Expression of glyceraldehyde 3-phosphate dehydrogenase is enhanced in Leishmania spp naturally resistant to nitric oxide.

Leishmania spp are the causative agents of a spectrum of diseases termed leishmaniasis that affect mammals, including humans and dogs. Although reactive nitrogen species are employed in the control of parasitism by the immune system, it is known that Leishmania can withstand this oxidative stress. As the mechanism by which these species are resistant to nitric oxide (NO) is poorly understood, the main objective of this study was to evaluate the expression of glyceraldehyde 3-phosphate dehydrogenase (GAPDH) in Leishmania amazonensis and Leishmania chagasi promastigotes showing natural resistance to NO. GAPDH transcript levels were quantified by real-time polymerase chain reaction amplification, and GAPDH activity (assessed by levels of NADH oxidation) was measured by spectrophotometry. The level of nitration in total protein was assessed by immunoblotting. The results demonstrated an increase in GAPDH expression in resistant isolates of both species compared to susceptible isolates. The increase in GAPDH expression led to an increase in the activity of GAPDH in L. amazonensis human isolates resistant to NO. The pattern of protein nitration did not differ between sensitive and resistant isolates. Our results suggest that changes in expression of GAPDH may be responsible, at least in part, to natural resistance to NO found in human and canine Leishmania spp.

Anti-inflammatory and cicatrizing activities of thymol, a monoterpene of the essential oil from Lippia gracilis, in rodents.

ETHNOPHARMACOLOGICAL RELEVANCE: Lippia gracilis Schauer (Verbenaceae) has long been recognized in folk medicine as a medicinal plant. The essential oil of Lippia gracilis has antimicrobial activity and is used externally to treat cutaneous diseases, burns, wounds, and ulcers. Recently, our research group demonstrated that the essential oil of Lippia gracilis leaves possesses antinociceptive and anti-inflammatory actions and its major component identified was thymol. The objective of this study was to assess the anti-inflammatory and wound healing activities of thymol in rodents. MATERIALS AND METHODS: For the anti-inflammatory analysis the paw oedema and peritonitis models were used, followed by the assessment of the mieloperoxidase (MPO) activity, total cell counting, and histological analysis. The animals were treated (i.p., n=6/group) with thymol (10, 30, and 100 mg/kg), dexamethasone (2 mg/kg), or vehicle (1% Tween 80). In order to assess the wound healing potential, thymol was vehiculated into collagen-based dressing films and a biological wound healing test was conducted. The retraction index of the wounds and histological analysis were performed on the 3rd, 7th, 14th, and 21th days, split into three groups: undressed wounds (CTR), dressed with collagen-based films (COL), and dressed with collagen-based containing thymol (COLTHY) films. RESULTS: Thymol reduced significantly the oedema (100 mg/kg, P<0.001) and, besides, diminished the influx of leukocytes to the injured area (10, 30, and 100 mg/kg), according to the assessment of MPO activity (P<0.001), total cell count (P<0.05), and histological analysis. Wounds dressed with COLTHY films showed significantly bigger wound retraction rates (7 and 14 day, P<0.05) and improved the granulation reaction, as well provided better collagenization density and arrangement during wound healing. CONCLUSIONS: This study suggests that thymol is a promising compound to be used in treatment of inflammatory processes as well as wound healing. The pharmacological actions of Lippia gracilis in popular medicine practices may be related, at least in part, to the presence of thymol in the essential oil.

Evaluation of the analgesic and anti-inflammatory effects of the essential oil of Lippia gracilis leaves.

AIM OF THE STUDY: The aim of the present study is to investigate the antinociceptive, anti-inflammatory, and antioxidant activities of essential oil (EO) of Lippia gracilis Schauer (Verbenaceae) leaves to support the medicinal uses claimed by folklore practitioners in the caatinga region (semi-arid) of Northeastern Brazil. MATERIALS AND METHODS: The chemical composition and antinociceptive and anti-inflammatory activities of the EO of Lippia gracilis leaves (50-200 mg/kg) were investigated. Antinociceptive activity of the EO was evaluated by writhing test. Anti-inflammatory activity of the EO was evaluated using paw oedema and peritonitis methods. RESULTS: Oral treatment with the EO of Lippia gracilis leaves elicited inhibitory activity on acetic acid effect at 50, 100, and 200 mg/kg (30.33+/-2.36, 25.20+/-1.48, and 21.00+/-1.54 abdominal writhes, respectively, P<0.05), as compared with the control group (36.73+/-1.92 writhes). The compound acetylsalicylic acid (ASA, 300 mg/kg) inhibited the acetic acid-induced writhing (12.67+/-0.50 abdominal writhes, P<0.001). Carrageenan-induced oedema formation was reduced with the EO of Lippia gracilis leaves at 200 mg/kg (0.72+/-0.06 mL h, P<0.001) and by the reference compound ASA (300 mg/kg, 0.85+/-0.04 mL h, P<0.001), as compared with the control group (1.76+/-0.06 mL h). Leukocyte migration into the peritoneal cavity induced by carrageenan was reduced with the EO of Lippia gracilis leaves at 50, 100, and 200 mg/kg (13.81+/-0.61, 11.77+/-0.91, and 10.30+/-0.60 leukocytes x 10(6)/mL, respectively, P<0.01), and by the compound dexamethasone (2 mg/kg, 5.34+/-0.33 leukocytes x 10(6)/mL, P<0.001), as compared with the control group (16.71+/-0.54 leukocytes x 10(6)/mL). The analyses of the essential oil allowed the identification of Lippia gracilis as a thymol-p-cymene chemotype (32.68% and 17.82%, respectively). CONCLUSIONS: The EO of Lippia gracilis leaves shows antinociceptive and anti-inflammatory activities.

