Atividade pró-trombótica da toxina ExoU de Pseudomonas aeruginosa detectada em modelos experimentais in vivo e in vitro / Prothrombotic activity of the Pseudomonas aeruginosa toxin ExoU detected in experimental models in vivo and in vitro

Pseudomonas aeruginosa é um importante agente de pneumonia, particularmente em pacientes submetidos à ventilação mecânica, que pode evoluir para sepse, com elevadas taxas de letalidade. Na sepse, o processo inflamatório sistêmico exacerbado favorece o desequilíbrio entre as vias de coagulação e fibrinólise e a instalação de um estado pró-coagulante, com o aparecimento de trombose microvascular, coagulação intravascular disseminada e falência de múltiplos órgãos. Conhecendo a potente atividade pró-inflamatória da toxina ExoU produzida por P. aeruginosa, decorrente de sua atividade fosfolipásica A2, o objetivo desta tese foi investigar seu potencial de indução de alterações hemostáticas relacionadas à patogênese da sepse. Utilizando modelo de sepse em camundongos inoculados, por via intratraqueal, com suspensões de P. aeruginosa produtora de ExoU (PA103) ou de cepa com deleção do gene exoU, não produtora da toxina, foi mostrado que ExoU determinou maior gravidade da infecção, maior taxa de letalidade, leucopenia, trombocitose, hiperpermeabilidade vascular e transudação plasmática, evidenciadas, respectivamente, pela maior concentração de proteínas nos lavados broncoalveolares (LBAs) e acúmulo do corante Azul de Evans, previamente inoculado nos animais, por via endovenosa, no parênquima renal. ExoU favoreceu, também, a ativação plaquetária, confirmada pela maior concentração de plaquetas expressando P-seletina em sua supefície, maior número de micropartículas derivadas de plaquetas e maior concentração plasmática de tromboxano A2. A histopatologia dos pulmões e rins dos animais infectados com PA103 confirmou a formação de microtrombos, que não foram detectados nos animais controles ou infectados com a cepa mutante. Nos pulmões, a produção de ExoU determinou intensa resposta inflamatória com maior concentração de leucócitos totais e polimorfonucleados, interleucina-6 e fator de necrose tumoral-alfa nos LBAs. A análise imunohistoquímica mostrou intensa deposição...

Pseudomonas aeruginosa is an important agent of pneumonia, mainly in patients undergoing mechanical ventilation, which can progress to sepsis with high mortality rates. In sepsis, the systemic inflammatory process favors exacerbated imbalance between the coagulation and fibrinolysis pathways and the installation of a procoagulant state, leading to microvascular thrombosis, disseminated intravascular coagulation and multiple organ failure. Knowing the powerful proinflammatory activity of the P. aeruginosa toxin ExoU, secondary to its phospholipase A2 activity, the goal of this study was to investigate the ExoU potential to induce hemostatic changes related to sepsis pathogenesis. By using a murine model of pneumosepsis, obtained by the intratracheal injection of suspensions of the ExoU-producing PA103 P. aeruginosa strain or of its isogenic mutant PA103 exoU, defective in the toxin synthesis, ExoU was shown to enhance the severity of the infection and to induce higher mice mortality rate as well as leukopenia, thrombocytosis, vascular hyperpermeability and plasma transudation, evidenced, respectively, by the higher protein concentration in the bronchoalveolar lavage fluids (BALF) and accumulation of Evans blue dye, previously intravenous infectioned, in mice renal parenchyma. ExoU also favored platelet activation, evidenced by the higher concentration of platelets expressing P-selectin on their surface, greater number of platelet-derived microparticles and increased plasma concentration of thromboxane A2. Histopathology of the lungs and kidneys of PA103 - infected animals confirmed the formation of microthrombi, which were not detected in controls or in animals infected with the bacterial mutant. In lungs, ExoU induced an intense inflammatory response with high concentrations of total and polymorphonuclear leukocytes, interleukin-6 and tumor necrosis factor-alfa in mice BALF. Immunohistochemical analysis showed intense fibrin deposition in the alveoli...

PAF antagonism modifies neuroprotective action of histone deacetylase and calcineurin phosphatase inhibitors in mice.

To evaluate the hypothesis that platelet activating factor (PAF) antagonism may affect the functional recovery following the nerve injuries and also to evaluate the effect of PAF receptor antagonism on the neuroprotective effect of tacrolimus and sodium valproate, effect of PAF receptor antagonist, WEB2086 was evaluated in animal models of sciatic nerve crush and endothelin-1 induced focal cerebral ischemia. WEB2086, per se, while attenuating spontaneous sensory motor recovery after sciatic nerve crush, enhanced functional recovery after focal cerebral ischemia. WEB2086 also attenuated the neuroprotective effect of tacrolimus and sodium valproate subsequent to peripheral nerve injury, while it significantly improved the neuroprotective action of tacrolimus and sodium valproate following cerebral ischemia reperfusion injury. These results suggest that PAF receptor antagonists alone and in combination with tacrolimus/sodium valproate could be used in the treatment of cerebral ischemia reperfusion injuries however, their use following peripheral nerve injuries could be detrimental.

