RESUMO
BACKGROUND: The addition of a nitric oxide (NO)-releasing moiety to prednisolone was shown to enhance the anti-inflammatory activity of this glucocorticoid in some experimental conditions, but its effectiveness in the context of eosinophilic inflammation remains to be elucidated. OBJECTIVE: This study compared the anti-inflammatory effect of prednisolone to a NO-releasing derivative of prednisolone, NCX-1015, using a model of allergen-evoked eosinophil recruitment in rats. The efficacy of a NO-donor compound, DETA-NONOate, was also assessed for comparison. METHODS: Wistar rats were actively sensitized with Al(OH)(3) plus ovalbumin and 14 days later challenged with antigen intrapleurally. Treatments were performed locally 1 h before challenge. Cysteinyl-leucotrienes (Cys-LT) and eotaxin were measured by ELISA. RESULTS: Antigen challenge induced an eosinophil infiltration at 12 h, maximal at 24 h. It also caused an increase in the levels of Cys-LTs in the pleural exudate and in the expression of 5-lipoxygenase (5-LO) in infiltrated leucocytes at 6 h, peaking at 12 h and persisting for at least 24 h. Treatment with equimolar doses of prednisolone and NCX-1015 inhibited the late eosinophil infiltration, although the dose required to produce maximal inhibition was about one-tenth that of prednisolone. Cys-LT generation and 5-LO expression were inhibited by NCX-1015 but not by prednisolone. Treatment with prednisolone combined with the NO-donor DETA-NONOate led to a greater inhibition of the eosinophilia and Cys-LT generation as compared with either drug alone. Administration of the steroid receptor antagonist RU 486, 1 h before prednisolone and NCX-1015, abolished the inhibitory effect of the former, under conditions where it only partially affected the latter. CONCLUSIONS: Our findings indicate that NCX-1015 provided a greater anti-inflammatory effect than prednisolone on the allergic eosinophil recruitment in rats, suggesting that NO-releasing steroids can be considered as a promising therapeutic approach to allergic diseases.
Assuntos
Eosinofilia/prevenção & controle , Hipersensibilidade/complicações , Doadores de Óxido Nítrico/uso terapêutico , Pleurisia/prevenção & controle , Prednisolona/análogos & derivados , Animais , Anti-Inflamatórios/uso terapêutico , Araquidonato 5-Lipoxigenase/metabolismo , Quimiocina CCL11/metabolismo , Cisteína/metabolismo , Modelos Animais de Doenças , Quimioterapia Combinada , Eosinofilia/etiologia , Eosinofilia/patologia , Eosinófilos/citologia , Hipersensibilidade/tratamento farmacológico , Leucócitos/citologia , Leucócitos/metabolismo , Leucócitos Mononucleares/citologia , Leucotrienos/metabolismo , Masculino , Mifepristona/farmacologia , Neutrófilos/citologia , Compostos Nitrosos/uso terapêutico , Ovalbumina/imunologia , Cavidade Pleural/metabolismo , Cavidade Pleural/patologia , Pleurisia/etiologia , Pleurisia/patologia , Prednisolona/uso terapêutico , Ratos , Ratos Wistar , Receptores de Glucocorticoides/antagonistas & inibidoresRESUMO
Eosinophils are supposed to play a critical role in the pathology of several allergic diseases because after activation they can release toxic and proinflammatory agents. In this study we have investigated whether IgE-mediated rat pleurisy could be affected by an ongoing pleural eosinophilic inflammatory response. IgE-passively sensitized rats were challenged with an intrapleural (i.pl.) injection of allergen (dinitrophenylated bovine serum albumin, 1 microgram/cavity) and exudation assessed by measuring the amount of protein extravasated into the pleural cavity within 4 h. We have confirmed that lipopolysaccharide (LPS) stimulation (250 ng/cavity i.pl.) was followed by a marked pleural neutrophilia, apparent at 3 h, which was followed by an eosinophil accumulation noted within 48-72 h postchallenge. We have also confirmed that a boiled sample of LPS pleural washing (LPS-PW, 200 microliters i.pl.) caused selective eosinophilia in recipient rats. Pleural exudation remained unaltered when the allergenic challenge was performed 3 h after LPS in a condition of intense pleural fluid neutrophilia. In contrast, this was significantly reduced (P < .001) when the challenge occurred 72 h after LPS or 24 h after LPS-PW in selective pleural fluid eosinophilia. In another series of experiments repeated daily i.pl. injections of platelet-activating factor (PAF; 1 microgram/cavity) resulted in a progressive increase in eosinophil number recovered from the pleural cavity. The values were 1.2 +/- 0.2, 3.0 +/- 0.2, and 5.8 +/- 0.5 x 10(6) eosinophils/cavity (mean +/- SEM) after 0, 1, and 4 injections, respectively. Allergen challenge performed after 0, 1, or 4 PAF stimulations led to pleural protein levels of 88.6 +/- 5.7, 33.7 +/- 0.7, and 19.4 +/- 2.3 mg/cavity, respectively, indicating that the allergic pleurisy is inhibited in a manner dependent on the magnitude of eosinophil accumulation. Furthermore, the impairment of PAF-induced eosinophil accumulation by cetirizine (30 mg/kg i.p.) restored the exudatory response. Exudation triggered by compound 48/80 (25 micrograms/cavity), histamine (200 micrograms/cavity), or 5-hydroxytryptamine (100 micrograms/cavity) was not affected by four previous PAF daily injections. The findings indicate that allergen-induced exudation is selectively down-regulated in the eosinophil-enriched pleural space of rats, a suppression that increased with increasing eosinophil number and disappeared after chemical impairment of the eosinophilia.
