Leguminous lectins as tools for studying the role of sugar residues in leukocyte recruitment.

The natural physiological ligands for selectins are oligosaccharides found in glycoprotein or glycolipid molecules in cell membranes. In order to study the role of sugar residues in the in vivo lectin anti-inflammatory effect, we tested three leguminous lectins with different carbohydrate binding affinities in the peritonitis and paw oedema models induced by carrageenin in rats. L. sericeus lectin was more anti-inflammatory than D. virgata lectin, the effects being reversed by their specific binding sugars (N-acetylglucosamine and alpha-methylmannoside, respectively). However, V. macrocarpa, a galactose-specific lectin, was not anti-inflammatory. The proposed anti-inflammatory activity of lectins could be due to a blockage of neutrophil-selectin carbohydrate ligands. Thus, according to the present data, we suggest an important role for N-acetylglucosamine residue as the major ligand for selectins on rat neutrophil membranes

The pharmacological profile of ovalbumin-induced paw oedema in rats.

Rats are commonly used in anaphylaxis models, mainly in intestinal anaphylaxis. Hypersensitivity mechanisms are complex and they are not clearly defined. Ovalbumin (OVA) is commonly used for studies on the hypersensitivity mechanism. However, the potential pro-inflammatory mediators induced by this antigen in the model of paw oedema in immunized rats are still not completely understood. This work examines the pharmacological modulation of several mediators involved in rat hind paw immune oedema induced by OVA. Wistar rats were previously immunized (14-18 days) with OVA (30 microg, intraperitoneally) or sham-sensitized with aluminum hydroxide (control). The paw volumes were measured before the antigenic stimuli and 1, 2, 3 and 4 h after the intraplantar injection of OVA (10 microg/paw). Subcutaneous injection of dexamethasone, diphenhydramine, cyproheptadine, chlorpromazine or methysergide significantly inhibited (p < 0.05) the allergic paw oedema. The dual inhibitor of cyclooxygenase and lipoxygenase (NDGA), the cyclooxygenase inhibitor (indomethacin), the lipoxygenase inhibitor (MK-886), the PAF antagonist (WEB 2086), the mast cell stabilizer (ketotifen), and the anti-histamine (meclizine) did not inhibit the immune oedema. In addition, thalidomide and pentoxifylline (anti-tumour necrosis factor drugs) were ineffective against OVA-induced oedema. The fact that indomethacin, MK-886, NDGA and WEB 2086 are unable to inhibit this allergic oedema indicates that the dexamethasone action seems not to be via phospholipase A2, but possibly due to the synthesis and/or the inhibitory activity of cytokines. The paw oedema inhibition by diphenhydramine, but not by meclizine, may suggest a different mechanism, which is independent of the effect of histamine. These data indicate that allergic oedema is more sensitive to anti-serotonin drugs, mainly anti-5-HT2, suggesting that the principal mediator of this inflammatory response is serotonin

Anti-inflammatory effect of glucose—mannose binding lectins isolated from Brazilian beans

Selectins are essential for leukocyte recruitment in inflammation. Because of a lectin domain present in the selectin structure, we investigated the anti-inflammtory activity of six mannose–glucose binding lectins from brazilian beans: Dioclea guianensis-DguiL; D. grandiflora-DgL; Cratylia floribunda-CfL; D. violacea-D.vL; D. virgata-DvirL and Canavalia brasiliensis-ConBr. The lectins were injected intravenously (i.v.) into rats (0.1 and 1.0 mg/kg; 30 min before irritants) and its activities compared to E. coli endotoxin (LPS,30 μg/kg i.v.). Three lectins (DvL, CfL and DguiL), although less intense than LPS, inhibited the neutrophil migration induced by carrageenan (Cg, 300 μg) in a dose-dependent manner (0.1 and 1.0 mg/kg). DvL activity was reversed by 0.1 M α-D-methyl-mannoside (α-CH3), but not by 0.1 M α-D-galactose. The fMLP (44 ng)-induced neutrophil migration was also reduced by these lectins. Endotoxin contamination of lectin samples could be excluded since α-CH3 treatment reversed the DvL effect, but did not modify LPS inhibitory activity. Carrageenan (300 μg)-induced paw oedema was also reduced by LPS or lectin treatments. Conversely, none of the tested lectins inhibited dextran (Dex, 300 μg)-induced paw oedema, a classical leukocyte independent model, or zymosan (Zy, 1.0 mg)-induced peritonitis and paw oedema. LPS showed no effect upon Dex-induced paw oedema and barely reduced (25%) the oedematogenic effects of zymosan. As proposed for LPS, the lectin inhibitory activity was better observed on neutrophil-mediated inflammatory reactions. We speculate that the plant lectin antiinflammatory activity is probably due to a competitive blockage of a common leukocyte and/or endothelial selectin carbohydrate ligand

