Sensibilização de hemácias de carneiro e restrição à lise pelo sistema complemento do soro eqüino e de carneiro

The effect of the degree of sheep red cell sensitisation by antibody in the resistance to lysis by sheep and horse complement was analysed. The results showed that, as the level of sensitisation increased, so did the homologous complement activity, eventually leading to one hundred percent of lysis; the same was observed for horse serum, generally considered as not efficient at lysing these cells. Therefore, the homologous and heterologous restriction can be overridden by this treatment. This may be of interest for the comprehension of the mechanisms involved in the modulation of the restriction phenomenon in health an in diseases involving complement in domestic animals and in human, as well as in laboratory diagnosis where hemolytic assays are used for measurement of complement activity and evaluation of presence of antibodies in serum samples.Neste trabalho foi analisado o efeito do grau de sensibilização de hemácias de carneiro por anticorpo, na resistência à lise pelo complemento do soro eqüino e de carneiro. Os resultados mostraram que, à medida que houve um aumento do nível de sensibilização, ocorreu também um aumento da atividade do complemento homólogo, levando a 100% de lise; o mesmo foi observado para o complemento do soro eqüino, geralmente considerado ineficiente na lise destas células. A restrição à lise pode, assim, ser sobrepujada por este tratamento. Estes dados podem ser de interesse para a compreensão dos mecanismos envolvidos na modulação do fenômeno de restrição, na saúde e em doenças envolvendo o sistema complemento em animais domésticos e no homem, bem como em diagnóstico laboratorial, onde são utilizados ensaios hemolíticos para medida de atividade do complemento e pesquisa de anticorpos em amostras de soro

Sensibilização de hemácias de carneiro e restrição à lise pelo sistema complemento do soro eqüino e de carneiro

Neste trabalho foi analisado o efeito do grau de sensibilização de hemácias de carneiro por anticorpo, na resistência à lise pelo complemento do soro eqüino e de carneiro. Os resultados mostraram que, à medida que houve um aumento do nível de sensibilização, ocorreu também um aumento da atividade do complemento homólogo, levando a 100% de lise; o mesmo foi observado para o complemento do soro eqüino, geralmente considerado ineficiente na lise destas células. A restrição à lise pode, assim, ser sobrepujada por este tratamento. Estes dados podem ser de interesse para a compreensão dos mecanismos envolvidos na modulação do fenômeno de restrição, na saúde e em doenças envolvendo o sistema complemento em animais domésticos e no homem, bem como em diagnóstico laboratorial, onde são utilizados ensaios hemolíticos para medida de atividade do complemento e pesquisa de anticorpos em amostras de soro

Inhibitory activity of liposomal flavonoids during oxidative metabolism of human neutrophils upon stimulation with immune complexes and phorbol ester

Context and objective: The massive production of reactive oxygen species by neutrophils during inflammation may cause damage to tissues. Flavonoids act as antioxidants and have anti-inflammatory effects. In this study, liposomes loaded with these compounds were evaluated as potential antioxidant carriers, in attempt to overcome their poor solubility and stability. Materials and methods: Liposomes containing quercetin, myricetin, kaempferol or galangin were prepared by the ethanol injection method and analyzed as inhibitors of immune complex (IC) and phorbol ester-stimulated neutrophil oxidative metabolism by luminol (CLlum) and lucigenin-enhanced (CLluc) chemiluminescence (CL) assays. The mechanisms involved this activity of liposomal flavonoids, such as cytotoxicity and superoxide anion scavenging capacity, and their effect on phagocytosis of ICs were also investigated. Results and discussion: The results showed that the inhibitory effect of liposomal flavonoids on CLlum and CLluc is inversely related to the number of hydroxyl groups in the flavonoid B ring. Moreover, phagocytosis of liposomes by neutrophils does not seem to necessarily promote such activity, as the liposomal flavonoids are also able to reduce CL when the cells are pretreated with cytochalasin B. Under assessed conditions, the antioxidant liposomes are not toxic to the human neutrophils and do not interfere with IC-induced phagocytosis. Conclusion: The studied liposomes can be suitable carriers of flavonoids and be an alternative for the treatment of diseases in which a massive oxidative metabolism of neutrophils is involved.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP, Brazil) [2006/04398-5, 2007/00161-3]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), BrazilConselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq) [306130/2009-2

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.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP), BrazilFundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP, Brazil) [2006/04398-5, 2007/00161-3