Assuntos
Proteínas Sanguíneas/metabolismo , Eosinófilos/fisiologia , Exsudatos e Transudatos/citologia , Imunoglobulina E/fisiologia , Derrame Pleural/patologia , Animais , Dinitrofenóis , Eosinofilia/induzido quimicamente , Eosinofilia/patologia , Eosinofilia/fisiopatologia , Eosinófilos/imunologia , Feminino , Histamina/toxicidade , Hipersensibilidade/imunologia , Hipersensibilidade/fisiopatologia , Imunoglobulina E/imunologia , Lipopolissacarídeos/toxicidade , Masculino , Mastócitos/citologia , Fator de Ativação de Plaquetas/toxicidade , Ratos , Ratos Wistar , Serotonina/toxicidade , Soroalbumina Bovina , p-Metoxi-N-metilfenetilamina/toxicidadeRESUMO
Selective platelet-activating factor (PAF) antagonists and autodesensitization to this lipid were used to investigate the role of PAF in antigen-induced pleurisy in the rat. Pleural inflammation was triggered by the intrathoracic (i.t.) injection of ovalbumin (12 micrograms/cavity) into animals actively sensitized 14 days before. Successive daily i.t. injections of PAF (1 microgram/cavity) led to selective autodesensitization, which was apparent after the third injection and maximal after the fifth. The PAF antagonists BN 52021 and WEB 2086 inhibited the late pleural eosinophil accumulation caused by antigen but, as also noted with WEB 2170, failed to modify the early antigen-induced plasma exudation and leukocyte infiltration. In contrast to the antagonists, desensitization to PAF was clearly effective against these early alterations. To further investigate this discrepancy, the antigenic challenge was performed 24 h after a single prestimulation with PAF, when sensitivity to the lipid was still intact. Under this condition, plasma exudation and cellular influx triggered by the antigen were also abrogated, indicating that this protective effect was accounted for by a mechanism other than refractoriness to PAF. Because 24 h after PAF injection only eosinophil counts remained elevated, an alternative eosinophilotactic substance was used to further study the mechanism of PAF versus antigen-induced pleural inflammation. Prior treatment with the peptide Ala-Gly-Ser-Glu (ECF-A, 20 micrograms/cavity) also inhibited the allergic pleurisy, whereas the noneosinophilotactic substances histamine (200 micrograms/cavity) and serotonin (100 micrograms/cavity) were inactive. Furthermore, drugs that share the ability to impair PAF-induced eosinophilia, including azelastine and cetirizine, prevented the inhibitory effect of PAF on the antigen-induced pleurisy. These findings suggest that PAF may account for the late eosinophilia, but not for the acute phase of the rat allergic pleurisy, which is clearly attenuated by PAF or ECF-A pretreatment.
Assuntos
Diterpenos , Hipersensibilidade a Drogas , Eosinófilos/efeitos dos fármacos , Ovalbumina/imunologia , Fator de Ativação de Plaquetas/fisiologia , Glicoproteínas da Membrana de Plaquetas , Pleurisia/imunologia , Receptores de Superfície Celular/fisiologia , Receptores Acoplados a Proteínas G , Análise de Variância , Animais , Azepinas/farmacologia , Fatores Quimiotáticos de Eosinófilos/farmacologia , Feminino , Adjuvante de Freund , Ginkgolídeos , Inflamação , Lactonas/farmacologia , Contagem de Leucócitos/efeitos dos fármacos , Masculino , Oligopeptídeos/farmacologia , Fator de Ativação de Plaquetas/antagonistas & inibidores , Fator de Ativação de Plaquetas/farmacologia , Pleurisia/sangue , Ratos , Ratos Wistar , Receptores de Superfície Celular/antagonistas & inibidores , Triazóis/farmacologiaRESUMO
Immunoglobulin E (IgE) has been shown to play a critical role in the allergic late-phase reaction, which is marked by intense leukocyte infiltration and edema. In this study we assessed the allergic pleural inflammation triggered by intrapleural (i.pl.) challenge in sensitized rats. We examined pleural effluent from actively sensitized rats following anti-IgE monoclonal antibody (mAb) (MARE-1) provocation for protein exudation, neutrophil as well as eosinophil accumulation. Inflammatory changes triggered by antigen after passive sensitization with IgE mAb was also assessed for comparison. Total serum level of IgE was found to be about threefold increased 7-8 days post-active sensitization, remaining augmented for at least 30 days. Increased levels of peritoneal leukocyte-bound IgE and serum IgE with specificity to ovalbumin were also detected. Nevertheless, the anti-IgE challenge in 14-day actively sensitized was shown to be a weak stimulus of neutrophil and eosinophil accumulation, despite being able to cause intense protein extravasation. Similarly, antigen challenge of IgE-passively sensitized rats caused protein leakage that was comparable to that induced by anti-IgE mAb in actively sensitized rats but led to a much lower neutrophil/eosinophil infiltration. Also, blockade of complement with recombinant human soluble C receptor-1 (sCR1) treatment prevented actively sensitized rats from reacting to antigen with neutrophil and eosinophil recruitment without modifying protein extravasation. These data suggest that IgE and complement-mediated mechanisms probably account for the exudation and leukocyte infiltration that is characteristic of the pleural inflammatory response observed in actively sensitized rats.