Antinociceptive and anti-inflammatory activities of Bowdichia virgilioides (sucupira).

ETHNOPHARMACOLOGICAL RELEVANCE: Bowdichia virgilioides Kunth (Leguminosae Papilonoideae) is a plant with anti-inflammatory activity used in folk medicine. The importance of this plant promoted its inclusion in Brazilian Pharmacopoeia. AIM OF THE STUDY: In order to evaluate the actions of this plant, studies were performed on antinociceptive and anti-inflammatory activities. MATERIALS AND METHODS: The aqueous extracts (AE) of Bowdichia virgilioides inner bark and leaves were used at 100, 200, and 400mg/kg. Antinociceptive activity of plant extract was evaluated by writhing, hot-plate and formalin tests. Anti-inflammatory activity was evaluated using paw oedema and peritonitis methods. RESULTS: Oral treatment with the AE of inner bark or leaves elicited inhibitory activity (P<0.01) on acetic acid effect at 200 and 400mg/kg, and reduced the formalin effect at the second-phase (200 and 400mg/kg, P<0.01), however it did not elicit any inhibitory effect on hot-plate test. The indomethacin inhibited the acetic acid-induced writhing and the formalin effect at the second-phase (P<0.001), and the morphine reduced the both phases of formalin test (P<0.001). Carrageenan-induced oedema formation and neutrophil migration into the peritoneal cavity were reduced with the AE of inner bark or leaves at 100, 200, and 400mg/kg (P<0.05), and by the reference compounds aspirin (P<0.001) and dexamethasone (P<0.001), respectively. CONCLUSIONS: The AE of Bowdichia virgilioides shows antinociceptive and anti-inflammatory activities, supporting the folkloric usage of the plant to treat various inflammatory diseases.

Role of nitric oxide on in vitro human eosinophil migration.

Eosinophils purified from the rat peritoneal cavity have been found to contain nitric oxide synthase (NOS) functionally coupled to a cyclic GMP transduction pathway that is involved in in vitro eosinophil migration, but no studies on cell locomotion have been done with purified human eosinophils. Therefore, this study was carried out to investigate the effects of N(omega) -nitro-L-arginine methyl ester (L-NAME; a non-selective NOS inhibitor), 1-(2-trifluoromethylphenyl) imidazole (TRIM; a type I/type II NOS inhibitor), 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT; a selective type II NOS inhibitor), and 1H-[1,2,4]-oxidiazolo[4,3-a] quinoxalin-1-one (ODQ; a soluble guanylate cyclase inhibitor) on human eosinophil migration induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP). Human eosinophils were purified from peripheral blood of healthy volunteers using a Percoll gradient followed by an immunomagnetic cell separator. Chemotaxis was evaluated using a 48-well microchemotaxis chamber. The fMLP (1.0 x 10(-7) M)-induced eosinophil migration was reduced significantly by l-NAME (0.1 and 1.0 mM), whereas the inactive enantiomer N(omega)-nitro-D-arginine methyl ester (D-NAME) had no effect. The inhibition by l-NAME was restored by sodium nitroprusside (0.25 mM). The NOS inhibitors AMT and TRIM (0.05 to 0.25 mM each) also markedly attenuated fMLP-induced chemotaxis. Additionally, ODQ (0.01 to 0.5 mM) concentration-dependently inhibited fMLP-induced migration, and the inhibition was restored by 2.0 mM dibutyryl cyclic GMP. In conclusion, this study demonstrates that human eosinophils present a nitric oxide-cyclic GMP pathway that is involved in the in vitro locomotion of this cell type.

