Laboratorio Of Farmacologia Aplicada

Rio de Janeiro, Brazil

Laboratorio Of Farmacologia Aplicada

Rio de Janeiro, Brazil

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Amaral J.J.,Instituto Oswaldo Cruz | Amaral J.J.,Brazilian National Institute of Technology | Antunes L.M.,University of British Columbia | de Macedo C.S.,Instituto Oswaldo Cruz | And 11 more authors.
PLoS Neglected Tropical Diseases | Year: 2013

Despite considerable efforts over the last decades, our understanding of leprosy pathogenesis remains limited. The complex interplay between pathogens and hosts has profound effects on host metabolism. To explore the metabolic perturbations associated with leprosy, we analyzed the serum metabolome of leprosy patients. Samples collected from lepromatous and tuberculoid patients before and immediately after the conclusion of multidrug therapy (MDT) were subjected to high-throughput metabolic profiling. Our results show marked metabolic alterations during leprosy that subside at the conclusion of MDT. Pathways showing the highest modulation were related to polyunsaturated fatty acid (PUFA) metabolism, with emphasis on anti-inflammatory, pro-resolving omega-3 fatty acids. These results were confirmed by eicosanoid measurements through enzyme-linked immunoassays. Corroborating the repertoire of metabolites altered in sera, metabonomic analysis of skin specimens revealed alterations in the levels of lipids derived from lipase activity, including PUFAs, suggesting a high lipid turnover in highly-infected lesions. Our data suggest that omega-6 and omega-3, PUFA-derived, pro-resolving lipid mediators contribute to reduced tissue damage irrespectively of pathogen burden during leprosy disease. Our results demonstrate the utility of a comprehensive metabonomic approach for identifying potential contributors to disease pathology that may facilitate the development of more targeted treatments for leprosy and other inflammatory diseases. © 2013 Amaral et al.


PubMed | Instituto Oswaldo Cruz, Laboratorio Of Farmacologia Aplicada and Osmania University
Type: | Journal: Pharmacological research | Year: 2016

Activation of toll-like receptors (TLRs) by pathogen-associated molecular patterns (PAMPs) triggers an innate immune response, via cytokine production and inflammasome activation. Herein, we have investigated the modulatory effect of the natural limonoid gedunin on TLR activation in vitro and in vivo. Intraperitoneal (i.p.) pre- and post-treatments of C57BL/6 mouse with gedunin impaired the influx of mononuclear cells, eosinophils and neutrophils, as well as the production of tumor necrosis factor (TNF)-, interleukin (IL)-6 and nitric oxide (NO), triggered by lipopolysaccharide (LPS) in mouse pleura. Accordingly, in vitro post-treatment of immortalized murine macrophages with gedunin also impaired LPS-induced production of such mediators. Gedunin diminished LPS-induced expression of the nucleotide-binding domain and leucine-rich repeat protein-3 (NLRP3) on pleural leukocytes in vivo and in immortalized macrophages in vitro. In line with this, gedunin inhibited LPS-induced caspase-1 activation and the production of IL-1 in vivo and in vitro. In addition, gedunin treatment triggered the generation of the anti-inflammatory factors IL-10 and heme oxigenase-1 (HO-1) at resting conditions or upon stimulation. We also demonstrate that gedunin effect is not restricted to TLR4-mediated response, since this compound diminished TNF-, IL-6, NO, NLRP3 and IL-1, as well as enhanced IL-10 and HO-1, by macrophages stimulated with the TLR2 and TLR3 agonists, palmitoyl-3-Cys-Ser-(Lys)4 (PAM3) and polyriboinosinic:polyribocytidylic acid (POLY I:C), in vitro. In silico modeling studies revealed that gedunin efficiently docked into caspase-1, TLR2, TLR3 and to the myeloid differentiation protein-2 (MD-2) component of TLR4. Overall, our data demonstrate that gedunin modulates TLR4, TLR3 and TLR2-mediated responses and reveal new molecular targets for this compound.


PubMed | Laboratorio Of Farmacologia Aplicada, University of Sao Paulo and State University Londrina
Type: Journal Article | Journal: Molecules (Basel, Switzerland) | Year: 2015