The inhibitory effect of methotrexate on PAF-induced neutrophil and eosinophil locomotion in asthmatic patients.

We have tested the effect of methotrexate (MTX) on platelet activating factor (PAF)-induced neutrophil and eosinophil locomotion, neutrophil leukotriene B4 (LTB4) generation and mononuclear cell DNA synthesis. Neutrophils from patients treated with low dose methotrexate showed reduced PAF-induced chemotactic responses (727.8 +/- 72.2/10 HPF vs 481.9 +/- 87.3/10 HPF, p < 0.05). Both MTX and the specific PAF antagonist BN-52021 significantly inhibited PAF-induced eosinophil and neutrophil locomotion in a dose-dependent manner. MTX also reduced calcium ionophore-driven LTB4 generation from the neutrophils of asthmatics (358.9 +/- 39.5 pg/10(6) cells vs 240.1 +/- 29.1 pg/10(6) cells, p < 0.05) and attenuated PHA-induced mononuclear DNA synthesis as shown by a reduction in 3H-thymidine uptake and propidium iodide staining. These findings support the view that the beneficial effects of MTX in asthma may be due not only to its anti-mitotic effects on the proliferation of mononuclear cells but also to direct effects on granulocyte locomotion and production of LTB4.

Enhanced eosinophil luminol-dependent chemiluminescence and complement receptor expression by platelet-activating factor and interleukin-5.

The cytokine interleukin-5 (IL-5) and the lipid mediator platelet-activating factor (PAF) have both been shown to be involved in eosinophil differentiation and activation. We have measured and compared the effect of PAF and IL-5 on human eosinophils in terms of their luminol-dependent chemiluminescence (CL) response and their expression of complement receptors, CR1 and CR3. Both IL-5 and PAF enhanced the eosinophil CL response. The optimal concentrations were 40 U/ml for IL-5, and 10(-6) M for PAF. The priming effect of IL-5 was slow and reached a maximal response after 90 minutes incubation. In contrast, the effect of PAF peaked early and declined during incubation. In the complement receptor study, only PAF was able to enhance CR3 expression (p < 0.05) while the effect of IL-5 on eosinophil complement receptor expression was negligible. These results provide evidence that both inflammatory mediator (PAF) and cytokine (IL-5) can activate eosinophils but the effects of IL-5 and PAF on eosinophil CL response appear to be distinct. The activation of eosinophils by PAF and IL-5 may occur through different mechanisms.

Anti-platelet activating factor property of Rubia cordifolia Linn.

Rubia cordifolia is clinically used for the purification of blood by the physicians of the Indian System of Medicine. For the first time, the effect of the partially purified fraction of this whole plant has been studied on rabbit platelets. It inhibits the platelet aggregation induced by PAF (platelet activating factor) but not thrombin. It also inhibits the binding of 3H-PAF to the platelets in the dose-dependent manner. Thus it appears that R. cordifolia inhibits action of PAF at its receptor level either by it's blocking or by desensitization.

Ginkgo biloba e radicais livres em camundongos / Ginkgo biloba and free radicals in mices

O extrato de Ginkgo biloba é um poderoso agente na terapia dos distúrbios vasculares (centrais ou apenas periféricos). É também um destruidor de radicais livres, em experiências com isquemia-reperfusäo intestinal, e um potente inibidor do PAF. No presente trabalho em camundongo normal, a sua açäo antilipoperoxidativa pode ser observada, quando ministrado com água

Inibidores da agregacao plaquetaria induzida pelo PAF-acether / Inhibitor of the platelets aggregation induced by the PAF-acether

O efeito do PAF-acether foi avaliado em plaquetas humanas de individuos adultos clinicamente saudaveis atraves de estudos em plasma rico em plaquetas (PRP) com inibidores plaquetarios "in vitro"(AAS, NDHA e CP/CPK) e "ex-vivo" (AAS). O PAF-acether induziu agregacao dose dependente em concentracoes de 5x10-9M a 5x10-7M, com dose limite ou threshold em 9x10-8M. Os resultados obtidos demonstraram que a fase primaria de agregacao e independente da formacao de metabolicos de acido araquidonico e liberacao de ADP endogeno visto que, com excessao do BN 52021, os demais inibidores nao influenciaram nessa fase. Por outro lado, a fase secundaria de agregacao irreversivel envolve interrelacao entre os mediadores ADP e Acido Araquidonico dependentes preferencialmente da formacao de metabolicos do acido araquidonico via ciclo-oxigenase.

