Maria das Graças Henriques

The anti-allergic activity of the acetate fraction of Schinus terebinthifolius leaves in IgE induced mice paw edema and pleurisy

Schinus is a genus of the Anacardiaceae family and contains Schinus terebinthifolius, the Brazilian pepper tree that is widely used in folk medicine. We investigate the anti-allergic activity of the ethyl acetate fraction of S. terebinthifolius Raddi (ST fraction). HPLC analysis reveled that gallic acid, methyl gallate and 1,2,3,4,6-pentagalloylglucose are the major aromatic components of the fraction. Oral pre-treatment with the ST fraction (100 mg/kg) significantly inhibited paw edema induced by compound 48/80 (100 ng/paw) and to a lesser extent, the allergic paw edema (OVA, 3 microg/paw). The ST fraction (100 and 200 mg/kg) also inhibited the edema induced by histamine (100 microg/paw), preventing mast cell degranulation and, consequently, histamine release in Wistar rat peritoneal mast cells induced by C 48/80 (5 microg/mL). This histamine inhibition was also observed after mast cell pre-treatment with both methyl gallate and 1,2,3,4,6-pentagalloylglucose (100 microg/mL), the isolated compounds from the ethyl acetate fraction. Pre-treatment with the ST fraction (100 mg/kg) significantly inhibited total leukocyte and eosinophil accumulation in pleural cavities 24 h after the intrathoracic injection of OVA (12.5 microg/cavity). This effect was related to the inhibition of CCL11/eotaxin and CCL5/RANTES in pleural lavage fluid. Pre-treatment with this fraction (100 mg/kg) failed to reduce the cell influx that was observed after LPS-injection into pleural cavity (250 ng/cavity). These findings demonstrate the anti-allergic effect of the ST fraction, which includes the inhibition of edema formation and histamine release caused by mast cell degranulation and eosinophil influx into the pleural cavity probably reflected by the decreased levels of chemokines in recovered pleural lavage fluid.

Immunomodulating and antiviral activities of Uncaria tomentosa on human monocytes infected with Dengue Virus-2

Uncaria tomentosa (Willd.) DC., a large woody vine native to the Amazon and Central American rainforests has been used medicinally by indigenous peoples since ancient times and has scientifically proven immunomodulating, anti-inflammatory, cytotoxic and antioxidant activities. Several inflammatory mediators that are implicated in vascular permeability and shock are produced after Dengue Virus (DENV) infection by monocytes, the primary targets for virus replication. Here we assessed the immunoregulatory and antiviral activities from U. tomentosa-derived samples, which were tested in an in vitro DENV infection model. DENV-2 infected human monocytes were incubated with U. tomentosa hydro-alcoholic extract or either its pentacyclic oxindole alkaloid-enriched or non-alkaloid fractions. The antiviral activity was determined by viral antigen (DENV-Ag) detection in monocytes by flow cytometry. Our results demonstrated an in vitro inhibitory activity by both extract and alkaloidal fraction, reducing DENV-Ag+ cell rates in treated monocytes. A multiple microbead immunoassay was applied for cytokine determination (TNF-alpha, IFN-alpha, IL-6 and IL-10) in infected monocyte culture supernatants. The alkaloidal fraction induced a strong immunomodulation: TNF-alpha and IFN-alpha levels were significantly decreased and there was a tendency towards IL-10 modulation. We conclude that the alkaloidal fraction was the most effective in reducing monocyte infection rates and cytokine levels. The antiviral and immunomodulating in vitro effects from U. tomentosa pentacyclic oxindole alkaloids displayed novel properties regarding therapeutic procedures in Dengue Fever and might be further investigated as a promising candidate for clinical application.

Targeting endothelin ETA and ETB receptors inhibits antigen-induced neutrophil migration and mechanical hypernociception in mice

Endothelin may contribute to the development of inflammatory events such as leukocyte recruitment and nociception. Herein, we investigated whether endothelin-mediated mechanical hypernociception (decreased nociceptive threshold, evaluated by electronic pressure-meter) and neutrophil migration (myeloperoxidase activity) are inter-dependent in antigen challenge-induced Th1-driven hind-paw inflammation. In antigen challenge-induced inflammation, endothelin (ET) ETA and ETB receptor antagonism inhibited both hypernociception and neutrophil migration. Interestingly, ET-1 peptide-induced hypernociception was not altered by inhibiting neutrophil migration or endothelin ETB receptor antagonism, but rather by endothelin ETA receptor antagonism. Furthermore, endothelin ETA, but not ETB, receptor antagonism inhibited antigen-induced PGE2 production, whereas either selective or combined blockade of endothelin ETA and/or ETB receptors reduced hypernociception and neutrophil recruitment caused by antigen challenge. Concluding, this study advances knowledge into the role for endothelin in inflammatory mechanisms and further supports the potential of endothelin receptor antagonists in controlling inflammation.

