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Eur J Pharmacol ; 809: 52-63, 2017 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-28501577


Probucol 4,4'- (Isopropylidenedithio)bis(2,6-di-tert-butylphenol) is a synthetic molecule clinically used for prevention and treatment of hypercholesterolemia and atherosclerosis. Recent studies have shown that the beneficial effects of probucol mainly derive from its anti-inflammatory and antioxidant properties. Gram-negative bacteria are common infectious agents and their wall components, e.g. lipopolysaccharide (LPS), are important elicitors of inflammation. LPS is sensed by tissue resident cells and it triggers a Toll-like receptor 4/MyD88-dependent signaling cascade resulting in endothelial activation, leukocyte recruitment and nociception. Therefore the present study aimed to investigate the anti-inflammatory and analgesic effects of probucol in models of LPS-induced acute inflammation. Probucol at 0.3-30mg/kg was administrated to male Swiss mice per oral 1h before intraplantar or intraperitoneal lipopolysaccharide stimulus. Probucol at 3mg/kg reduced lipopolysaccharide-induced mechanical and thermal hyperalgesia. These effects were accompanied by reduced leukocyte influx and cytokine production in both paw skin and peritoneum exudate. Unexpectedly, probucol did not alter lipopolysaccharide-induced tissue oxidative stress at anti-inflammatory /analgesic dose. On the other hand, probucol inhibited lipopolysaccharide-induced nuclear factor kappa B (NF-кB) activation in paw tissue as well as NF-кB activity in cultured macrophages in vitro, reinforcing the inhibitory effect of probucol over the NF-кB signaling pathway. In this sense, we propose that probucol acts on resident immune cells, such as macrophages, targeting the NF-кB pathway. As a result, it prevents the amplification and persistence of the inflammatory response by attenuating NF-кB-dependent cytokine production and leukocyte recruitment explaining its analgesic effects as well.

Citocinas/biossíntese , Hiperalgesia/tratamento farmacológico , Leucócitos/efeitos dos fármacos , Leucócitos/imunologia , Lipopolissacarídeos/farmacologia , NF-kappa B/metabolismo , Probucol/farmacologia , Animais , Hiperalgesia/complicações , Hiperalgesia/imunologia , Hiperalgesia/metabolismo , Inflamação/complicações , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Masculino , Camundongos , Cavidade Peritoneal , Probucol/uso terapêutico , Células RAW 264.7
BMC Immunol ; 17(1): 22, 2016 07 04.
Artigo em Inglês | MEDLINE | ID: mdl-27377926


BACKGROUND: Mast cells are hematopoietically derived cells that play a role in inflammatory processes such as allergy, as well as in the immune response against pathogens by the selective and rapid release of preformed and lipid mediators, and the delayed release of cytokines. The native homotetrameric lectin ArtinM, a D-mannose binding lectin purified from Artocarpus heterophyllus seeds, is one of several lectins that are able to activate mast cells. Besides activating mast cells, ArtinM has been shown to affect several biological responses, including immunomodulation and acceleration of wound healing. Because of the potential pharmacological application of ArtinM, a recombinant ArtinM (rArtinM) was produced in Escherichia coli. The current study evaluated the ability of rArtinM to induce mast cell degranulation and activation. RESULTS: The glycan binding specificity of rArtinM was similar to that of jArtinM. rArtinM, via its CRD, was able to degranulate, releasing ß-hexosaminidase and TNF-α, and to promote morphological changes on the mast cell surface. Moreover, rArtinM induced the release of the newly-synthesized mediator, IL-4. rArtinM does not have a co-stimulatory effect on the FcεRI degranulation via. The IgE-dependent mast cell activation triggered by rArtinM seems to be dependent on NFkB activation. CONCLUSIONS: The lectin rArtinM has the ability to activate and degranulate mast cells via their CRDs. The present study indicates that rArtinM is a suitable substitute for the native form, jArtinM, and that rArtinM may serve as an important and reliable pharmacological agent.

Mastócitos/imunologia , Lectinas de Plantas/imunologia , Proteínas Recombinantes/imunologia , Animais , Artocarpus/imunologia , Degranulação Celular , Linhagem Celular , Clonagem Molecular , Escherichia coli/genética , Histamina/metabolismo , Imunoglobulina E/metabolismo , Imunomodulação , Interleucina-4/metabolismo , Manose/metabolismo , NF-kappa B/metabolismo , Lectinas de Plantas/isolamento & purificação , Ligação Proteica , Ratos , Proteínas Recombinantes/isolamento & purificação , beta-N-Acetil-Hexosaminidases/metabolismo
Int J Biol Macromol ; 82: 22-30, 2016 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-26433176


Recent advances in glycobiology have revealed the essential role of lectins in deciphering the glycocodes at the cell surface to generate important biological signaling responses. ArtinM, a d-mannose-binding lectin isolated from the seeds of jackfruit (Artocarpus heterophyllus), is composed of 16 kDa subunits that are associated to form a homotetramer. Native ArtinM (n-ArtinM) exerts immunomodulatory and regenerative effects, but the potential pharmaceutical applicability of the lectin is highly limited by the fact that its production is expensive, laborious, and impossible to be scaled up. This led us to characterize a recombinant form of the lectin obtained by expression in Saccharomyces cerevisiae (y-ArtinM). In the present study, we demonstrated that y-ArtinM is similar to n-ArtinM in subunit arrangement, oligomerization and carbohydrate binding specificity. We showed that y-ArtinM can exert n-ArtinM biological activities such as erythrocyte agglutination, stimulation of neutrophil migration and degranulation, mast cell degranulation, and induction of interleukin-12 and interleukin-10 production by macrophages. In summary, the expression of ArtinM in yeast resulted in successful production of an active, recombinant form of ArtinM that is potentially useful for pharmaceutical application.

