Ana Elisa Caleiro Seixas Azzolini

Effect of Tityus serrulatus scorpion venom and its major toxin, TsTX-I, on the complement system in vivo

The effects of Tityus serrulatus venom and TsTX-I (Ts1 or gamma-toxin) on the lytic activity of the complement system (CS) were investigated in vivo. Serum classical pathway (CP) and alternative pathway (AP) activities were determined in sera of rats (200+/-10 g) injected i.p. with soluble venom (150 microg/kg), TsTX-I (150 microg/kg) or saline (control). The animals were sacrificed 0.5, 1, 2, 4, 24 and 48 h after injection. The results showed an increase in serum lytic activity of animals injected with venom, reaching values up to 70% above controls in CP activity and 120% in AP activity. These effects were biphasic with maximum values 1 and 24 h after venom injection. Similar effects were obtained for TsTX-I, but with lower intensity. Hematocrit values of all tested animals were determined to evaluate the effect of hemoconcentration on the lytic activity of the CS. It was observed that the maximum hematocrit value was obtained 1 h after injection and returned to normal values within 24 h. These data indicate that hemoconcentration can play a relevant role in the first peak of complement activity, but we cannot discard a direct action of the venom on the system during this period, since the serum venom concentration is maximal 15-30 min after envenomation. The high lytic activity of the serum observed after 24 h, period in which the hematocrit values are normal and no venom can be detected, may be consequence of the inflammatory process induced by the venom or toxin. The lytic activity of the serum of rats injected with venom, TsTX-I or saline was abolished when the serum was previously adsorbed on zymosan. These data confirm that the increase of the lytic activity of the serum induced by the venom or toxin is dependent on CS. These results show that CS is involved in the inflammatory process induced by the venom or toxin and consequently in the lung edema, hemolysis, leukocytosis, among other clinical manifestations of severe envenomation.

Elastase Release by Stimulated Neutrophils Inhibited by Flavonoids: Importance of the Catechol Group

Pathogenesis of chronic inflammatory diseases is associated with excessive elastase release through neutrophil degranulation. In the present study, inhibition of human neutrophil degranulation by four flavonoids (myricetin, quercetin, kaempferol, galangin) was evaluated by using released elastase as a biomarker. Inhibitory potency was observed in the following order: quercetin > myricetin > kaempferol = galangin. Quercetin, the most potent inhibitor of elastase release also had a weak inhibitory effect on the enzyme catalytic activity. Furthermore, the observed effects were highly dependent on the presence of a catechol group at the flavonoid B-ring. The results of the present study suggest that quercetin may be a promising therapeutic agent in the treatment of neutrophil-dependent inflammatory diseases.

Modulation of human neutrophil oxidative metabolism and degranulation by extract of Tamarindus indica L. fruit pulp.

The tamarind (Tamarindus indica L.) is indigenous to Asian countries and widely cultivated in the American continents. The tamarind fruit pulp extract (ExT), traditionally used in spices, food components and juices, is rich in polyphenols that have demonstrated anti-atherosclerotic, antioxidant and immunomodulatory activities. This study evaluated the modulator effect of a crude hydroalcoholic ExT on some peripheral human neutrophil functions. The neutrophil reactive oxygen species generation, triggered by opsonized zymosan (OZ), n-formyl-methionyl-leucyl-phenylalanine (fMLP) or phorbol myristate acetate (PMA), and assessed by luminol- and lucigenin-enhanced chemiluminescence (LumCL and LucCL, respectively), was inhibited by ExT in a concentration-dependent manner. ExT was a more effective inhibitor of the PMA-stimulated neutrophil function [IC50 (in microg/10(6)cells)=115.7+/-9.7 (LumCL) and 174.5+/-25.9 (LucCL)], than the OZ- [IC50=248.5+/-23.1 (LumCL) and 324.1+/-34.6 (LucCL)] or fMLP-stimulated cells [IC50=178.5+/-12.2 (LumCL)]. The ExT also inhibited neutrophil NADPH oxidase activity (evaluated by O2 consumption), degranulation and elastase activity (evaluated by spectrophotometric methods) at concentrations higher than 200 microg/10(6)cells, without being toxic to the cells, under the conditions assessed. Together, these results indicate the potential of ExT as a source of compounds that can modulate the neutrophil-mediated inflammatory diseases.

