthy and inflamed human skin and analysed by gc-ms. The mean PAF andlyso-PAF values for skin of healthy subjects were 1.86±0.92 (n=7) and 160±21 (n=8) nM, respectively, in suction blister exudates, and 0.38±0.08 (n=14) and 11.0±1.9 (n=13) pmol/abrasion, respectively. PAF levels were not altered in psoriasis, nickel contact dermatitis, delayed pressure urticaria, mastocytosis or immediate and late phase reactions to antigen. Significantly, lesslyso-PAF was found in uninvolved skin of psoriasis. Increasedlyso-PAF was found in nickel contact dermatitis compared with untreated skin of the same subjects or skin or normal healthy volunteers. We conclude that, if PAF is a significant mediator of inflammation in human skin, it may remain associated with the synthesising cell or act in its immediate proximity.

     

Blocking action of platelet activating factor (PAF) on atrial fiber calcium currents in frogs and guinea pigs

Bulletin of Experimental Biology and Medicine -     

过敏性哮喘豚鼠血小板激活因子的变化及川芎嗪的影响

本研究采用生物定量测定方法观察了豚鼠实验性哮喘时血浆ｌｙｓｏ－ＰＡＦ和肺泡灌洗（ＢＡＬＦ）中ＰＡＦ的变化。结果显示：致敏级豚鼠哮喘发作时血浆ｌｙｓｏ－ＰＡＦ水平显著高于对照组（Ｐ〈０．０１），ＢＡＬＦ中ＰＡＦ水平也明显高于对照组（Ｐ〈０．０５），同时伴有血浆及ＢＡＬＦ中ＴＸＢ２、ＴＸＢ２／６－酮－ＰＧＦ１α水平的升高（Ｐ〈０．０１）。致敏＋川芎嗪组豚鼠血浆ｌｙｓｏ－ＰＡＦ明显低于致敏组豚鼠（Ｐ〈０     

Platelet-activating factor in late asthmatic response

The role of platelet-activating factor (PAF) in the late asthmatic responses was studied. The concentrations of lyso-form of PAF in plasma were measured at 0 and 20 min, and 6 and 24 h after the antigen inhalation challenge among patients with bronchial asthma. PAF activities were measured by their aggregating ability of washed rabbit platelets after acetylation of lyso-PAF into the biological active form of PAF, when there were no detectable amounts of PAF in plasma. The concentrations of lyso-PAF were found to be significantly increased in patients with the late asthmatic response compared with patients with the single immediate response at 6 h after the antigen challenge. In contrast, lyso-PAF levels were not significantly different at 20 min after the antigen challenge between these two groups. PAF inactivator activity in plasma increased when there was a decrease in the lyso-PAF level. These results suggest that PAF may participate in the late asthmatic response and may provide a new insight into the pathogenesis and the treatment of bronchial asthma.     

Activation of human platelets by platelet activating factor (PAF) derived from sensitized rabbit basophils.

Rabbit basophil-derived platelet activating factor (PAF), a mediator of anaphylaxis, induces the aggregation and release of serotonin from rabbit platelets. In the present study, we report that PAF obtained by challenge of specifically sensitized rabbit basophils induced the noncytotoxic release of serotonin from human platelets; maximal extent of release ranged between 34-46%. This release was unaltered in the presence of indomethacin, indicating that such secretion was not a consequence of contaminating arachidonic acid; further, as previously demonstrated with platelets of rabbit origin, it was markedly independent of a requirement for an intact prostaglandin biosynthetic pathway. In contrast to its effect upon rabbit platelets, rabbit PAF did not induce aggregation of human platelets, suggesting that the aggregation and secretion reactions induced by this agent are separable and that this cross-species activation may be incomplete. Whether this is a result of the differential ability of rabbit PAF to bind to and activate rabbit as compared to human platelets or to the existence of a family of PAF molecules is not yet known. The capacity of PAF to participate in a secretory event involving human platelets lends support to the belief that PAF may play an important ubiquitous role in the cooperative, leucocyte-dependent, release of vasoactive amines which results in increased vascular permeability.     