Carboidratos/química , Lectinas de Ligação a Manose/química , Estrutura Molecular , Proteínas Recombinantes , Animais , Degranulação Celular/efeitos dos fármacos , Degranulação Celular/imunologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/imunologia , Citocinas/biossíntese , Hemaglutinação , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Macrófagos/metabolismo , Masculino , Lectinas de Ligação a Manose/metabolismo , Lectinas de Ligação a Manose/farmacologia , Mastócitos/efeitos dos fármacos , Mastócitos/imunologia , Mastócitos/metabolismo , Camundongos , Neutrófilos/efeitos dos fármacos , Neutrófilos/imunologia , Neutrófilos/metabolismo , Polissacarídeos/química , Ligação Proteica , Receptor 2 Toll-Like , Leveduras/genética , Leveduras/metabolismo
PLoS One ; 10(12): e0144507, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26659253


Toxoplasma gondii is an obligate intracellular protozoan parasite found worldwide that is able to chronically infect almost all vertebrate species, especially birds and mammalians. Chitinases are essential to various biological processes, and some pathogens rely on chitinases for successful parasitization. Here, we purified and characterized a chitinase from T. gondii. The enzyme, provisionally named Tg_chitinase, has a molecular mass of 13.7 kDa and exhibits a Km of 0.34 mM and a Vmax of 2.64. The optimal environmental conditions for enzymatic function were at pH 4.0 and 50 °C. Tg_chitinase was immunolocalized in the cytoplasm of highly virulent T. gondii RH strain tachyzoites, mainly at the apical extremity. Tg_chitinase induced macrophage activation as manifested by the production of high levels of pro-inflammatory cytokines, a pathogenic hallmark of T. gondii infection. In conclusion, to our knowledge, we describe for the first time a chitinase of T. gondii tachyzoites and provide evidence that this enzyme might influence the pathogenesis of T. gondii infection.

Quitinases/imunologia , Ativação de Macrófagos/imunologia , Macrófagos Peritoneais/imunologia , Proteínas de Protozoários/imunologia , Toxoplasma/imunologia , Sequência de Aminoácidos , Animais , Quitinases/genética , Quitinases/metabolismo , Cromatografia Líquida , Citocinas/imunologia , Citocinas/metabolismo , Citoplasma/enzimologia , Interações Hospedeiro-Parasita/imunologia , Concentração de Íons de Hidrogênio , Mediadores da Inflamação/imunologia , Mediadores da Inflamação/metabolismo , Cinética , Macrófagos Peritoneais/metabolismo , Macrófagos Peritoneais/parasitologia , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Microscopia Confocal , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Proteínas de Protozoários/química , Espectrometria de Massas em Tandem , Temperatura Ambiente , Toxoplasma/enzimologia , Toxoplasma/fisiologia
Cell Tissue Res ; 357(3): 719-30, 2014 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-24842046


ArtinM is a D-mannose-binding lectin extracted from Artocarpus heterophyllus that promotes interleukin-12 production by macrophages and dendritic cells. This property is considered responsible for T helper 1 immunity induced in vivo after ArtinM administration. In this study, we investigated the effect of native (jArtinM) and recombinant (rArtinM) forms of lectin on murine spleen cells and isolated T lymphocytes. We found that ArtinM binds to the surface of spleen cells. This interaction, which was blocked by D-mannose, induced cell activation, as manifested by increased mitochondrial activity, interleukin-2 production, and cell proliferation. We verified that a 30-times higher concentration of rArtinM was required to trigger optimal activation of spleen cells compared with that needed with jArtinM, although these proteins have identical sugar recognition properties and use the same signaling molecules to trigger cell activation. Because the distinction between native and recombinant is restricted to their tertiary structure (tetrameric and monomeric, respectively), we postulated that the multi-valence of jArtinM accounts for its superiority in promoting clustering of cell surface glycoreceptors and activation. The jArtinM and rArtinM activation effect exerted on spleen cells was reproduced on purified CD4(+) T cells. Our results suggest that ArtinM interaction with T cells leads to responses that may act in concert with the interleukin-12 produced by antigen-presenting cells to modulate immunity toward the T helper 1 axis. Further studies are necessary to dissect ArtinM/T-cell interactions to more fully understand the immunomodulation induced by carbohydrate recognition.

Fatores Imunológicos/farmacologia , Lectinas de Ligação a Manose/farmacologia , Baço/citologia , Animais , Artocarpus/química , Complexo CD3/metabolismo , Linfócitos T CD4-Positivos/efeitos dos fármacos , Linfócitos T CD4-Positivos/metabolismo , Metabolismo dos Carboidratos/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Interleucina-2/biossíntese , Ativação Linfocitária/efeitos dos fármacos , Masculino , Lectinas de Ligação a Manose/metabolismo , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Receptor 2 Toll-Like/metabolismo , Receptor 4 Toll-Like/metabolismo