Comparison of antioxidant activities of tocopherols alone and in pharmaceutical formulations

The objective of the present investigation was to compare the antioxidant effect of different forms of Vitamin E (DL-alpha-tocopherol, Mixed Tocopherols, Ronoxan MAP and alpha-tocopherol acetate) and of topical formulations containing these active pharmaceutical ingredients, using chemiluminescence and the stable free radical 1,1-diphenyl-2-picrylhydrazyl (DPPH) assays. Inhibition of the intensity of chemiluminescence, using the H2O2-luminol-horseradish peroxidase (HRP) enzyme system, was measured for 10 min at room temperature in 10 microl samples of each vitamin at different concentrations, and of formulations containing these vitamins. H-donor ability in the DPPH assay, was measured in 10 microl samples at different concentrations of each vitamin, as well as in formulation in ethanol solution; the decrease of absorbency was read at 517 nm. DL-alpha-tocopherol, Mixed Tocopherols and Ronoxan MAP alone or in formulations, markedly inhibited chemiluminescence intensity and decreased absorbency in the DPPH assay in a concentration-dependent manner. Alpha-tocopherol acetate and formulations containing this vitamin did not show antioxidant activity in either assay. Other components of the formulations did not interfere with the measurements, indicating that the methods employed can be used to evaluate antioxidant activity in topical formulations.

A simple method to study the activity of natural compounds on the chemiluminescence of neutrophils upon stimulation by immune complexes

INTRODUCTION Neutrophils (PMNs) are the main effector cells involved in the immune response to microorganisms. However, in various noninfectious states, such as autoimmune and immune complex (ICs) diseases, ICs are found to be deposited in various organs, leading to recruitment and activation of PMNs at these sites of deposition. Consequently, reactive oxygen species (ROS) and lysosomal enzymes are extensively released by activated PMNs into the extracellular milieu, leading to host tissue injury. METHODS In the present study, we discuss some experimental conditions of a luminol-enhanced chemiluminescence (LECL) assay to study the effect of natural compounds on the production of ROS by rabbit PMNs stimulated with precipitated ICs. Moreover, we evaluated the activities of quercetin and 7-allyloxycoumarin on this ROS-producing system and their toxicity to PMNs. RESULTS Both compounds had concentration-dependent inhibitory effects on LECL. Quercetin at concentration of 5 micromol/l inhibited 94.5+/-1.0% of LECL, whereas 7-allyloxycoumarin at concentration of 200 micromol/l inhibited 53.8+/-2.4% of LECL. Neither compound was toxic to PMNs under the tested conditions. DISCUSSION The proposed method may be useful for the screening of nontoxic compounds that can modulate ROS production by IC-stimulated PMNs. Special attention should be devoted to natural compounds from higher plants, since their potential as sources of new drugs is still largely unexplored.

Galectin-3 plays a modulatory role in the life span and activation of murine neutrophils during early Toxoplasma gondii infection.

Galectins are beta-galactoside-binding lectins involved in several biological processes and galectin-3 (Gal-3) is related to modulation of immune and inflammatory responses. This study aimed to evaluate the role of Gal-3 in the life span and biological functions of murine neutrophils during in vitro infection by virulent Toxoplasma gondii RH strain. Inflammatory peritoneal neutrophils (Nphi) from C57BL/6 wild-type (WT) and Gal-3 knockout (KO) mice were cultured in the presence or absence of parasites and analyzed for phosphatidylserine (PS) exposure and cell death using Annexin-V and propidium iodide staining, and cell viability by MTT assay. Cell toxicities determined by lactate dehydrogenase (LDH), degranulation by lysozyme release, and cytokine production were measured in Nphi culture supernatants. Phorbol myristate acetate (PMA)- or zymosan-dependent reactive oxygen species (ROS) were measured in Nphi cultures. Our results demonstrated that Gal-3 is involved in the increase of the viable Nphi number and the decrease of PS exposure and cell death following T. gondii infection. We also observed that Gal-3 downmodulates T. gondii-induced Nphi toxicity as well as Nphi degranulation regardless of infection. Furthermore, Gal-3 expression by Nphi was associated with increased levels of IL-10 in the beginning and decreased levels of TNF-alpha later on, regardless of parasite infection, as well as with decreased levels of IL-6 and increased IL-12 levels, following early parasite infection. Our results also showed that Gal-3 suppresses PMA- but not zymosan-induced ROS generation in Nphi following T. gondii infection. In conclusion, Gal-3 plays an important modulatory role by interfering in Nphi life span and activation during early T. gondii infection.