Synthesis and antitubercular activity of 7-chloro-4-quinolinylhydrazones derivatives

A series of twenty-one 7-chloro-4-quinolinylhydrazones (3a-u) have been synthesized and evaluated for their in vitro antibacterial activity against Mycobacterium tuberculosis H(37)Rv. The compounds 3f, 3i and 3o were non-cytotoxic and exhibited an important minimum inhibitory concentration (MIC) activity (2.5 microg/mL), which can be compared with that of the first line drugs, ethambutol (3.12 microg/mL) and rifampicin (2.0 microg/mL). These results can be considered an important start point for the rational design of new leads for anti-TB compounds.

Endothelins modulate inflammatory reaction in zymosan-induced arthritis: participation of LTB4, TNF-α, and CXCL-1

Endothelins (ETs) are involved in inflammatory events, including pain, fever, edema, and cell migration. ET‐1 levels are increased in plasma and synovial membrane of rheumatoid arthritis (RA) patients, but the evidence that ETs participate in RA physiopathology is limited. The present study investigated the involvement of ETs in neutrophil accumulation and edema formation in the murine model of zymosan‐induced arthritis. Intra‐articular (i.a.) administration of selective ETA or ETB receptor antagonists (BQ‐123 and BQ‐788, respectively; 15 pmol/cavity) prior to i.a. zymosan injection (500 μg/cavity) markedly reduced knee‐joint edema formation and neutrophil influx to the synovial cavity 6 h and 24 h after stimulation. Histological analysis showed that ETA or ETB receptor blockade suppressed zymosan‐induced neutrophil accumulation in articular tissue at 6 h. Likewise, dual blockade of ETA/ETB with bosentan (10 mg/kg, i.v.) also reduced edema formation and neutrophil counts 6 h after zymosan stimulation. Pretreatment with BQ‐123 or BQ‐788 (i.a.; 15 pmol/cavity) also decreased zymosan‐induced TNF‐α production within 6 h, keratinocyte‐derived chemokine/CXCL1 production within 24 h, and leukotriene B4 at both time‐points. Consistent with the demonstration that ET receptor antagonists inhibit zymosan‐induced inflammation, i.a. injection of ET‐1 (1–30 pmol/cavity) or sarafotoxin S6c (0.1–30 pmol/cavity) also triggered edema formation and neutrophil accumulation within 6 h. Moreover, knee‐joint synovial tissue expressed ETA and ETB receptors. These findings suggest that endogenous ETs contribute to knee‐joint inflammation, acting through ETA and ETB receptors and modulating edema formation, neutrophil recruitment, and production of inflammatory mediators.

Effects of endothelin ETA receptor antagonism on granulocyte and lymphocyte accumulation in LPS‐induced inflammation

Endothelin peptides play active roles in different aspects of inflammation. This study investigates the contribution of endogenous endothelins to lipopolysaccharide (LPS) pulmonary inflammation by assessing the influence of ETA receptor antagonism on leukocyte accumulation, granulocyte adhesion molecule expression, and chemokine/cytokine modulation. Local pretreatment with BQ‐123 or A‐127722 (150 pmol), two selective and chemically unrelated endothelin ETA receptor antagonists, inhibits neutrophil and eosinophil accumulation in LPS‐induced pleurisy at 24 h but not neutrophil migration at 4 h. The effect of endothelin antagonism on neutrophil accumulation at 24 h was concomitant with inhibition of eosinophil and CD4 and CD8 T lymphocyte influx. It is surprising that the ETA receptor blockade did not inhibit the accumulation of γδ T lymphocytes, cells that are important for granulocyte recruitment in this model. Blockade of ETA receptors did not influence the expression of adhesion molecules (CD11b, CD49d) on granulocytes but abrogated the increase in tumor necrosis factor α levels 4 h after LPS stimulation and also markedly inhibited increases in levels of interleukin‐6 and keratinocyte‐derived chemokine/CXC chemokine ligand 1 but not eotaxin/chemokine ligand 11. Thus, acting via ETA receptors, endogenous endothelins play an important role in early cytokine/chemokine production and on granulocyte and lymphocyte mobilization in LPS‐induced pleurisy.

Effects of inhibitors of inflammatory mediators and cytokines on eosinophil and neutrophil accumulation induced by Mycobacterium bovis bacillus Calmette-Guérin in mouse pleurisy.