Experimental periodontitis promotes transient vascular inflammation and endothelial dysfunction

AbstractObjectivesThis study aimed to evaluate the systemic inflammatory response and cardiovascular changes induced by experimental periodontitis in rats.DesignExperimental periodontitis was induced by placing a cotton ligature around the cervix of both sides of mandibular first molars and maxillary second molars in each male rat. Sham-operated rats had the ligature removed immediately after the procedure. Seven, 14 or 28 days after procedure, the effects of acetylcholine, sodium nitroprusside and phenylephrine were evaluated on blood pressure, aortic rings and isolated and perfused mesenteric bed. The blood was obtained for plasma Interleukin-6 (IL-6), C-reactive protein (CRP) and lipid evaluation. The mesenteric vessels were obtained to evaluate superoxide production and nitric oxide synthase 3 (NOS-3) expression.ResultsLigature induced periodontitis reduced endothelium-dependent vasodilatation, a hallmark of endothelial dysfunction. This effect was associated with an increase in systemic inflammatory markers (IL-6 and CRP), worsens on lipid profile, increased vascular superoxide production and reduced NOS-3 expression. It is interesting to note that many of these effects were transitory.ConclusionPeriodontitis induced a transient systemic and vascular inflammation which leads to endothelial dysfunction, an initial step for cardiovascular diseases. Moreover, the animal model of periodontitis used here may represent a valuable tool for studying the relationship between periodontitis and endothelial dysfunction

The ATP-sensitive potassium channel blocker glibenclamide prevents renal ischemia/reperfusion injury in rats

The ATP-sensitive potassium channel blocker glibenclamide prevents renal ischemia/reperfusion injury in rats.BackgroundRenal ischemia/reperfusion (I/R) is a complex neutrophil-mediated syndrome. Adenosine-triphosphate (ATP)-sensitive potassium (KATP) channels are involved in neutrophil migration in vivo. In the present study, we have investigated the effects of glibenclamide, a KATP channel blocker, in renal I/R injury in rats.MethodsThe left kidney of the rats was excised through a flank incision and ischemia was performed in the contralateral kidney by total interruption of renal artery flow for 45 minutes. Renal perfusion was reestablished, and the kidney and lungs were removed for analysis of vascular permeability, neutrophil accumulation, and content of cytokines [tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, and IL-10] 4 and 24 hours later. Renal function was assessed by measuring creatinine, Na+, and K+ levels in the plasma and by determination of creatinine clearance. Drugs were administered subcutaneously after the onset of ischemia.ResultsReperfusion of the ischemic kidney induced local (kidney) and remote (lung) inflammatory injury and marked renal dysfunction. Glibenclamide (20 mg/kg) significantly inhibited the reperfusion-associated increase in vascular permeability, neutrophil accumulation, increase in TNF-α levels and nuclear factor-κB (NF-κB) translocation. These inhibitory effects were noticed in the kidney and lungs. Moreover, glibenclamide markedly ameliorated the renal dysfunction at 4 and 24 hours.ConclusionTreatment with glibenclamide is associated with inhibition of neutrophil recruitment and amelioration of renal dysfunction following renal I/R. Glibenclamide may have a therapeutic role in the treatment of renal I/R injury, such as after renal transplantation