Gedunin, a natural limonoid from Meliaceae species, has been previously described as an antiinflammatory compound in experimental models of allergic inflammation. Here, we report the antiinflammatory and antinociceptive effects of gedunin in an acute model of articular inflammation induced by zymosan (500 g/cavity; intra-articular) in C57BL/6 mice. Intraperitoneal (i.p.) pretreatment with gedunin (0.005-5 mg/kg) impaired zymosan-induced edema formation, neutrophil accumulation and hypernociception in mouse knee joints, due to decreased expression of preproET-1 mRNA and production of LTB4, PGE2, TNF- and IL-6. Mouse post-treatment with gedunin (0.05 mg/kg; i.p.) 1 and 6 h after stimulation also impaired articular inflammation, by reverting edema formation, neutrophil accumulation and the production of lipid mediators, cytokines and endothelin. In addition, gedunin directly modulated the functions of neutrophils and macrophages in vitro. The pre-incubation of neutrophil with gedunin (100 M) impaired shape change, adhesion to endothelial cells, chemotaxis and lipid body formation triggered by different stimuli. Macrophage pretreatment with gedunin impaired intracellular calcium mobilization, nitric oxide production, inducible nitric oxide synthase expression and induced the expression of the antiinflammatory chaperone heat shock protein 70. Our results demonstrate that gedunin presents remarkable antiinflammatory and anti-nociceptive effects on zymosan-induced inflamed knee joints, modulating different cell populations.


Ferraris F.K.,Laboratorio Of Farmacologia Aplicada | Ferraris F.K.,Oswaldo Cruz Institute | Rodrigues R.,Laboratorio Of Quimica Of Produtos Naturais | Rodrigues R.,State University of Norte Fluminense | And 4 more authors.
International Immunopharmacology | Year: 2011

We have previously described the anti-allergic activities of a pooled fraction of tetranortriterpenoids (TNTPs) containing 6α-acetoxygedunin, 7-deacetoxy-7-oxogedunin, andirobin and methyl angolensate isolated from the seeds of Carapa guianensis. In the present study, we performed in vitro studies in order to elucidate the mechanisms by which TNTPs present their anti-allergic effects and to identify the bioactive compound(s) present in such fraction. Here, we show that in vitro incubation of eosinophils with the pooled TNTP fraction, as well as with each one of the five isolated tetranortriterpenoids, impaired the adhesion of eosinophils to tumor necrosis factor-α (TNF-α)-primed tEND.1 endothelial cells. Furthermore, the individual or pooled TNTPs impaired CCL11/eotaxin-mediated chemotaxis. By contrast, pooled TNTPs failed to inhibit adhesion and chemotaxis of T lymphocytes. However, TNTPs were able to impair anti-CD3 monoclonal antibody-induced T cell proliferation and the expression of CD25 and CD69. These data suggest that TNTPs prevent T cell activation. Pretreatment of splenocytes with the pooled TNTP fraction, as well as with each one of the five isolated TNTPs, inhibited ovalbumin (OVA)-induced in vitro production of interleukin-2, chemokine (C-C motif) ligand 11 (CCL11) and regulated on activation normal T cell expressed and secreted (RANTES, also known as CCL5). TNTPs (except 6α-acetoxygedunin) also impaired nuclear factor-κB (NFκB) nuclear translocation in OVA-challenged splenocytes. Taken together, these results demonstrate that the anti-allergic effects of TNTPs isolated from C. guianensis might rely on their ability to inhibit eosinophil migration, as well as the activation of T lymphocytes, which is shared by the five isolated TNTPs. © 2010 Elsevier B.V. All rights reserved.


Farias J.A.C.,Federal University of Alagoas | Ferro J.N.S.,Federal University of Alagoas | Silva J.P.,Federal University of Alagoas | Agra I.K.R.,Federal University of Alagoas | And 7 more authors.
Inflammation | Year: 2012

The present study was carried out to investigate the anti-inflammatory effect of the hexane extract of the leaves from Clusia nemorosa G. Mey, called HECn, using carrageenan-induced mice pleurisy and cotton pellet-induced mice granuloma. Additionally, the ability of HECn to affect both neutrophil migration as viability was investigated by use of the Boyden chamber assay and flow cytometry, respectively. The HECn significantly inhibited exudation, total leukocytes and neutrophils influx, as well as TNFα levels in carrageenan-induced pleurisy. However, the extract not suppressed the granulomatous tissue formation in the cotton pellet-induced granuloma test. Experiments performed in vitro revealed that HECn on human neutrophils inhibited a dose-dependent manner the CXCL1-induced neutrophil chemotaxis. Furthermore, HECn also inhibited the chemoattraction of human neutrophils induced by formyl-methionyl-leucyl-phenylalanine (fMLP), leukotriene B4 (LTB4) and platelet activating factor (PAF) in a Boyden chamber. However, this same treatment not was able to induce apoptosis. The results obtained in this study showed that the extract from leaves of C. nemorosa possess a potent inhibitory activity in acute model of inflammation, being the effects mediated, in part, by inhibition of neutrophil responsiveness. These results indicate that C. nemorosa could be a good source for anti-inflammatory compounds. © 2011 Springer Science+Business Media, LLC.