Nitric oxide regulates human eosinophil adhesion mechanisms in vitro by changing integrin expression and activity on the eosinophil cell surface.

1. The nitric oxide synthase (NOS) inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME), inhibits both rat and human eosinophil chemotaxis in vitro. Here, the role of nitric oxide (NO) in human eosinophil cell surface integrin expression and function was investigated. 2. Human peripheral blood eosinophils were treated with L-NAME (0.01 - 1.0 mM) and their adhesion to human fibronectin and serum observed. Adhesion of cells to fibronectin and serum increased by 24.0+/-4.6 and 43.8+/-4.7%, respectively, when eosinophils were treated with 1.0 mM L-NAME. Increased adhesion by L-NAME could be abolished when cells were co-incubated with VLA-4- and Mac-1-specific monoclonal antibodies (mAbs). 3. The NO donor, sodium nitroprusside (2.5 mM), significantly inhibited eosinophil adhesion to fibronectin and serum by 34.3+/-4.5 and 45.2+/-5.6%, respectively. This inhibition was accompanied by a 4 fold increase in the levels of intracellular cyclic GMP. 4. Flow cytometrical analysis demonstrated that L-NAME induced an increased expression of CD11b (Mac-1) on the eosinophil cell surface of 36.3+/-7.4%. L-NAME had no effect upon CD49d (VLA-4) expression. 5. Treatment of human eosinophils, in vitro, with H-[1,2,4] oxadiazolo quinoxalin-1-one (ODQ) (0.1 mM), an inhibitor of soluble guanylate cyclase, also significantly increased eosinophil adhesion to fibronectin and serum by 73.5+/-17.9 and 91.7+/-12.9%, respectively. This increase in adhesion could also be inhibited by co-incubation with the Mac-1 and VLA-4-specific mAbs. 6. In conclusion, results indicate that NO, via a cyclic GMP-dependent mechanism, inhibits the adhesion of human eosinophils to the extracellular matrix (ECM). This inhibition is accompanied by a decrease in the expression and function of the eosinophil's adhesion molecules, in particular, the expression of the Mac-1 integrin and the function of the VLA-4 integrin.

Involvement of resident macrophages and mast cells in the writhing nociceptive response induced by zymosan and acetic acid in mice.

Intraperitoneal administration of zymosan and acetic acid induced a dose-dependent nociceptive writhing response in mice. Lavage of the peritoneal cavities with saline reduced the number of total resident peritoneal cells and caused a proportional decrease in the nociceptive responses induced by these stimuli. Furthermore, the specific reduction of the peritoneal mast cell population by intraperitoneal administration of compound 48/80 also reduced the nociceptive responses induced by zymosan and acetic acid. In contrast, enhancement of the peritoneal macrophage population by pretreatment of the cavities with thioglycollate caused an increase in the number of writhes induced by both stimuli. These data suggest that the nociceptive responses induced by zymosan and acetic acid are dependent upon the peritoneal resident macrophages and mast cells. These cells modulate the nociceptive response induced by zymosan and acetic acid via release of tumour necrosis factor alpha (TNF-alpha), interleukin 1beta and interleukin 8. This suggestion is supported by the following observations: (a) pretreatment of the peritoneal cavities with antisera against these cytokines reduced the nociceptive responses induced by these stimuli; (b) peritoneal cells harvested from cavities injected with zymosan or acetic acid released both interleukin 1beta and TNF-alpha; (c) although individual injection of TNF-alpha, interleukin 1beta or interleukin 8 did not induce the nociceptive effect, intraperitoneal injection of a mixture of these three recombinant cytokines caused a significant nociceptive writhing response. In conclusion, our results suggest that the nociceptive activity of zymosan and acetic acid in the writhing model is due to the release of TNF-alpha, interleukin 1beta and interleukin 8 by resident peritoneal macrophages and mast cells.

Tumor necrosis factor, interleukin-1 and interleukin-8 mediate the nociceptive activity of the supernatant of LPS-stimulated macrophages.

