Inflammation and NO(X)-induced nitration: assay for 3-nitrotyrosine by HPLC with electrochemical detection

The identification of 15N-labeled 3-nitrotyrosine (NTyr) by gas chromatography/mass spectroscopy in protein hydrolyzates from activated RAW 264.7 macrophages incubated with 15N-L-arginine confirms that nitric oxide synthase (NOS) is involved in the nitration of protein-bound tyrosine (Tyr). An assay is presented for NTyr that employs HPLC with tandem electrochemical and UV detection. The assay involves enzymatic hydrolysis of protein, acetylation, solvent extraction, O-deacetylation, and dithionite reduction to produce an analyte containing N-acetyl-3-aminotyrosine, an electrochemically active derivative of NTyr. We estimate the level of protein-bound NTyr in normal rat plasma to be approximately 0-1 residues per 10(6) Tyr with a detection limit of 0.5 per 10(7) Tyr when > 100 nmol of Tyr is analyzed and when precautions are taken to limit nitration artifacts. Zymosan-treated RAW 264.7 cells were shown to have an approximately 6-fold higher level of protein-bound NTyr compared with control cells and cells treated with N(G)-monomethyl-L-arginine, an inhibitor of NOS. Intraperitoneal injection of F344 rats with zymosan led to a marked elevation in protein-bound NTyr to approximately 13 residues per 10(6) Tyr, an approximately 40-fold elevation compared with plasma protein of untreated rats; cotreatment with N(G)-monomethyl-L-arginine inhibited the formation of NTyr in plasma protein from blood and peritoneal exudate by 69% and 53%, respectively. This assay offers a highly sensitive and quantitative approach for investigating the role of reactive byproducts of nitric oxide in the many pathological conditions and disease states associated with NO(X) exposure such as inflammation and smoking.

Role of sensory innervation in the rat pulmonary neutrophil recruitment induced by staphylococcal enterotoxins type A and B

Rat airways exposure to Staphylococcal enterotoxin A (SEA) and B (SEB) induces marked neutrophil influx. Since sensory neuropeptides play important roles in cell infiltration, in this study we have investigated its contribution in triggering SEA- and SEB-induced pulmonary neutrophil infiltration. Male Wistar rats were exposed intratracheally with SEA (3 ng/trachea) or SEB (250 ng/trachea). Animals received different in vivo pretreatments, after which the neutrophil counts and levels of substance P and IL-1 in bronchoalveolar lavage fluid were evaluated. Alveolar macrophages and peritoneal mast cells were incubated with SEA and SEB to determine the IL-1 and TNF-alpha levels. Capsaicin pretreatment significantly reduced SEA- and SEB-induced neutrophil influx in bronchoalveolar lavage fluid, but this treatment was more effective to reduce SEA responses. Treatments with SR140333 (tachykinin NK(1) receptor antagonist) and SR48968 (tachykinin NK(2) receptor antagonist) decreased SEA-induced neutrophil influx, whereas SEB-induced responses were inhibited by SR140333 only. Cyproheptadine (histamine/5-hydroxytriptamine receptor antagonist) and MD 7222 (5-HT(3) receptor antagonist) reduced SEA- and SEB-induced neutrophil influx. The substance P and IL-1 levels in bronchoalveolar lavage fluid of SEA-exposed rats were significantly hi.-her than SEB. In addition, SEA (but not SEB) significantly released mast cell TNF-alpha. Increased production of TNF-alpha and IL-1 in alveolar macrophages was observed in response to SEA and SEB. In conclusion, sensory neuropeptides contribute significantly to SEA- and SEB-induced pulmonary neutrophil recruitment, but SEA requires in a higher extent the airways sensory innervation, and participation of mast cells and alveolar macrophage products. (C) 2009 Elsevier B.V. All rights reserved.

Compound 48/80 induces endothelium-dependent and histamine release-independent relaxation in rabbit aorta