Curcumin-loaded lipid and polymeric nanocapsules stabilized by nonionic surfactants: An In Vitro and In Vivo antitumor activity on B16-F10 melanoma and macrophage uptake comparative study

Curcumin is a polyphenol obtained from the plant Curcuma longa (called turmeric) that displays several pharmacological activities, including anti-inflammatory, antioxidant, antimicrobial and antitumoral activity, but clinical use has been limited by its poor solubility in water and, consequently, minimal systemic bioavailability. We have therefore formulated the drug into nanocarrier systems in an attempt to improve its therapeutic properties. This study evaluates the effect of intraperitoneally administered nanocapsules containing curcumin on subcutaneous melanoma in mice inoculated with B16-F10 cells, and on the cytotoxicity activity against B16-F10 cells in vitro. Phagocytic uptake of formulations was also evaluated upon incubation with macrophage J774 cells by fluorescence microscopy. Lipid and polymeric nanocapsules were prepared by the phase inversion and nanoprecipitation methods, respectively. The uptake of the lipid nanocapsules prepared using Solutol HS15 was significantly reduced in J774 cells. Curcumin, as free drug or as drug-loaded nanocapsules, was administrated at a dose of 6 mg/kg twice a week for 21 days. Free drug and curcuminloaded nanocapsules significantly reduced tumor volume (P < 0.05 vs. control), but no difference was found in the antitumor activity displayed by lipid and polymeric nanocapsules. This assumption was supported by the in vitro study, in which free curcumin as well as loaded into nanocapsules caused significant reduction of cell viability in a concentration- and time-dependent manner.Fil: Mazzarino, Letícia. Universidade Federal de Santa Catarina; BrasilFil: Silva, Luís F. C.. Universidade Federal de Santa Catarina; BrasilFil: Curta, Juliana C.. Universidade Federal de Santa Catarina; BrasilFil: Licínio, Marley A.. Universidade Federal de Santa Catarina; BrasilFil: Costa, Aline. Universidade Federal de Santa Catarina; BrasilFil: Pacheco, Letícia K.. Universidade Federal de Santa Catarina; BrasilFil: Siqueira, Jarbas M.. Universidade Federal de Santa Catarina; BrasilFil: Martinetti Montanari, Jorge Anibal. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Romero, Eder. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnología; ArgentinaFil: Assreuy, Jamil. Universidade Federal de Santa Catarina; BrasilFil: Santos Silva, Maria C.. Universidade Federal de Santa Catarina; BrasilFil: Lemos Senna, Elenara. Universidade Federal de Santa Catarina; Brasi

Bronchial Responsiveness Is Related to Increased Exhaled NO (FENO) in Non-Smokers and Decreased FENO in Smokers

Rationale Both atopy and smoking are known to be associated with increased bronchial responsiveness. Fraction of nitric oxide (NO) in the exhaled air (FENO), a marker of airways inflammation, is decreased by smoking and increased by atopy. NO has also a physiological bronchodilating and bronchoprotective role. Objectives To investigate how the relation between FENO and bronchial responsiveness is modulated by atopy and smoking habits. Methods Exhaled NO measurements and methacholine challenge were performed in 468 subjects from the random sample of three European Community Respiratory Health Survey II centers: Turin (Italy), Gothenburg and Uppsala (both Sweden). Atopy status was defined by using specific IgE measurements while smoking status was questionnaire-assessed. Main Results Increased bronchial responsiveness was associated with increased FENO levels in non-smokers (p = 0.02) and decreased FENO levels in current smokers (p = 0.03). The negative association between bronchial responsiveness and FENO was seen only in the group smoking less &lt;10 cigarettes/day (p = 0.008). Increased bronchial responsiveness was associated with increased FENO in atopic subjects (p = 0.04) while no significant association was found in non-atopic participants. The reported interaction between FENO and smoking and atopy, respectively were maintained after adjusting for possible confounders (p-values&lt;0.05). Conclusions The present study highlights the interactions of the relationship between FENO and bronchial responsiveness with smoking and atopy, suggesting different mechanisms behind atopy- and smoking-related increases of bronchial responsiveness