Assuntos
Imunoglobulina E/imunologia , Pleurisia/imunologia , Proteínas/imunologia , Cloreto de Alumínio , Compostos de Alumínio , Animais , Anticorpos Monoclonais/imunologia , Cloretos , Eosinófilos/efeitos dos fármacos , Feminino , Imunoglobulina E/sangue , Contagem de Leucócitos/efeitos dos fármacos , Masculino , Neutrófilos/efeitos dos fármacos , Ovalbumina/imunologia , Proteínas/metabolismo , Ratos , Ratos Wistar , Receptores de Complemento/imunologiaRESUMO
1. Recent evidence has implicated eosinophils in the inhibition of allergen-induced rat pleurisy, but the mechanism of this negative modulation is not completely understood. This study was undertaken in order to define the potential role of prostaglandins in this phenomenon. 2. Wistar rats were actively sensitized by subcutaneous injection of a mixture of ovalbumin and AI(OH)3 and challenged with an intrapleural (i.pl.) injection of ovalbumin (12 micrograms/cavity) 14 days later. 3. Analysis of the pleural fluid effluent revealed a massive mast cell degranulation and plasma protein extravasation 4 h post-challenge. We confirmed that concurrently with selective pleural fluid eosinophilia caused by platelet-activating factor (PAF), the pleural cavity became hyporesponsive to allergen-induced protein exudation and to the parallel reduction in the number of intact mast cells. 4. These hyporesponsive animals presented a significant augmentation in the pleural effluent level of prostaglandin E2 (PGE2), which increased with increasing numbers of eosinophils in the pleural cavity. Furthermore, pretreatment with either indomethacin or aspirin failed to modify allergen-induced exudation but reversed the exudatory hyporesponsiveness associated with eosinophil recruitment. 5. Allergic exudation was clearly down-regulated by the following pretreatments: (i) PGE2 (10 micrograms/cavity, i.pl.) plus rolipram (40 micrograms/cavity, i.pl.), (ii) misoprostol (200 micrograms kg-1, p.o.) or (iii) dibutyryl cyclic AMP (80 micrograms/cavity, i.pl.). 6. We conclude that prostaglandins may be implicated in the eosinophil-mediated inhibition of allergic pleurisy, probably acting via cyclic AMP signalling pathway activation.
Assuntos
Dinoprostona/farmacologia , Regulação para Baixo/fisiologia , Eosinófilos/metabolismo , Hipersensibilidade/metabolismo , Pleurisia/metabolismo , Animais , Aspirina/farmacologia , Feminino , Indometacina/farmacologia , Masculino , Ratos , Ratos WistarRESUMO
1. The effect of topical or systemic treatment with the histamine H1-receptor antagonist, cetirizine, on the rat pleural eosinophil accumulation induced by PAF or compound 48/80 was investigated. The number of pleural resident eosinophils increased 6 h after the intrathoracic (i.t.) injection of PAF (1 microgram/cavity), peaked within 24 h and persisted significantly augmented for at least 96 h. Compound 48/80 (25 micrograms/cavity) also produced a long lasting pleural eosinophilia but this was first noted only 24 h after stimulation. 2. Intraperitoneal pretreatment with cetirizine inhibited eosinophilia induced by either PAF (ED50 = 19 mg kg-1) or compound 48/80 (ED50 = 14 mg kg-1) whereas meclizine, another histamine H1-receptor antagonist, was inactive. 3. Administered locally, cetirizine (5 and 15 micrograms/cavity) also dose-dependently inhibited both PAF- and compound 48/80-induced eosinophilia, providing evidence that its inhibitory effect is not due to any action upon circulating eosinophils. Consistent with this result, incubation of isolated peritoneal eosinophils with cetirizine failed to modify in vitro eosinophil migration caused by PAF. 4. Since the late eosinophilia induced by PAF may depend on the synthesis of a transferable protein mediator, cetirizine was administered to donor or recipient rats in order to determine whether it interferes with the generation or with the expression of this protein. We showed that only the treatment of recipient rats abolished the transfer of the eosinophilotactic activity, indicating that cetirizine does not modify the generation but inhibits the expression of this activity. 5. Our findings indicate that cetirizine is able to inhibit eosinophil accumulation by acting locally. The mechanism is neither related to its recognized ability to antagonize histamine H,-receptors nor to a direct action upon circulating eosinophils.