Compound 48/80 (C48/80) is a synthetic condensation product of N-methyl-p-methoxyphenethyl am me with formaldehyde and is an experimental drug used since the 1950s to induce anaphylactic shock through histamine release. This study was carried out to further elucidate the mechanism by which this drug induces nitric oxide (NO) release. Our specific goals were: (a) to verify if C48/80`s relaxation occurs through the stimulation of histamine receptors; (b) to evaluate the endothelium-dependent relaxation induced by C48/80; (c) to identify NO as the endothelium-relaxing factor released by C48/80; (d) to identify the NO synthase (NOS) responsible for NO release; and (e) to verify if the relaxation induced by C48/80 is calcium and cyclic guanidine monophosphate (cGMP) dependent. Rabbit aorta segments, with and without endothelium, were suspended in organ chambers (25 ml) filled with Krebs solution maintained at 37 degrees C, bubbled with 95% O-2/5% CO2 (pH 7.4). Phenylephrine was used to contract the segments. Other protocol drugs included H-1- and H-2-receptor antagonists, cyclooxygenase, NOS, guanylyl cyclase and phospholipase C (PLC) inhibitors. Endothelium-dependent relaxation induced by C48/80 was also studied in calcium-free Krebs solution associated with a calcium chelator. In summary, our investigation demonstrated that the C48/80 vasodilating action: (a) does not depend on H-1 and H-2 histamine receptors; (b) is NO endothelium-dependent; (c) is dependent on the endothelial constitutive NOS (NOS-3) isoform activation; (d) is cGMP-dependent; and that NOS-3 activation by C48/80: (a) is independent of PLC up to 25 mu g/ml and (b) is partially dependent of this lipase in higher doses. (C) 2007 Elsevier Inc. All rights reserved.

Anaphylaxis with Schistosoma mansoni extracts in normal and infected mice

Methods generally utilized for studies on anaphylaxis to protein antigens such as determination of histamine release to the blood, hemoconcentration, histamine release from peritoneal mast cells and passive cutaneous anaphylaxis (PCA) were used to investigate some aspects of the anaphylaxis to parasite antigens in Schistosoma mansoni infected mice. The release of histamine to the blood and significant rates of hemoconcentration were induced by intravenous injection of schistosomula or cercarial extracts into 10-13 weeks infected mice. Cercarial, schistosomula, worm tegument and soluble egg antigens were able to trigger histamine release from peritoneal mast cells from chronically infected mice. In spite of the PCA reaction beeing detected within 2 hours of sensitization (IgG1antibodies) in 6 of 8 tested sera from chronically infected mice, no detectable reactions were obtained after 48 hours sensitization (IgE antibodies). Although IgE was not detected in the circulation, by the PCA technique, the results indicate that the infected mice contained IgE antibodies bound to their mast cells.

Requirement for CARMA1 in antigen receptor-induced NF-kappa B activation and lymphocyte proliferation.

Ligation of antigen receptors (TCR, BCR) on T and B lymphocytes leads to the activation of new transcriptional programs and cell cycle progression. Antigen receptor-mediated activation of NF-kappa B, required for proliferation of B and T cells, is disrupted in T cells lacking PKC theta and in B and T cells lacking Bcl10, a caspase recruitment domain (CARD)-containing adaptor protein. CARMA1 (also called CARD11 and Bimp3), the only lymphocyte-specific member in a family of membrane-associated guanylate kinase (MAGUK) scaffolding proteins that interact with Bcl10 by way of CARD-CARD interactions, is required for TCR-induced NF-kappa B activation in Jurkat T lymphoma cells. Here we show that T cells from mice lacking CARMA1 expression were defective in recruitment of Bcl10 to clustered TCR complexes and lipid rafts, in activation of NF-kappa B, and in induction of IL-2 production. Development of CD5(+) peritoneal B cells was disrupted in these mice, as was B cell proliferation in response to both BCR and CD40 ligation. Serum immunoglobulin levels were also markedly reduced in the mutant mice. Together, these results show that CARMA1 has a central role in antigen receptor signaling that results in activation and proliferation of both B and T lymphocytes.

Lipoxin A₄ prevents the progression of de novo and established endometriosis in a mouse model by attenuating prostaglandin E₂ production and estrogen signaling.