NADPH Phagocyte Oxidase Knockout Mice Control Trypanosoma cruzi Proliferation, but Develop Circulatory Collapse and Succumb to Infection

•NO is considered to be a key macrophage-derived cytotoxic effector during Trypanosoma cruzi infection. On the other hand, the microbicidal properties of reactive oxygen species (ROS) are well recognized, but little importance has been attributed to them during in vivo infection with T. cruzi. In order to investigate the role of ROS in T. cruzi infection, mice deficient in NADPH phagocyte oxidase (gp91phox−/− or phox KO) were infected with Y strain of T. cruzi and the course of infection was followed. phox KO mice had similar parasitemia, similar tissue parasitism and similar levels of IFN-γ and TNF in serum and spleen cell culture supernatants, when compared to wild-type controls. However, all phox KO mice succumbed to infection between day 15 and 21 after inoculation with the parasite, while 60% of wild-type mice were alive 50 days after infection. Further investigation demonstrated increased serum levels of nitrite and nitrate (NOx) at day 15 of infection in phox KO animals, associated with a drop in blood pressure. Treatment with a NOS2 inhibitor corrected the blood pressure, implicating NOS2 in this phenomenon. We postulate that superoxide reacts with •NO in vivo, preventing blood pressure drops in wild type mice. Hence, whilst superoxide from phagocytes did not play a critical role in parasite control in the phox KO animals, its production would have an important protective effect against blood pressure decline during infection with T. cruzi

Resistance of Leishmania (Viannia) braziliensis to nitric oxide: correlation with antimony therapy and TNF-α production

<p>Abstract</p> <p>Background</p> <p>Nitric oxide (NO) produced in macrophages plays a pivotal role as a leishmanicidal agent. A previous study has demonstrated that 20% of the <it>L. (V.) braziliensis </it>isolated from initial cutaneous lesions of patients from the endemic area of Corte de Pedra, Bahia, Brazil, were NO resistant. Additionally, 5 to 11% of the patients did not respond to three or more antimony treatments" (refractory patients). The aim of this study is to investigate if there is an association between the resistance of <it>L. (V.) braziliensis </it>to NO and nonresponsiveness to antimony therapy and cytokine production.</p> <p>Methods</p> <p>We evaluated the <it>in vitro </it>toxicity of NO against the promastigotes stages of <it>L. (V.) braziliensis </it>isolated from responsive and refractory patients, and the infectivity of the amastigote forms of these isolates against human macrophages. The supernatants from <it>Leishmania </it>infected macrophage were used to measure TNF-α and IL-10 levels.</p> <p>Results</p> <p>Using NaNO₂(pH 5.0) as the NO source, <it>L. (V.) braziliensis </it>isolated from refractory patients were more NO resistant (IC50 = 5.8 ± 4.8) than <it>L. (V.) braziliensis </it>isolated from responsive patients (IC50 = 2.0 ± 1.4). Four isolates were selected to infect human macrophages: NO-susceptible and NO-resistant <it>L. (V.) braziliensis </it>isolated from responsive and refractory patients. NO-resistant <it>L. (V.) braziliensis </it>isolated from refractory patients infected more macrophages stimulated with LPS and IFN-γ at 120 hours than NO-susceptible <it>L. (V.) braziliensis </it>isolated from refractory patients. Also, lower levels of TNF-α were detected in supernatants of macrophages infected with NO-resistant <it>L. (V.) braziliensis </it>as compared to macrophages infected with NO-susceptible <it>L. (V.) braziliensis </it>(p < 0.05 at 2, 24 and 120 hours), while no differences were detected in IL-10 levels.</p> <p>Conclusion</p> <p>These data suggest that NO resistance could be related to the nonresponsiveness to antimony therapy seen in American Tegumentary Leishmaniasis.</p