It has been suggested that the supernatant of LPSstimulated macrophages (macrophage nociceptive factor, MNF) promotes nociception in mice. Intraperitoneal administration of MNF induced dose-related writhing, which reached a plateau between 18 and 26 min after injection and decreased within 60 min. The release of MNF was inhibited by the pretreatment of the macrophages with cycloheximide, a protein synthesis inhibitor, or with the glucocorticoid dexamethasone. Cyclooxygenase inhibitors, such as indomethacin or paracetamol, had no effect. The MNF-induced nociception was inhibited in a dose-related manner by pretreatment of the animals with indomethacin, paracetamol or dexamethasone. Pretreatment of the animals with the sympatholytics guanethidine and atenolol partially reduced the MNF nociception, which was abolished by the combination of guanethidine or atenolol with indomethacin. The preincubation of MNF with antisera against TNF-alpha, IL-1 or IL-8 partially inhibited its nociceptive effect. Intraperitoneal injection of a mixture of the recombinants cytokines TNF-alpha, IL-1 and IL-8 mimicked MNF nociception. The individual injection of these cytokines was unable to induce the nociceptive effect. In conclusion, our data suggest that the nociceptive activity of the supernatant of LPSstimulated macrophages is explained by the presence of TNF-alpha, IL-1 and IL-8, the nociceptive activity of which (in mice) seems to be due to the release of cyclooxygenase and sympathetic metabolites.

Pertussis toxin from Bordetella pertussis blocks neutrophil migration and neutrophil-dependent edema in response to inflammation.

Pertussis toxin (Ptx) is a hexameric protein with classical AB architecture produced by Bordetella pertussis. The aim of this study was to investigate the effect of Ptx on migration of polymorphonuclear leukocytes to site of inflammation and on cell-dependent edema. Ptx was purified from the supernatant of the culture medium of B. pertussis using hydroxylapatite chromatography and fetuin affinity chromatography. Ptx induced a maximal clustering of Chinese hamster ovary cells at concentrations as low as 0.1 ng/ml. Intravenous injection of Ptx (400 ng) significantly blocked the neutrophil migration induced by 200 ng of lipopolysaccharide (LPS from E. coli O111:B4; 2.27 +/- 0.13 vs 0.61 +/- 0.16 per 10(6) neutrophils/ml; P < 0.001; N = 5) and by 200 ng of formyl-methionyl-leucyl-phenylalanine (fMLP; 2.53 +/- 0.45 vs 0.75 +/- 0.14 per 10(6) neutrophils/ml; P < 0.01; N = 6) into the peritoneal cavities of male Wistar rats (weighing 150-180 g). In addition, Ptx (400 ng) pretreatment also blocked the edema induced by intraplantar injection of 100 micrograms carrageenin (delta increase in volume: 0.667 +/- 0.087 vs 0.313 +/- 0.058 ml; P < 0.01; N = 5) but not the edema induced by 100 micrograms dextran (delta increase in volume: 0.537 +/- 0.06 vs 0.385 +/- 0.076 ml; P > 0.05; N = 5). These data demonstrate that Ptx blocked neutrophil migration induced by a direct fMLP stimulus of a site of inflammation. In addition, this toxin blocks the indirect stimulus of LPS on neutrophil migration.(ABSTRACT TRUNCATED AT 250 WORDS)

Pertussis toxin from Bordetella pertussis blocks neutrophil migration and neutrophil-dependent edema in response to inflammation

Pertussis toxin (Ptx) is a hexameric protein with classical AB architecture produced by Bordetella pertussis. The aim of this study was to investigate the effect og Ptx on migration of polymorphonuclear leukocytes to site of inflamation and on cell- dependent edema. Ptx was purified from the supernatant of the culture medium of B. pertussis using hydroxylapatite chromatography and fetuin affinity chromatography. Ptx induced a maximal clusterin of Chinese hamster ovary cells at concentration as low as 0.1 ng/ ml. Intravenous inection of Ptx (400 ng) significantly blocked the neutrophil migration induced by 200 ng of lipopolysaccharide (LPS from E. coli O111:B4; 2.27 ñ 0.13 vs 0.61 ñ 0.16 per 10**6 neutrophils/ml; P < 0.001; N = 5) and by 200 ng of formylmethionyl-leucyl-phenylalanine(fMLP; 2.53 ñ 0.45 vs 0.75 ñ 0.14 per 10**6 neutrophils/ml; P < 0.01; N=6) into the peritoneal cavities of male Wistar rats (eighing 150-180). In addition, Ptx (400ng) pretreatment also blocked the edema induced by intraplantar injection of 100 µg carrageenin ( increase in volume: 0.667 ñ 0.087 vs 0.313 ñ 0.058 ml; P < 0.01; N = 5) but not the edema induced by 100 µg dextran ( increase in volume: 0.537 ñ 0.06 vs 0.385 ñ 0.076 ml; P > 0.05; N = 5). These data demonstrate that Ptx blocked neutrophil migration induced by a direct f MLP stimulus of a site of inflammation. In addition, this toxin blocks the indirect stimulus of LPS on neutrophil migration. Furthermore, Ptx also inhibits the neutrophil-dependent edema induced by carrageenin, but not the edema induced by dextran that is in part dependent on basophil cells. These results warrant further studies on the mechanisms of Ptx inhibition of neutrophil-dependent edema and cell migration