Endometriosis, a leading cause of pelvic pain and infertility, is characterized by ectopic growth of endometrial-like tissue and affects approximately 176 million women worldwide. The pathophysiology involves inflammatory and angiogenic mediators as well as estrogen-mediated signaling and novel, improved therapeutics targeting these pathways are necessary. The aim of this study was to investigate mechanisms leading to the establishment and progression of endometriosis as well as the effect of local treatment with Lipoxin A4 (LXA₄), an anti-inflammatory and pro-resolving lipid mediator that we have recently characterized as an estrogen receptor agonist. LXA₄ treatment significantly reduced endometriotic lesion size and downregulated the pro-inflammatory cytokines IL-1β and IL-6, as well as the angiogenic factor VEGF. LXA₄ also inhibited COX-2 expression in both endometriotic lesions and peritoneal fluid cells, resulting in attenuated peritoneal fluid Prostaglandin E₂ (PGE₂) levels. Besides its anti-inflammatory effects, LXA₄ differentially regulated the expression and activity of the matrix remodeling enzyme matrix metalloproteinase (MMP)-9 as well as modulating transforming growth factor (TGF)-β isoform expression within endometriotic lesions and in peritoneal fluid cells. We also report for first time that LXA₄ attenuated aromatase expression, estrogen signaling and estrogen-regulated genes implicated in cellular proliferation in a mouse model of disease. These effects were observed both when LXA₄ was administered prior to disease induction and during established disease. Collectively, our findings highlight potential targets for the treatment of endometriosis and suggest a pleotropic effect of LXA₄ on disease progression, by attenuating pro-inflammatory and angiogenic mediators, matrix remodeling enzymes, estrogen metabolism and signaling, as well as downstream proliferative pathways.

Emprego de compostos organometálicos mononucleares de paládio(II) na ativação de macrófagos peritoneais de camundongos

The immune responses are mediated by a variety of cells that, when activated, produce a number of molecules. Macrophages are the first cells to take part in the immune response releasing many compounds in the extracellular environment such as H2O2. Taking into account this aspect we evaluated the activation of an immunological system, in vitro, by determining the H2O2 released in cultures of peritoneal macrophage cells from Swiss mice in the presence of organopalladated compounds of the type [Pd(dmba)(X)(dppp)], dmba = N,N-dimethylbenzylamine, dppp = 1,3-bis(diphenylphosphine)propane, X = Cl, N3, NCO, NCS. An excellent activation of macrophages by the [Pd(dmba)(X)(dppp)] compounds was observed and the influence of the X ligand on the immune response could be verified.

The Influence of Diet and Microbes on Colonic Immune Regulation and their Implications on Type 1 Diabetes

Dietary and microbial factors are thought to contribute to the rapidly increasing prevalence of T1D in many countries worldwide. The impact of these factors on immune regulation and diabetes development in non-obese diabetic (NOD) mice are investigated in this thesis. Diabetes can be prevented in NOD mice through dietary manipulation. Diet affects the composition of intestinal microbiota, which may subsequently influence intestinal immune homeostasis. However, the specific effects of anti-diabetogenic diets on gut immunity and the explicit associations between intestinal immune disruption and type 1 diabetes onset remain unclear. The research presented herein demonstrates that newly weaned NOD mice suffer from a mild level of colitis, which shifts the colonic immune cell balance towards a proinflammatory status. Several aberrations can also be observed in the peritoneal B cells of NOD mice; an increase in activation marker expression, increased trafficking to the pancreatic lymph nodes and significantly higher antigen presenting cell (APC) efficiency towards insulin-specific T cells. A shift towards inflammation is likewise observed in the colon of germ-free NOD mice, but signs of peritoneal B cell activation are lacking in these mice. Remarkably, most of the abnormalities in the colon, peritoneal macrophages and the peritoneal B cell APC activity of NOD mice are abrogated when NOD mice are maintained on a diabetes-preventive, soy-based diet (ProSobee) from the time of weaning. Dietary and microbial factors hence have a significant impact on colonic immune regulation and peritoneal B cell activation and it is suggested that these factors influence diabetes development in NOD mice.

Pharmacological study of anti-allergic activity of Syzygium cumini (L.) Skeels

Myrtaceae is a plant family widely used in folk medicine and Syzygium and Eugenia are among the most important genera. We investigated the anti-allergic properties of an aqueous leaf extract of Syzygium cumini (L.) Skeels (SC). HPLC analysis revealed that hydrolyzable tannins and flavonoids are the major components of the extract. Oral administration of SC (25-100 mg/kg) in Swiss mice (20-25 g; N = 7/group) inhibited paw edema induced by compound 48/80 (50% inhibition, 100 mg/kg; P <= 0.05) and, to a lesser extent, the allergic paw edema (23% inhibition, 100 mg/kg; P <= 0.05). SC treatment also inhibited the edema induced by histamine (58% inhibition; P <= 0.05) and 5-HT (52% inhibition; P <= 0.05) but had no effect on platelet-aggregating factor-induced paw edema. SC prevented mast cell degranulation and the consequent histamine release in Wistar rat (180-200 g; N = 7/group) peritoneal mast cells (50% inhibition, 1 µg/mL; P <= 0.05) induced by compound 48/80. Pre-treatment of BALB/c mice (18-20 g; N = 7/group) with 100 mg/kg of the extract significantly inhibited eosinophil accumulation in allergic pleurisy (from 7.662 ± 1.524 to 1.89 ± 0.336 x 10(6)/cavity; P <= 0.001). This effect was related to the inhibition of IL-5 (from 70.9 ± 25.2 to 12.05 ± 7.165 pg/mL) and CCL11/eotaxin levels (from 60.4 ± 8.54 to 32.8 ± 8.4 ng/mL) in pleural lavage fluid, using ELISA. These findings demonstrate an anti-allergic effect of SC, and indicate that its anti-edematogenic effect is due to the inhibition of mast cell degranulation and of histamine and serotonin effects, whereas the inhibition of eosinophil accumulation in the allergic pleurisy model is probably due to an impairment of CCL11/eotaxin and IL-5 production.