Assuntos
Eosinofilia/prevenção & controle , Antagonistas dos Receptores Histamínicos H1/farmacologia , Hidroxizina/análogos & derivados , Fator de Ativação de Plaquetas/antagonistas & inibidores , Pleurisia/prevenção & controle , p-Metoxi-N-metilfenetilamina/farmacologia , Administração Tópica , Animais , Cetirizina , Fatores Quimiotáticos de Eosinófilos/antagonistas & inibidores , Hidroxizina/farmacologia , Técnicas In Vitro , Masculino , Ratos , Ratos Endogâmicos , Fatores de TempoRESUMO
1 The intrathoracic injection of platelet activating factor (PAF) into rats induced a decrease in the pleural leucocyte numbers within 15 min, accompanied by a marked exudation, maximal 1 h later. After 6 h, concomitantly with the reduction of exudation, a marked increase in the number of mononuclear cells, neutrophils and eosinophils was observed. Within 24 h, the pleural eosinophil accumulation peaked and persisted up to 96 h. 2 Topical treatment with nedocromil sodium affected pleural exudation by PAF under conditions where systemic meclizine was ineffective. Nedocromil sodium blocked, dose-dependently, the increase in the pleural content of mononuclear cells, neutrophils and eosinophils, observed 6 h after PAF administration, as well as the eosinophilia 24 h later. Moreover, the co-incubation of peritoneal eosinophils with nedocromil sodium did not interfere with the migration triggered by PAF. 3 The transfer of the 6 h-PAF pleural washings from donor to recipient rats caused a selective pleural eosinophilia, which was clearly inhibited when nedocromil sodium was administered to donor, but not to recipient animals, showing that this drug interferes with the generation rather than with the expression of the eosinophilotactic activity(ies). 4 These findings indicate that the nedocromil sodium interferes with PAF-induced exudation and leucocyte accumulation, by a mechanism other than its ability to reduce the local effects of histamine and which may relate to suppression of the eosinophilotactic principle generation.
Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Linfocinas/biossíntese , Fator de Ativação de Plaquetas/farmacologia , Quinolonas/farmacologia , Animais , Quimiotaxia de Leucócito/efeitos dos fármacos , Eosinófilos/efeitos dos fármacos , Feminino , Histamina/farmacologia , Contagem de Leucócitos , Masculino , Nedocromil , Fator de Ativação de Plaquetas/antagonistas & inibidores , Pleurisia/induzido quimicamente , Pleurisia/metabolismo , Proteínas/metabolismo , Ratos , Ratos EndogâmicosRESUMO
1. Inhibitory effects of the hetrazepinic derivative BN 50730 on the rat pleural inflammatory response, triggered by PAF or lipopolysaccharides (LPS), were examined. The type of pharmacological blockade exerted by this antagonist in in vitro assays of eosinophil chemotaxis and platelet aggregation were also investigated. 2. Intrathoracic injection of PAF (1 microgram per cavity) caused a 4 fold increase in the extravasated protein within 15 min and led to a marked eosinophil accumulation 24 h post-challenge. BN 50730 (0.5-10 micrograms per cavity) inhibited exudation by PAF dose-dependently without modifying the response induced by histamine, bradykinin or 5-hydroxytryptamine (5-HT). 3. The kinetics of the inhibitory effect on exudation revealed that the actions of WEB 2086 and BN 52021 (10 micrograms per cavity) were over within 2 and 4 h respectively, whereas BN 50730 (10 micrograms per cavity) retained 80% of its inhibitory activity for 4 days. 4. Oral treatment with BN 50730 (10-20 mg kg-1, 1 h beforehand) suppressed the leucocyte accumulation and late eosinophilia observed 6 and 24 h after PAF respectively, but did not modify the eosinophilia induced by leukotriene B4 (LTB4) or bradykinin. BN 50730 also failed to reduce the eosinophil accumulation induced by LPS but drastically inhibited the neutrophil influx. 5. The pre-incubation of rat peritoneal eosinophils for 10 min with BN 50730 (30 nM-1 microM) dose-dependently inhibited the chemotaxis induced by PAF (0.1 microM) in vitro. The IC50 values for BN 52021, WEB 2086 and BN 50730 in this system were 5, 5 and 0.05 microM respectively. 6. In separate assays, rat peritoneal eosinophils and rabbit washed platelets were preincubated with BN 50730 or WEB 2086 (1 pM) then subjected to a series of at least two consecutive washings in order to remove the antagonist from the receptor environment. Under such conditions, only the cells pretreated with WEB 2086 recovered the sensitivity to the lipid.7. We conclude that BN 50730 is a potent, specific and long-acting PAF antagonist and its effect seems to result from a high affinity and non-competitive interaction of the drug with the PAF receptor.
Assuntos
Azepinas/farmacologia , Lipopolissacarídeos/farmacologia , Fator de Ativação de Plaquetas/antagonistas & inibidores , Triazóis/farmacologia , Animais , Movimento Celular/efeitos dos fármacos , Quimiotaxia de Leucócito/efeitos dos fármacos , Exsudatos e Transudatos/efeitos dos fármacos , Feminino , Leucócitos/efeitos dos fármacos , Leucócitos/fisiologia , Masculino , Fator de Ativação de Plaquetas/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Coelhos , Ratos , Ratos Wistar , TienopiridinasRESUMO
Extra-cellular and cell-associated Ca(2+)-dependent phospholipases A2 and released thromboxane B2 were correlated to exudation and cell migration during rat pleurisy induced by carrageenan or zymosan. Extra-cellular phospholipase A2 was delayed with respect to acute inflammation, while cell-associated phospholipase A2 closely correlated with cell migration and thromboxane B2 levels. This confirms that the subcellular localization of phospholipases A2 is linked to their physiological action and, in particular, suggests that the cell-associated, rather than the extracellular enzyme, accounts for the production of eicosanoids.