Procoagulant property of platelet activating factor (PAF)

In this report we demonstrate that ET-2, in addition to its powerful vasoconstrictor properties and its role in hypertension, is capable of creating a hypercoagulable (prothrombotic) state. This study was facilitated by the utilization of a sensitive modified recalcification time (MRT) test that can measure coagulation at all points in the coagulation spectrum. The MRT was determined on aliquots (1 ml) of citrated blood which were added to saline (MRTS), to 10 μg of endotoxin (MRTE), and to 10 pg of ET-2 (MRT ET-2). The mean values of MRTS, MRTE, and MRT ET-2 were 4.3 ±0.8, 3.7 ± 1.0, and 3.9 ± 0.8 min, respectively. There was a statistically significant difference between MRTE vs. MRTS and MRT ET-2 vs. MRTS using the twotailedt-test (p<0.02 andp<0.005, respectively).

     

Multiple antigen challenge produces pulmonary eosinophilia but not pulmonary hyperresponsiveness in actively sensitized guinea pigs

Exposure of actively sensitized boosted guinea pigs to aerosolized antigen, 3 times on alternate days, produced pulmonary eosinophilia but not pulmonary hyperresponsiveness to methacholine Cl measured 3 days after the last antigen challenge. These data suggest that the presence of large numbers of eosinophils in the airways and tissues of the lungs is not sufficient to produce nonspecific pulmonary hyperresponsivenese. These data also suggest that actively sensitized and boosted guinea pigs respond differently to repeated antigen exposure than do asthmatics or wild caught allergic cynomolgus monkeys.     

Classical conditioning of anaphylaxis in sensitized guinea pigs]

An attempt of classical conditioning of anaphylaxis by odor was carried out, using actively sensitized guinea pigs with an inhalation of ovalbumin (OA). One month after sensitization, animals were divided into the conditioned group; group C, and the unconditioned group; group U, consisted of 6 animals, respectively. Dimethylsulfied (DMS: sulfur odor), was inhaled in group C as a conditioned stimulus with OA which is an unconditioned stimulus, while only OA was inhaled in group U. Four days after these procedures, saline was inhaled in group C and DMS was solely inhaled in group U in order to equalize the total inhaled dose of OA and DMS in both groups. These sessions were repeated once a week for seven weeks. After final sessions, all animals were inhaled DMS, saline and OA separately, and blood samples were drawn after each inhalation to measure plasma histamine levels. After an inhalation of DMS only, plasma histamine levels of group C and U were 47.5 +/- 9.7 and 25.7 +/- 1.2 ng/ml, respectively. In group C, plasma histamine levels were 32.9 +/- 4.7 at the inhalation of saline and 59.0 +/- 9.2 ng/ml at OA inhalation. Plasma histamine level after an inhalation of DMS only was significantly higher in group C than that in group U (p < 0.05). These results suggest that the conditional stimulus (DMS inhalation) may induce histamine release in the absence of any antigenic stimulus and support the evidence for mast cell-neuron interaction.     

Effects of an ATP-sensitive K+ channel activator, JTV-506, on antigen-induced early and late asthmatic responses in sensitized guinea pigs]

Effects of an ATP-sensitive K+ channel activator, JTV-506, on dual asthmatic responses and airway inflammation after antigen inhalation challenge were investigated in asthma model of guinea pigs. The animals were given an oral dose of 1 mg/kg of JTV-506 or vehicle (0.5% carboxymethyl cellulose sodium) 1 hour before and 3 hours after antigen inhalation challenge. Measurement of pulmonary resistance for 6 h was followed by bronchoalveolar lavage. After antigen challenge, all guinea pigs in the vehicle group displayed dual-phase airway obstruction and accumulation of eosinophils in the airways. After the treatment with JTV-506, the early asthmatic response was significantly inhibited, although the late asthmatic response or the recruitment of eosinophils into the airways were not inhibited. Therefore, we suggested that JTV-506 may inhibit airway smooth contraction induced by chemical mediators, but not function of CD4+ T lymphocytes.     