The influence of antibody functional affinity on the effector functions involved in the clearance of circulating immune complexes anti-BSA IgG/BSA.

A systematic study was carried out to investigate the role of antibody functional affinity in the capacity of immune complexes (IC) to activate the complement system and to trigger subsequently the molecular events involved in the handling of IC by providing a clearance mechanism. For this purpose, two populations of polyclonal anti-BSA IgG antibodies of different affinities were prepared, with values of 1.89x10(8) M(-1) and 4.94x10(8) M(-1). First we studied the capacity of IC formed at equivalence with both antibodies to activate the classical and the alternative pathways of human complement and the ability of the complexes to bind to erythrocyte C3b-C4b receptors (CR1; CD35). The data showed that the highest affinity antibodies were more efficient in activating complement by both pathways. However, their binding to erythrocyte CR1 was significantly lower compared to the binding of the lowest affinity IgG. Second we compared these IC in terms of their ability to stimulate the respiratory burst of neutrophils (PMN) and to induce the release of PMN lysosomal enzymes. In general, both of these PMN functions were better stimulated by the IC prepared with the IgG antibodies having a highest affinity, although the effects were variable for different IC concentrations. The suggestion to be drawn from the data is that the antibody affinity has an influence on the formation of the immune complex lattice, modulating its three-dimensional structure and the arrangement of the antibody Fc fragments, interfering with complement activation and access to the neutrophil IgG receptors. The significance of these observations for the understanding of how affinity influences the precise biological mechanism that participates in the fate of IC is discussed.

Study of quercetin-loaded liposomes as potential drug carriers: in vitro evaluation of human complement activation

Liposomes have been employed as potential drug carriers. However, after their in vivo administration, they can be destabilized by proteins of complement system, contributing to the clearance of vesicles from blood circulation. Antioxidant flavonoids such as quercetin have been reported to be beneficial to human health, but their low water solubility and bioavailability limit their enteric administration. Therefore, the development of appropriate flavonoid-carriers could be of great importance to drug therapy. The aim of the present study was to evaluate the activation of human complement system proteins by liposomes composed of soya phosphatidylcholine (SPC) and cholesterol (CHOL) or cholesteryl ethyl ether (CHOL-OET) loaded with quercetin or not. The consumption of complement, via classical (CP) and alternative (AP) pathways, by different vesicles was evaluated using a hemolytic assay and quantitative determination of iC3b and natural antibodies deposited on empty liposomal surfaces by ELISA. The main results showed that empty liposomes composed of large amounts of CHOL consumed more complement components than the others for both CP and AP. Furthermore, replacement of CHOL with CHOL-OET reduced complement consumption via both CP and AP. Incorporation of quercetin did not change CP and AP consumption. Deposition of iC3b, IgG and IgM in vesicles composed of SPC:CHOL-OET at a molar ratio of 1.5:1 was lower compared to the others. Taken together, these observations suggest that liposomes composed of SPC:CHOL-OET at a molar ratio of 1.5:1 are the most appropriate among the vesicles studied herein to be used as a drug carrier system in further investigations.

Superoxide anion production by neutrophils is associated with prevalent clinical manifestations in systemic lupus erythematosus

To determine the relation between neutrophil function and the clinical characteristics of systemic lupus erythematosus (SLE), the superoxide anion (