In this work we characterize the Mycobacterium bovis bacillus Calmette‐Guérin(BCC) ‐induced pleurisy and investigate the role of chemical mediators and cytokines in BCG‐induced granulocyte accumulation at 24 h. Intrathoracic injection of BCG in C57B1/6 mice induces a biphasic inflammatory reaction with intense leukocyte accumulation at 24 h and 15 days. Neutrophils were observed in the pleural cavity at 4‐24 h, mononuclear cells and eosinophils after 24 h. A new wave of mononuclear cells and neutrophils were observed after 15 days. Pretreatments with dexamethasone, BW 755C, BW A4C, WEB 2170, L‐NAME, and monoclonal antibody (mAb) anti‐interleukin‐5 (IL‐5; TRFK‐5) had inhibited the eosinophil accumulation. On the other hand, only the pretreatments with dexamethasone, L‐NAME, or mAb antitumor necrosis factor a (TNF‐α; MP6‐XT3) had inhibited the neutrophil influx. These results suggest the involvement of leukotrienes, platelet‐activating factor, nitric oxide, and IL‐5 in the eosinophil accumulation, and a role for nitric oxide and TNF‐α in the neutrophil influx induced by BCG. J. Leukoc. Biol. 62: 778–785; 1997.

Leukotriene B4 mediates γδ T lymphocyte migration in response to diverse stimuli

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 LTB4 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. LTB4/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.

Mefloquine-oxazolidine derivatives, derived from mefloquine and arenecarbaldehydes: In vitro activity including against the multidrug-resistant tuberculosis strain T113.

Ten new mefloquine-oxazolidine derivatives, 4-[(1S,8aR)-3-(aryl)hexahydro[1,3]oxazolo[3,4-a]pyridin-1-yl]-2,8-bis(trifluoromethyl)quinoline (1: aryl=substituted phenyl) and 4-[(1S,8aR)-3-(heteroaryl)hexahydro[1,3]oxazolo[3,4-a]pyridin-1-yl]-2,8-bis(trifluoromethyl)quinoline [2: heteroaryl=5-nitrothien-2-yl (2a); 5-nitrofuran-2-yl (2b) and 4H-imidazol-2-yl) (2c)], have been synthesized and evaluated against Mycobacterium tuberculosis. Compounds 1f (aryl=3-ethoxyphenyl), 1g (Ar=3,4,5-(MeO)(3)-C(6)H(2)) and 2c were slightly more active than mefloquine (MIC=33μM) with MICs=24.5, 22.5 and 27.4, respectively, whereas compounds 1e (aryl=3,4-(MeO)(2)-C(6)H(3)) and 2a (MICs=11.9 and 12.1μM, respectively) were ca. 2.7 times more active than mefloquine, with a better tuberculostatic activity than the first line tuberculostatic agent ethambutol (MIC=15.9). The compounds were also assayed against the MDR strain T113 and the same MICs were observed. Thus the new derivatives have advantages over such anti-TB drugs as isoniazid, rifampicin, ethambutol and ofloxacin, for which this strain is resistant. The most active compounds were not cytotoxic to Murine Macrophages Cells in a concentration near their MIC values.

AT1 receptor-mediated angiotensin II activation and chemotaxis of T lymphocytes.

Angiotensin II (Ang II), a central renin-angiotensin system (RAS) effector molecule, and its receptors, AT(1) and AT(2), have been shown to be involved in the inflammatory aspects of different diseases, however the cellular mechanisms underlying the regulation of immunity are not fully understood. In this work, using spleen-derived CD4(+) and CD8(+) T lymphocytes activated in vitro, we tested the influence of Ang II on different aspects of the T cell function, such as activation and adhesion/transmigration through endothelial basal membrane proteins. The addition of 10(-8)M Ang II did not change any of the parameters evaluated. However, 10(-6)M losartan, an AT(1) receptor antagonist: (i) reduced the percentage of CD25(+) and CD69(+) cells of both subsets; (ii) inhibited adhesion of these cells to fibronectin or laminin by 53% or 76%, respectively and (iii) significantly reduced transmigration through fibronectin or laminin by 57% or 43%, respectively. In addition, 10(-6)M captopril, an angiotensin-converting enzyme inhibitor had similar effects to Ang II, however its effects were reverted by exogenous Ang II (10(-8)M). None of these responses was modified by 10(-7)M PD123319, an AT(2) antagonist. These data reinforce the notion of endogenous production of Ang II by T cells, which is important for T cell activation, and adhesion/transmigration induced on interaction with basal membrane proteins, possibly involving AT(1) receptor activation. Moreover, AT(1) receptor expression is 10-fold higher in activated T lymphocytes compared with naive cells, but AT(2) receptor expression did not change after T cell receptor triggering.