Effect of San-Ao-Tang on immediate and late airway response and leukocyte infiltration in asthmatic guinea pigs

San-Ao-Tang (SAT), a traditional Chinese medicines, has been used to treat patients with the bronchial asthma for several centuries. However, the therapeutic mechanisms of this Chinese medicine are still far from clear. To understand the mechanism of antiasthmatic property of SAT, a guinea pig model of allergic asthma was used to investigate the effects of SAT on Dermatophagoides pteronyssinus-induced. immediate and late asthmatic responses and airway inflammation. Our results showed that administration of SAT (10g/kg) extracts singificantly inhibited the antigen induced immediate asthmatic responses (IAR) in actively sensitized guinea pig. Examination of bronchoalveolar lavage fluid (BALF) revealed that SAT significantly inhibited the increase in neutrophil in the airway at 1, 2, 4, 6, 8 hr after antigen challenge. Histopathologic examination showed SAT suppressed the neutrophil infiltration into lung tissue. These results suggest that the antiasthmatic effect of SAT be mainly due to its bronchodilator effect and its ability to inhibit the neutrophil into the airway. The precise mechanism of action of SAT in asthma remains to be elucidated.     

Platelet activating factor (PAF) increases plasma protein extravasation and induces lowering of interstitial fluid pressure (Pif) in rat skin

Aim: To investigate the ability of the microdialysis technique to measure capillary selectivity of different sized plasma proteins induced by local administration of platelet activating factor (PAF). Methods: We used hollow plasmapheresis fibres with 3 cm membrane (cut off 3000 kDa) placed on the back of anaesthetized rats. Results: Platelet activating factor (50 μg mL^?1) administered locally via the fibre, increased extravasation of radiolabelled ¹²⁵I‐HSA from plasma to the microdialysis fibre by approximately 900% compared both to baseline and the control fibre within 70 min (n = 6, P < 0.05). The extravasation in the control fibre did not change over time. HPLC measurement of plasma proteins in the microdialysis perfusate also demonstrated decreased capillary selectivity for proteins in the diameter range of 73 Å, 56 Å and 39 Å after local administration of PAF (n = 6, P < 0.05). PAF also significantly lowered interstitial fluid (P_if) pressure after subcutaneous administration (50 μg mL^?1). Mean arterial pressure (MAP) after intravenous injection of PAF (0.4 μg kg^?1) fell instantly by about 50 mmHg, and stabilized at 50 mmHg after 15 min (n = 6). MAP was unaltered when PAF was given through the microdialysis fibre (n = 4). Both total tissue water (TTW) and extravasation of albumin, measured as the plasma‐to‐tissue clearance (E‐alb) showed a significant increase after PAF (n = 7, P < 0.05). Conclusions: The present study demonstrates that PAF induces plasma protein extravasation and decrease capillary selectivity of different sized plasma proteins. It also increases transcapillary fluid flux, and lowers P_if, indicating a role for PAF in the interstitium for generation of transcapillary transport of water and large molecules followed by formation of oedema.     

Experimental asthma in guinea pigs sensitized with mites (Dermatophagoides farinae)

Guinea pigs were intraperitoneally immunized with 1, 10, 100 or 1,000 micrograms protein of an extract from mites (Dermatophagoides farinae) incorporated with aluminium hydroxide gel 3 times every 28 days. Ten days after the last injection, a bronchial provocation test was performed by the use of the mite extract. The group immunized with 1 microgram protein produced very low levels of IgE-, IgG1- and IgG2 antibodies, and did not show asthmatic response upon the challenge. Although the group immunized with 10 micrograms protein showed higher rates of asthmatic onset than other groups, the sera of this group contained lower levels of IgE antibodies than the group immunized with 100 or 1,000 micrograms protein. However, the sera of the group immunized with 10 micrograms protein showed higher ratios of IgE/IgG1, IgE/IgG2 and lower ratios of IgG1/IgG2 antibodies than the sera of the group immunized with 100 or 1,000 micrograms protein.     