Assuntos
Fosfolipases A/metabolismo , Pleurisia/enzimologia , Animais , Biomarcadores , Carragenina , Ativação Enzimática/efeitos dos fármacos , Espaço Extracelular/enzimologia , Exsudatos e Transudatos/enzimologia , Cinética , Masculino , Fosfolipases A2 , Pleurisia/induzido quimicamente , Prostaglandina-Endoperóxido Sintases/metabolismo , Radioimunoensaio , Ratos , Ratos Wistar , Tromboxano B2/metabolismo , ZimosanRESUMO
Intrathoracic injection of endotoxin lipopolysaccharide, LPS into rats induced a dose-dependent increase in the number of eosinophils recovered from the pleural cavity. The pleural eosinophil accumulation peaked within 24-48 h, and returned to basal levels within 120 h. This phenomenon was accompanied by mononuclear cell infiltration, and preceded by massive neutrophil accumulation. Pretreatment with indomethacin, BW 755C (a dual cyclo/lipoxygenase inhibitor), BW A4C (a specific lipoxygenase inhibitor) or the platelet activating factor (PAF) antagonists WEB 2086 and PCA 4248 failed to inhibit the endotoxin-induced pleural eosinophilia, whilst dexamethasone (5-10 micrograms/cavity) or cycloheximide (14-28 micrograms/cavity) abolished this phenomenon. Transfer of the cell-free pleural washing from LPS-treated donor rats to normal recipient rats led to a two-fold increase in the eosinophil counts. Treatment of donors, but not recipients, with cycloheximide or dexamethasone inhibited the eosinophil accumulation induced by the pleural washings, indicating that the generation of the eosinophilotactic activity, but not its effects, depends on protein synthesis. This eosinophilotactic activity was maintained after lyophilization and heating (100 degrees C for 30 min), but was destroyed by trypsin. This substance has a molecular weight ranging between 10 and 50 kDa. The available data suggest that the late eosinophil accumulation induced by LPS is independent of arachidonic acid metabolites and PAF, and probably depends on a newly generated heat-stable soluble protein.
Assuntos
Eosinófilos/efeitos dos fármacos , Escherichia coli/metabolismo , Lipopolissacarídeos/farmacologia , Pleura/citologia , Proteínas/fisiologia , Animais , Cicloeximida/farmacologia , Dexametasona/farmacologia , Feminino , Temperatura Alta , Hidrólise , Contagem de Leucócitos/efeitos dos fármacos , Inibidores de Lipoxigenase/farmacologia , Masculino , Peso Molecular , Fator de Ativação de Plaquetas/antagonistas & inibidores , Pleura/efeitos dos fármacos , Biossíntese de Proteínas , Proteínas/química , Ratos , Ratos Wistar , Tripsina , UltrafiltraçãoRESUMO
The local effect of salbutamol and N6,2'-O-dibutyryl adenosine 3':5'-cyclic monophosphate (Bt2 cyclic AMP) on the rat pleural inflammation caused by allergen was investigated. Antigen (ovalbumin, 12 micrograms/cavity) intrathoracically administered to immunized rats led to a marked pleural protein extravasation and leukocyte infiltration, as attested by the quantification of protein and enumeration of leukocytes recovered from the pleural cavity. Salbutamol (10-40 micrograms/cavity) and the cell-permeable cyclic AMP analogue, Bt2 cyclic AMP (20-160 micrograms/cavity), injected 1 h and 5 min before the antigen, respectively, inhibited the exudation occurring within 30 min, and neutrophil and eosinophil accumulation occurring 4 and 24 h, respectively. The late eosinophilia was also markedly attenuated by salbutamol administered 10 min post-challenge, when mast cells had already been degranulated. Pretreatment with the beta-adrenoceptor antagonist propranolol (1 mg/kg, i.v.) failed to modify the inhibitory effect of Bt2 cyclic AMP, but abolished the blockade caused by salbutamol of leukocyte infiltration under conditions where the salbutamol anti-exudatory activity was impaired to about 80%. In another set of experiments, salbutamol (20 and 40 micrograms/cavity) markedly inhibited the exudation caused by histamine and 5-hydroxytryptamine (5-HT) which, though to a lesser extent, was also sensitive to Bt2 cyclic AMP (80 micrograms/cavity). As observed with allergic pleurisy, propranolol impaired the inhibition by salbutamol of histamine- and 5-HT-induced exudation, whereas the Bt2 cyclic AMP inhibition was not affected. We conclude that salbutamol and Bt2 cyclic AMP share the ability to inhibit pleural exudation and leukocyte recruitment caused by allergen in immunized rats, suggesting that the anti-inflammatory effect of salbutamol may be mediated by a cyclic AMP signaling pathway, probably via beta 2-adrenoceptor activation.