Ferraris F.K.,Laboratorio Of Farmacologia Aplicada | Moret K.H.,Laboratorio Of Farmacologia Aplicada | Figueiredo A.B.C.,Laboratorio Of Farmacologia Aplicada | Penido C.,Laboratorio Of Farmacologia Aplicada | Henriques M.D.G.M.O.,Laboratorio Of Farmacologia Aplicada
International Immunopharmacology | Year: 2012

T lymphocytes are critical cells involved in allergy. Here, we report that the natural tetranortriterpenoid gedunin impaired allergic responses primarily by modulating T lymphocyte functions. The intraperitoneal (i.p.) administration of gedunin inhibited pleural leukocyte accumulation triggered by intra-pleural (i.pl.) challenge with ovalbumin (OVA) in previously sensitized C57BL/6 mice; this inhibition was primarily due to the impairment of eosinophil and T lymphocyte influx. Likewise, i.pl. pre-treatment with gedunin inhibited eosinophil and T lymphocyte migration into mouse lungs 24 h after OVA intra-nasal (i.n.) instillation. Pre-treatment with gedunin diminished the levels of CCL2, CCL3, CCL5, CCL11, Interleukin-5 and leukotriene B4 at the allergic site. In vitro pre-treatment with gedunin failed to inhibit T lymphocyte adhesion and chemotaxis towards pleural washes recovered from OVA-challenged mice, suggesting that gedunin inhibits T lymphocyte migration in vivo via the inhibition of chemotactic mediators in situ. In vivo pre-treatment with gedunin reduced the numbers of CD69+ and CD25+ T lymphocytes in the pleura and CD25+ cells in the thoracic lymph nodes 24 h after OVA i.pl. challenge. In accordance, in vitro treatment of T lymphocytes with gedunin inhibited α-CD3 mAb-induced expression of CD69 and CD25, proliferation, Interleukin-2 production and nuclear translocation of NFκB and NFAT. Notably, post-treatment of mice with gedunin reverted OVA-induced lung allergic inflammation by decreasing the T lymphocyte and eosinophil counts and the levels of eosinophilotactic mediators in bronchoalveolar lavage fluid. Our results demonstrate a remarkable anti-allergic effect of gedunin due to its capability to modulate T cell activation and trafficking into the airways. © 2012 Elsevier B.V.


De Souza Costa M.F.,Laboratorio Of Farmacologia Aplicada | De Souza-Martins R.,Laboratorio Of Farmacologia Aplicada | De Souza M.C.,Laboratorio Of Farmacologia Aplicada | Benjamim C.F.,Federal University of Rio de Janeiro | And 6 more authors.
Journal of Leukocyte Biology | Year: 2010

Herein, we investigated the involvement of the 5-LO-derived lipid mediator LTB4 in γδ T cell migration. When injected into the i.pl. space of C57BL/6 mice, LTB4 triggered γδ T lymphocyte mobilization in vivo, a phenomenon also observed in in vitro chemotaxis assays. The i.pl. injection of Escherichia coli endotoxin (LPS) triggered increased levels of LTB4 in pleural cavities. The in vivo inhibition of LTB4 biosynthesis by the 5-LO inhibitor zileuton or the FLAP inhibitor MK886 attenuated LPS-induced γδ T cell accumulation into pleural cavities. Accordingly, 5-LO KO mice failed to recruit γδ T cells into the inflammatory site after i.pl. LPS. Antagonists of the high-affinity LTB 4 receptor BLT1, CP105,696, and LY292476 also attenuated LPS-induced γδ T cell accumulation in pleural cavities as well as in vitro chemotaxis toward pleural washes obtained from LPS-simulated mice. LTB 4/BLT1 also accounted for γδ T cell migration induced by i.pl. administration of Mycobacterium bovis BCG or antigen in sensitized mice. BLT1 was expressed on naïve, resident as well as LPS-recruited γδ T cells. Isolated γδ T cells were found to undergo F-actin cytoskeleton reorganization when incubated with LTB4 in vitro, confirming that γδ T lymphocytes can respond directly to LTB4. In addition to its direct effect on γδ T cells, LTB4 triggered their accumulation indirectly, via modulation of CCL2 production in mouse pleural cavities. These data show that γδ T cell migration into the pleural cavity of mice during diverse inflammatory responses is dependent on LTB4/BLT1. © Society for Leukocyte Biology.