Platelet activating factor (PAF) increases plasma protein extravasation and induces lowering of interstitial fluid pressure (P) in rat skin

AIM: To investigate the ability of the microdialysis technique to measure capillary selectivity of different sized plasma proteins induced by local administration of platelet activating factor (PAF). METHODS: We used hollow plasmapheresis fibres with 3 cm membrane (cut off 3000 kDa) placed on the back of anaesthetized rats. RESULTS: Platelet activating factor (50 microg mL(-1)) administered locally via the fibre, increased extravasation of radiolabelled 125I-HSA from plasma to the microdialysis fibre by approximately 900% compared both to baseline and the control fibre within 70 min (n = 6, P < 0.05). The extravasation in the control fibre did not change over time. HPLC measurement of plasma proteins in the microdialysis perfusate also demonstrated decreased capillary selectivity for proteins in the diameter range of 73 A, 56 A and 39 A after local administration of PAF (n = 6, P < 0.05). PAF also significantly lowered interstitial fluid (P(if)) pressure after subcutaneous administration (50 microg mL(-1)). Mean arterial pressure (MAP) after intravenous injection of PAF (0.4 microg kg(-1)) fell instantly by about 50 mmHg, and stabilized at 50 mmHg after 15 min (n = 6). MAP was unaltered when PAF was given through the microdialysis fibre (n = 4). Both total tissue water (TTW) and extravasation of albumin, measured as the plasma-to-tissue clearance (E-alb) showed a significant increase after PAF (n = 7, P < 0.05). CONCLUSIONS: The present study demonstrates that PAF induces plasma protein extravasation and decrease capillary selectivity of different sized plasma proteins. It also increases transcapillary fluid flux, and lowers P(if), indicating a role for PAF in the interstitium for generation of transcapillary transport of water and large molecules followed by formation of oedema.     

The effect of formoterol on the late asthmatic phenomena in guinea pigs.

We investigated the effects of formoterol, a new, long-acting, selective beta 2-adrenoceptor agonist, on the antigen-induced late asthmatic response (LAR) and airway inflammation in guinea pigs. Animals were sensitized by exposure to aerosolized ovalbumin (2% in saline). After antigen challenge, preceded by administration of an H1-receptor antagonist, specific airway conductance was measured with a two-chambered whole-body plethysmograph. An aerosolized solution of formoterol, isoproterenol, or saline was inhaled 15 minutes before challenge. Bronchoalveolar lavage (BAL) was performed 24 hours after challenge. The provocative concentrations of histamine required to decrease specific airway conductance by 50% were obtained before challenge, at 24 hours, and at 72 hours after challenge. The LAR (52.7% +/- 7.7% of the baseline; p less than 0.02) was observed 6 to 8 hours after antigen challenge. An increased cellular influx in BAL (mainly eosinophils and macrophages) and an increased bronchial responsiveness to histamine occurred 24 hours after antigen challenge. Formoterol completely inhibited the LAR and the cellular increase in BAL; however, isoproterenol failed to prevent either the cellular infiltration or the LAR. Formoterol also decreased the antigen-induced increase in bronchial reactivity. These findings suggest that formoterol has inhibitory effects on the underlying inflammatory processes in antigen-induced asthma in addition to prolonged bronchodilation.     

Leukotriene B4, administered via intracerebroventricular injection, attenuates the antigen-induced asthmatic response in sensitized guinea pigs

Background  

Despite intensive studies focused on the pathophysiology of asthmatic inflammation, little is known about how cross-talk between neuroendocrine and immune systems regulates the inflammatory response during an asthmatic attack. We recently showed corresponding changes of cytokines and leukotriene B₄ (LTB₄) in brain and lung tissues of antigen-challenged asthmatic rats. Here, we investigated how LTB₄ interacts with the neuroendocrine-immune system in regulating antigen-induced asthmatic responses in sensitized guinea pigs.