Assuntos
Agonistas Adrenérgicos beta/farmacologia , Albuterol/farmacologia , Antígenos/imunologia , Bucladesina/farmacologia , Quimiotaxia de Leucócito/efeitos dos fármacos , Pleurisia/prevenção & controle , Antagonistas Adrenérgicos beta/farmacologia , Animais , AMP Cíclico/metabolismo , Feminino , Hipersensibilidade/prevenção & controle , Masculino , Pleurisia/etiologia , Pleurisia/metabolismo , Propranolol/farmacologia , Ratos , Ratos Wistar , Transdução de SinaisRESUMO
The efficacy of cetirizine in comparison with meclizine, another piperazine H1 receptor antagonist, in rat pleurisy caused by allergen or autacoid was investigated. Sensitization was achieved by subcutaneous injection of a mixture of ovalbumin and aluminium hydroxide. Fourteen days later, the animals were challenged with an intrathoracic injection of ovalbumin (12 micrograms/cavity), which caused drastic mast cell degranulation, followed by pleural oedema and leucocyte influx. Cetirizine and meclizine (2.5-30 mg/kg i.p.), 1 h before challenge, inhibited the exudatory response evoked by antigen, under conditions where neutrophil and eosinophil accumulation was affected only by the former. When administered intrathoracically 22 h after allergen, i.e. using a curative approach, cetirizine (15 micrograms/cavity) drastically reduced the pleural eosinophilia noted 24 h post-challenge, indicating that this drug can reverse an already established eosinophilia. Cetirizine (15 mg/kg i.p.) also restored, to about 39% (P < 0.001), the number of uninjured mast cells recovered from the pleural cavity following allergen stimulation. In normal rats, cetirizine (5-15 micrograms/cavity) completely inhibited the pleural exudation elicited by histamine and only partially the exudation caused by 5-hydroxytryptamine or bradykinin, but was quite inactive against platelet-activating factor. We conclude that the pleural exudation triggered by allergen, vasoactive amines or bradykinin is clearly sensitive to cetirizine. In addition, the ability of the drug to interfere with pleural neutrophil or eosinophil mobilization and mast cell degranulation seems not to be associated with its ability to block the histamine H1 receptor.
Assuntos
Cetirizina/uso terapêutico , Antagonistas dos Receptores Histamínicos H1/uso terapêutico , Pleurisia/tratamento farmacológico , Animais , Antígenos/imunologia , Bradicinina/farmacologia , Grânulos Citoplasmáticos/efeitos dos fármacos , Feminino , Histamina/farmacologia , Contagem de Leucócitos , Masculino , Mastócitos/efeitos dos fármacos , Meclizina/farmacologia , Ovalbumina/imunologia , Fator de Ativação de Plaquetas/farmacologia , Pleurisia/imunologia , Ratos , Ratos Wistar , Serotonina/farmacologiaRESUMO
The pericarp and seeds from fruits of Garcinia brasiliensis were subjected to extraction with hexane and ethanol. The pericarp hexane extract (PHE) and seed ethanol extract (SEE) were purified by silica gel column chromatography, which permitted isolation of the prenylated benzophenones 7-epiclusianone (1) and guttiferone-A (2), respectively. The antimicrobial activity of PHE, SEE, and compounds 1 and 2 were evaluated against Candida albicans, Staphylococcus aureus, Escherichia coli, and Bacillus cereus cultures. The minimum inhibitory concentration and minimum bactericidal concentration were established. The substances presented activity against S. aureus and B. cereus as follows: PHE, 4.0 microg/mL and 2.4 microg/mL; SEE, 10.0 microg/mL and 12.6 microg/mL; 7-epiclusianone, 1.2 microg/mL and 0.6 microg/mL; and guttiferone-A, 2.4 microg/mL and 2.4 microg/mL, respectively. The direct relationship between the lipophilic character of the structure and activity in Gram-positive bacteria was specifically observed. Therefore these extracts and prenylated benzophenones represent an interesting topic for further studies and open possibilities for an alternative control of diseases associated with Gram-positive bacteria.
Assuntos
Antibacterianos/farmacologia , Antifúngicos/farmacologia , Benzofenonas/farmacologia , Benzoquinonas/farmacologia , Garcinia , Floroglucinol/farmacologia , Extratos Vegetais/farmacologia , Antibacterianos/isolamento & purificação , Antifúngicos/isolamento & purificação , Bactérias/efeitos dos fármacos , Benzofenonas/isolamento & purificação , Benzoquinonas/isolamento & purificação , Candida albicans/efeitos dos fármacos , Frutas , Garcinia/química , Testes de Sensibilidade Microbiana , Floroglucinol/análogos & derivados , Floroglucinol/isolamento & purificação , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , SementesRESUMO
We previously reported that alloxan-induced diabetes results in reduction in the number and reactivity of mast cells at different body sites. In this study, the influence of diabetes on thymic mast cells was investigated. Thymuses from diabetic rats showed marked alterations including shrinkage, thymocyte depletion, and increase in the extracellular matrix network, as compared to those profiles seen in normal animals. Nevertheless, we noted that the number and reactivity of mast cells remained unchanged. These findings indicate that although diabetes leads to critical alterations in the thymus, the local mast cell population is refractory to its effect. This suggests that thymic mast cells are under a different regulation as compared to those located in other tissues.