Involvement of PAF-acether and eicosanoids in adrenaline-induced pulmonary edema in mice

The participation of platelet-activating factor (PAF,PAF-acether) in a mouse model of pulmonary edema was studied using specific antagonists.Mice were treated before induction of edema with the PAF antagonists BN52021 (10mg/kg, ip), PCA 4248 (10 mg/kg, po) or WEB2170 (10mg/kg, ip),the lipoxygenase inhibitor EP10161 (10 mg/kg,ip),the cyclo-oxygenase inhibitor aspirin (250 mg/kg,po), or with the mixed cyclo-lipoxygenase inhibitor BW755C(50 mg/kg, ip).The test drugs were administered to animals either 30 min (When the ip route was used) or 60 min (when given po) prior to the induction of pulmonary edema.Pulmonary edema was induced by intravenous administration of adrenaline (2 mg/kg). When the lung-body index was usedas thecriterion for comparision between groups,BN52021, PCA4248 and WEB2170 were found to have no significant effect on pulmonary edema. In contrast, EP10161, aspirinand BW755C significantly inhibited pulmonary edemaby 49%,30% and 27%,respectively. The results suggest that arachidonate metabolites are likely to play a major roe in adrenaline-induced pulmonary edema in mice, whereas PAF-acether does not seem to play an important role in this model

Paf antagonists do not modify IgE antibody production in mice

The effect of selective PAF antagonists on the in vivo production of IgE antibodies was investigated. The anti-ovalbumin IgE antibody content was estimated by passive cutaneous anaphylactic reaction (PCA) in the plasma of Balb/c mice 10 days after immunization with ovalbumin and alum. The PAF antagonists, BN 52021 (5 mg/kg, ip), BN 50730(20 mg/kg, ip) BN 50730 (20 mg/kg, po), WEB 2086 (2 mg/kg, ip) and WEB 2170 (5 mg/kg, ip) were administered 1 h before immunization and twice a day for 8 days thereafter. The effect of the antagonists on the PAF-induced vasopermeability was also assayed. In the immunized mice the level of antiovalbumin IgE antibody, estimated by PCA titer, was 1/640. The treatment with the PAF antagonists did not change this level. At the concentrations employed, the antagonists BN 50730, WEB 2086 and WEB 2170 significantly reduced the PAF-induced vascular permeability. These results suggest that PAF does not seem to have a relevant effect on the production of IgE antibodies in vivo in the system used in the present study

Enterolobin induces rat paw oedema independently of PAF-acether

The potential participation of PAF-acether (PAF) on the paw oedema triggered by enterolobin was investigated. Intraplantar injections of enterolobin )5-20 µg/paw) yielded a dose response curve for edema which appeared after 30 min, peaked in the interval between 2-4 h and faded after 24h. The pre-treatment with BN 52021, but not with other PAF antagonists such as PCA 4248 or WEB 2086, significantly blocked enterolobin-induced oedema. To clarify better the discrepant results obtained with the PAF antagonists, desensitization to PAF was performed. The oedema triggered by enterolobin was not modified in paf desensitized animals. It was concluded that the paw inflammation induced by enterolobin does not require PAF mechanism.

PAF-acether and the acute inflammatory reaction

PAF-aceter é um fosfolipídio que exerce um papel importante na reaçäo inflamatória aguda. No modelo experimental do edema da para de rato e pleurisia, a reaçäo induzida pelo PAF era acompanhada de hemoconcentraçäo, embora esses processos pareçam ser mediados por mecanismos distintos. Antagonistas específicos do PAF, como BN 52021 e WEB 2086, inibem 60% do edema induzido pelo PAF. Leucotrienos e mecanismos histaminérgicos envolvendo receptores H2 parecem participar no processo. A participaçäo do sistema adrenérgico e de metabólitos da ciclooxigenase é controversa. Infiltraçäo de leucócitos, observada após a injeçäo de PAF, e plaquetas näo säo necessárias para a formaçäo de edema. Auto-dessensibilizaçäo induzida pelo PAF pôde ser observada, sendo seletiva e dependente a interaçäo do PAF com receptores específicos em um processo de regulaçäo depressiva ("dow-regulation"). Nenhuma participaçäo deo PAF foi observada na reaçäo inflamatória induzida pela carregenina, contrariamente áquela provocada pelo zimosan, que é principalmente dependente do PAF-aceter

Chemotactic effect of PAF-acether on peritoneal eosinophils from normal rats

The chemotactic activity of PAF-acether was compared with that of tetrapeptide eosinophil chemotactic factors of anaphylaxis (ECF-A, Ala-Gly-Ser Glu and Val-Gly-Ser-Glu) using eosinophils obtained from the peritoneal cavity of normal rats. Cells were isolated by separation over discontinuous metrizamide gradients which resulted in eosinophil suspensions of 80 to 90% purity. PAF-acether produced 7-fold greater than the maximal activity obtained with the ECF-A-tetrapeptides. BN 52021 and WEB 2086 inhibited PAF-acether-induced eosinophil chemotaxis in a dose-dependent manner, suggesting that this phenomenon is mediated by specific PAF-acether receptors