In vitro evaluation of the antioxidant activity of liposomal flavonols by the HRP--H(2)O(2)--luminol system

Considering that antioxidant flavonols have been reported to be beneficial to human health, but that their low water solubility and bioavailability limit their administration through systemic route, the development of suitable flavonol-carriers is of great importance for clinical therapeutics. The aim of this study was to prepare liposomes containing flavonols or not and evaluate their antioxidant activity. Vesicles were obtained by ethanol injection method and characterized in terms of entrapment efficiency, size and zeta potential. Inhibitory activity of liposomal flavonols on reactive oxygen species generation was assessed in vitro using luminol--H(2)O(2)--horseradish peroxidase technique. Antioxidant activity of liposomal flavonols is dependent on concentration and chemical structure of active compound. Quercetin and myricetin are the most active flavonols (IC(50) == 0.6--0.9 mu A mu mol/L), followed by kaempferol (IC(50) == 3.0--4.5 mu A mu mol/L) and galangin (IC(50) == 4.0--7.0 mu A mu mol/L). Our results suggest that antioxidant-loaded liposomes may be promising tools for therapy of diseases where oxidative stress is involved.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP, Brazil)[2006/04398-5]Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP, Brazil)[2007/00161-3

Influence of Fc gamma RIIIb polymorphism on its ability to cooperate with Fc gamma RIIa and CR3 in mediating the oxidative burst of human neutrophils

Considering that human neutrophil Fc gamma RIIa and Fc gamma RIIIb receptors interact synergistically with CR3 in triggering neutrophil functional responses, allelic polymorphisms in these receptors might influence such interactions. We assessed whether Fc gamma RIIIb polymorphisms affect Fc gamma R/CR cooperation in mediating the neutrophil oxidative burst (OB), in particular the Fc gamma RIIIb/CR3 cooperation that occurs via lectin-saccharide-like interactions. The OB of human neutrophil antigen (HNA)-1a-, HNA-1b-, and HNA-1a/-1b-neutrophils stimulated with immune complexes, opsonized or not with serum complement, was measured by the luminol-enhanced chemiluminescence assay. Compared with HNA-1a-neutrophils, HNA-1b-neutrophils exhibited reduced Fc gamma R-stimulated OB, but increased Fc gamma R/CR-stimulated OB. It suggests that (i) Fc gamma R and CR cooperate more effectively in HNA-1b-neutrophils, and (ii) the HNA-1b allotype influences the Fc gamma RIIIb cooperation with Fc gamma RIIa, but not with CR3. HNA-1a- and HNA-1b-neutrophils exhibited similar OB responses elicited via CR3 alone or via Fc gamma R/CR-independent pathways. In addition, the level of Fc gamma RIIIb, Fc gamma RIIa, and CR3 expression did not differ significantly among the neutrophil groups studied. Together, these results demonstrate that the HNA-1b allotype influences the functional cooperation between Fc gamma RIIIb and Fc gamma RIIa, and suggest that the difference in the glycosylation pattern between HNA-1a and HNA-1b does not affect the Fc gamma RIIIb cooperation with CR3. (C) 2014 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Incorporation of <i>Baccharis dracunculifolia</i> DC (Asteraceae) leaf extract into phosphatidylcholine-cholesterol liposomes improves its anti-inflammatory effect <i>in vivo</i>

<p>The aerial parts of <i>Baccharis dracunculifolia</i> (BdE) is used in the Brazilian folk medicine to treat inflammatory conditions. Here we examined the ability of free and liposomal BdE to modulate reactive oxygen species generation in human neutrophils <i>in vitro</i> and zymosan-induced acute joint inflammation in Wistar rats. We prepared biocompatible liposomes of soya phosphatidylcholine and cholesterol with low diameter, homogeneous size distribution, and neutral surface charge. Free BdE decreased joint swelling, total leucocyte and neutrophil infiltration, and the synovial levels of tumour necrosis factor-α and interleukins 6 and 1β. Incorporation of BdE into liposomes preserved its capacity to inhibit the neutrophil superoxide anion and total reactive oxygen species generation, and improved its anti-inflammatory effect <i>in vivo</i> by decreasing the effective BdE dose by nearly sixfold. The same liposome type lowered the effective dose of caffeic acid by nearly sixteenfold. Therefore, incorporation of BdE into phosphatidylcholine-cholesterol liposomes improves its anti-inflammatory effect.</p