Assuntos
Diabetes Mellitus Experimental/patologia , Mastócitos/patologia , Timo/patologia , Aloxano , Animais , Contagem de Células , Masculino , Ratos , Ratos WistarRESUMO
The flagellate Herpetomonas roitmani is a symbiont-bearing trypanosomatid that spontaneously differentiates from promastigote to para- and opisthomastigote forms when maintained in axenic culture medium. Thus, after cultivation for 72 h at 28 degrees C, 37% of the total number of cells are in the opisthomastigote form. In the present study, light microscopy observations of Giemsastained H. roitmani cells demonstrated that in early cultures (12 h at 28 degrees C) the percentage of opisthomastigotes was markedly high (about 98%). Furthermore, proliferative opisthomastigote forms (dividing cells with the kinetoplast posteriorly located relative to the nucleus) were frequently seen in these cultures. The latter observation was confirmed by analysis of routinely fixed parasites by transmission electron microscopy.
Assuntos
Dípteros/parasitologia , Trypanosomatina/ultraestrutura , Animais , Trypanosomatina/crescimento & desenvolvimentoRESUMO
Herpetomonas roitmani, a non-pathogenic trypanosomatid was grown in chemically defined media either containing proline or glucose as carbon source. Using transmission electron microscopy we observed that cells grown in the presence of proline present more lipid inclusions, and a larger mitochondrion with more cristae and higher activity of succinate cytochrome c reductase. On the other hand, cells grown with glucose as carbon source had more glycosomes, which were preferentially located close to the bacterium endosymbiont, and a much higher activity of hexokinase, a typical glycosome marker. Three-dimensional reconstruction and morphometrical analysis confirm these observations. The number of promastigotes of H. roitmani increased in the presence of proline. Taken together these results indicate that the growth conditions markedly influenced the ultrastructure and the metabolism of H. roitmani.
Assuntos
Metabolismo Energético/fisiologia , Trypanosomatina , Animais , Meios de Cultura/farmacologia , Metabolismo Energético/efeitos dos fármacos , Flagelos/metabolismo , Flagelos/ultraestrutura , Glucose/metabolismo , Glucose/farmacologia , Processamento de Imagem Assistida por Computador , Microscopia Eletrônica , Mitocôndrias/metabolismo , Mitocôndrias/ultraestrutura , Prolina/metabolismo , Prolina/farmacologia , Trypanosomatina/crescimento & desenvolvimento , Trypanosomatina/metabolismo , Trypanosomatina/ultraestruturaRESUMO
Cell surface saccharide composition and surface charge of promastigote (PRO) and opisthomorph (OPM) forms of Herpetomonas roitmani were analyzed using labeled lectins and flow cytometry and cell electrophoresis. The FITC signals for concanavalin A, Helix pomatia agglutinin and wheat germ agglutinin were stronger in PRO forms, whereas for Limulus polyphemus agglutinin (LPA) and Wisteria floribunda agglutinin they were stronger in OPM forms. Prior treatment of the cells with neuraminidase decreased the FITC signal for LPA in OPM but not in PRO forms. Furthermore OPMs displayed a high negative charge (-15.45+/-1.10 mV) than PROs (-9.47+/-1.01 mV). Neuraminidase and phospholipase C treatment of the parasites significantly reduced the surface charge, especially in OPM forms. TLC analysis of the acidic components of H. roitmani showed the presence of N-acetyl-neuraminic acid. The results presented in this work indicate that changes in exposed cell surface components occur between PRO and OPM forms of H. roitmani obtained by growing the cells under different conditions.
Assuntos
Trypanosomatina/química , Animais , Membrana Celular/química , Ácido N-Acetilneuramínico/análiseRESUMO
Alterations in the local mast cell population and the eosinophil accumulation in the rat pleural cavity were studied using pleurisy induced by compound 48/80, a standard mast-cell-degranulating agent. Twenty-four hours after the intrathoracic injection of compound 48/80 (1-50 micrograms/cavity), a dose-dependent eosinophil enrichment of the exudate was noted, concomitantly with a drastic reduction in the total number of undamaged mast cells recovered from the pleural washing. At 24 h, neutrophil counts were not modified, and the number of mononuclear cells was increased, but only at the highest dose of compound 48/80. The temporal analysis showed that mast cell degranulation, exudation and neutrophil infiltration were maximal at the interval of 1-6 h after compound 48/80 (25 micrograms/cavity), whereas eosinophil accumulation peaked within 24 h, persisting elevated at least until 96 h. Since compound 48/80 was itself unable to induce eosinophil migration in vitro, attempts were made to investigate the potential involvement of recognized eosinophil chemo-attractants, such as histamine, leukotriene B4 (LTB4) and platelet-activating factor (PAF-acether). The intraperitoneal pretreatment with either cyproheptadine (2 mg/kg), meclizine (40 mg/kg), BW755C (25 mg/kg) or with the PAF-acether receptor antagonist WEB 2086 (20 mg/kg) had no effect on the eosinophil recruitment induced by compound 48/80 (25 micrograms/cavity). However, the treatment with the corticosteroid dexamethasone or the local inhibition of protein biosynthesis with cycloheximide (0.04-200 nmol/cavity) blocked the eosinophil pleural accumulation, but not the mast cell degranulation induced by compound 48/80. Our findings indicate that the pleural eosinophil accumulation induced by compound 48/80 is sensitive to dexamethasone, requires local protein biosynthesis and is independent of histamine, LTB4 and PAF-acether.