PubMed | Laboratorio Of Farmacologia Aplicada
Type: Journal Article | Journal: International immunopharmacology | Year: 2012

T lymphocytes are critical cells involved in allergy. Here, we report that the natural tetranortriterpenoid gedunin impaired allergic responses primarily by modulating T lymphocyte functions. The intraperitoneal (i.p.) administration of gedunin inhibited pleural leukocyte accumulation triggered by intra-pleural (i.pl.) challenge with ovalbumin (OVA) in previously sensitized C57BL/6 mice; this inhibition was primarily due to the impairment of eosinophil and T lymphocyte influx. Likewise, i.pl. pre-treatment with gedunin inhibited eosinophil and T lymphocyte migration into mouse lungs 24 h after OVA intra-nasal (i.n.) instillation. Pre-treatment with gedunin diminished the levels of CCL2, CCL3, CCL5, CCL11, Interleukin-5 and leukotriene B(4) at the allergic site. In vitro pre-treatment with gedunin failed to inhibit T lymphocyte adhesion and chemotaxis towards pleural washes recovered from OVA-challenged mice, suggesting that gedunin inhibits T lymphocyte migration in vivo via the inhibition of chemotactic mediators in situ. In vivo pre-treatment with gedunin reduced the numbers of CD69(+) and CD25(+) T lymphocytes in the pleura and CD25(+) cells in the thoracic lymph nodes 24 h after OVA i.pl. challenge. In accordance, in vitro treatment of T lymphocytes with gedunin inhibited -CD3 mAb-induced expression of CD69 and CD25, proliferation, Interleukin-2 production and nuclear translocation of NFB and NFAT. Notably, post-treatment of mice with gedunin reverted OVA-induced lung allergic inflammation by decreasing the T lymphocyte and eosinophil counts and the levels of eosinophilotactic mediators in bronchoalveolar lavage fluid. Our results demonstrate a remarkable anti-allergic effect of gedunin due to its capability to modulate T cell activation and trafficking into the airways.


PubMed | Federal University of Rio de Janeiro, Laboratorio Of Imunologia, Laboratorio Of Farmacologia Aplicada and Instituto Oswaldo Cruz
Type: | Journal: BMC immunology | Year: 2015

Lung inflammation is a major consequence of the systemic inflammatory response caused by severe sepsis. Increased migration of T lymphocytes into the lungs has been previously demonstrated during experimental sepsis; however, the involvement of the T cell subtype V4 has not been previously described.Severe sepsis was induced by cecal ligation and puncture (CLP; 9 punctures, 21G needle) in male C57BL/6 mice. and V4 T lymphocyte depletion was performed by 3A10 and UC3-10A6 mAb i.p. administration, respectively. Lung infiltrating T lymphocytes, IL-17 production and mortality rate were evaluated.Severe sepsis induced by CLP in C57BL/6 mice led to an intense lung inflammatory response, marked by the accumulation of T lymphocytes (comprising the V4 subtype). T lymphocytes present in the lungs of CLP mice were likely to be originated from peripheral lymphoid organs and migrated towards CCL2, CCL3 and CCL5, which were highly produced in response to CLP-induced sepsis. Increased expression of CD25 by V4 T lymphocytes was observed in spleen earlier than that by T cells, suggesting the early activation of V4 T cells. The V4 T lymphocyte subset predominated among the IL-17(+) cell populations present in the lungs of CLP mice (unlike V1 and T lymphocytes) and was strongly biased toward IL-17 rather than toward IFN- production. Accordingly, the in vivo administration of anti-V4 mAb abrogated CLP-induced IL-17 production in mouse lungs. Furthermore, anti-V4 mAb treatment accelerated mortality rate in severe septic mice, demonstrating that V4 T lymphocyte play a beneficial role in host defense.Overall, our findings provide evidence that early-activated V4 T lymphocytes are the main responsible cells for IL-17 production in inflamed lungs during the course of sepsis and delay mortality of septic mice.


PubMed | Laboratorio Of Farmacologia Aplicada
Type: Journal Article | Journal: European journal of immunology | Year: 2012

Herein, we provide evidence that during allergic inflammation, CCL25 induces the selective migration of IL-17(+) T cells mediated by (4) (7) integrin. Intrapleural injection of CCL25 into ovalbumin (OVA)-immunized C57BL/6 mice triggered the accumulation of T lymphocytes expressing CCR9 (CCL25 receptor) and (4) (7) integrin in the pleura, but failed to attract T lymphocytes. CCL25 attracted CCR6(+) T cells producing IL-17 (but not IFN- or IL-4). OVA challenge triggered increased production of CCL25 followed by the accumulation of CCR9(+) , (4) (7) (+) , and CCR6(+) /IL-17(+) T cells into the pleural cavities of OVA-immunized mice, which was inhibited by the in vivo neutralization of CCL25. The in vivo blockade of (4) (7) integrin also inhibited the migration of IL-17(+) T lymphocytes (but not of T lymphocytes) into mouse pleura after OVA challenge, suggesting that the CCL25/(4) (7) integrin pathway is selective for T cells. In addition, (4) (7) integrin blockade impaired the in vitro transmigration of T cells across endothelium (which expresses (4) (7) ligands VCAM-1 and MadCAM-1), which was induced by CCL25 and by cell-free pleural washes recovered from OVA-challenged mice. Our results reveal that during an allergic reaction, CCL25 drives IL-17(+) T-cell mobilization to inflamed tissue via (4) (7) integrin and modulates IL-17 levels.

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