Assuntos
Dexametasona/farmacologia , Eosinófilos/efeitos dos fármacos , Mastócitos/efeitos dos fármacos , Pleura/patologia , Biossíntese de Proteínas , p-Metoxi-N-metilfenetilamina/farmacologia , 4,5-Di-Hidro-1-(3-(Trifluormetil)Fenil)-1H-Pirazol-3-Amina/farmacologia , Animais , Azepinas/farmacologia , Cicloeximida/farmacologia , Eosinófilos/patologia , Feminino , Contagem de Leucócitos , Masculino , Mastócitos/patologia , Fator de Ativação de Plaquetas/farmacologia , Ratos , Ratos Endogâmicos , Triazóis/farmacologiaRESUMO
The injection of platelet-activating factor (PAF) into the rat pleural cavity resulted in a marked increase in the number of eosinophils, neutrophils and mononuclear cells recovered from pleural washings after 6 hr. Within 24 hr, neutrophils and mononuclear cell counts returned to control values, whereas the eosinophilia peaked and persisted up to 96 hr. Treatment with the PAF antagonist BN 52021 [3-(1,1-dimethylethyl)hexahydro-1,4,7b-trihydroxy-8-alpha-methyl-9H-1,7- alpha-(epoxymethanol)1H,6aH-cyclopenta (c) furo (2,3-5) (3',2':3,4) cyclopenta (1,2-d) furan-5,9,12(4H)trione)] (5-20 micrograms/cavity) or with dexamethasone (1-50 micrograms/cavity) inhibited the early (6 hr) and late (24 hr) pleural eosinophil accumulation induced by PAF. Dexamethasone, but not BN 52021, was still effective in inhibiting the late eosinophilia if administered 5 hr after the lipid, suggesting that the delayed eosinophilia may involve a secondary mechanism, still responsive to the glucocorticoid, but independent of PAF itself. The coinjection of the protein synthesis inhibitor cycloheximide or of the inhibitor of mRNA synthesis DNA-dependent actinomycin D selectively suppressed the eosinophil mobilization at 24 hr. Transfer of the cell-free 6-hr PAF pleural washing from donor to normal recipient rats led to a selective and delayed pleural eosinophilia. This activity was destroyed by heating (+100 degrees C) or freezing (-20 degrees C) the material recovered from the donor pleural fluid. Treatment of donors, but not recipients, with either BN 25021, dexamethasone, actinomycin D or cycloheximide inhibited the late eosinophil accumulation triggered by transferred PAF pleural washing, indicating that the generation of this eosinophilotactic activity, but not its effects, is suppressed by those agents.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Fatores Quimiotáticos de Eosinófilos/metabolismo , Quimiotaxia de Leucócito/efeitos dos fármacos , Diterpenos , Fator de Ativação de Plaquetas/farmacologia , Pleura/metabolismo , Animais , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Dexametasona/farmacologia , Estabilidade de Medicamentos , Eosinófilos/efeitos dos fármacos , Ginkgolídeos , Lactonas/farmacologia , Masculino , Fator de Ativação de Plaquetas/antagonistas & inibidores , Pleura/citologia , Pleura/efeitos dos fármacos , Ratos , Ratos Endogâmicos , TemperaturaRESUMO
In the present study, we have performed a comparative analysis of the effect of selective inhibitors of phosphodiesterase (PDE) type III, IV and V on eosinophil chemotaxis triggered by platelet activating factor (PAF) and leukotriene B4 (LTB4) in vitro. The effect of the analogues N6-2'-O-dibutyryladenosine 3':5'cyclic monophosphate (Bt2 cyclic AMP) and N2-2'-O-dibutyrylguanosine 3':5' cyclic monophosphate (Bt2 cyclic GMP) has also been determined. The eosinophils were obtained from the peritoneal cavity of naive Wistar rats and purified in discontinuous Percoll gradients to 85-95% purity. We observed that pre-incubation of eosinophils with the PDE type IV inhibitor rolipram suppressed the chemotactic response triggered by PAF and LTB4' in association with an increase in the intracellular levels of cyclic AMP. In contrast, neither zaprinast (type V inhibitor) nor type III inhibitors milrinone and SK&F 94836 affected the eosinophil migration. Only at the highest concentration tested did the analogue Bt2 cyclic AMP suppress the eosinophil chemotaxis, under conditions where Bt2 cyclic GMP was ineffective. We have concluded that inhibition of PDE IV, but not PDE III or V, was able to block the eosinophil chemotaxis in vitro, suggesting that the suppressive activity of selective PDE IV inhibitors on tissue eosinophil accumulation may, at least, be partially dependent on their ability to directly inhibit the eosinophil migration.