Characterization of the mechanisms underlying the inflammatory response to Polistes lanio lanio (paper wasp) venom in mouse dorsal skin

Stings by Polistes wasps can cause life-threatening allergic reactions, pain and inflammation. We examined the changes in microvascular permeability and neutrophil influx caused by the venom of Polistes lanio a paper wasp found in southeastern Brazil. The intradermal injection of wasp venom caused long-lasting paw oedema and dose-dependently increased microvascular permeability in mouse dorsal skin. SR140333, an NK(1) receptor antagonist, markedly inhibited the response, but the NK(2) receptor antagonist SR48968 was ineffective. The oedema was reduced in capsaicin-treated rats, indicating a direct activation of sensory fibres. Dialysis of the venom partially reduced the oedema and the remaining response was further inhibited by SR140333. Mass spectrometric analysis of the venom revealed two peptides (QPPTPPEHRFPGLM and ASEPTALGLPRIFPGLM) with sequence similarities to the C-terminal region of tachykinin-like peptides found in Phoneutria nigniventer spider venom and vertebrates. Wasp venom failed to release histamine from mast cells in vitro and spectrofluorometric assay of the venom revealed a negligible content of histamine in the usual dose of P.l. lanio venom (1 nmol of histamine/7 mu g of venom)that was removed by dialysis. The histamine H(1) receptor antagonist pyrilamine, but not bradykinin B(1) or B(2) receptor antagonists, inhibited venom-induced oedema. In conclusion, P. l. lanio venom induces potent oedema and increases vascular permeability in mice, primarily through activation of tachykinin NK(1) receptors by substance P released from sensory C fibres, which in turn releases histamine from dermal mast cells. This is the first description of a neurovascular mechanism for P. l. lanio venom-mediated inflammation. The extent to which the two tachykinin-like peptides identified here contribute to this neurogenic inflammatory response remains to be elucidated. (c) 2008 Elsevier Ltd. All rights reserved.

Efeito antiinflamatório de fucana extraída da alga Parda spatoglossum schroederii em modelos experimentais de Peritonite, choque não séptico e colite

Fucans is a name used for sulfated polysaccharides, which is most characteristic structure of the presence of sulfated L-fucose, are found in brown seaweed (Phaeophyceae) and echinoderms (sea urchins and sea cucumbers). These polysaccharides have been reported to possess anticoagulant, antitumor, anti-viral, anti-proliferative and anti-inflammatory activities. Therefore, in the present study was evaluate the effect of the fucan from the brown seaweed Spatoglossum schroederii in models of peritonitis and non-septic shock induced by zymosan, as well as in a murine model of colitis induces by DSS. So, the mice treatment by intravenous route with the fucan was able to reduce the exudate formation and the cell migration in the model of acute peritonitis induced by zymosan during the kinetic of 6, 24 and 48 hours. Similarly, in the model of non-septic shock induced by zymosan the fucan demonstrated a protector effect to inhibited the cellular migration to the peritoneo, to decrease the levels of IL-6 in the serum and in the peritoneal exudate, to attenuate the lose of weight in the mice; beside to reduce the serum levels of hepatic transaminases and as well as the liver injury. In the model of murine colitis, the treatment with the fucan reduced the lose of weight of the animals, decreased the levels of IL-17 and IFN- produced in the gut and decrease the intestinal lesion induced by DSS. In conclusion, the fucan used in this study presented a significant protector effect in the murine models of inflammation

Intermediate reactive oxygen and nitrogen from macrophages induced by Brazilian lichens

The activity of ten compounds isolated from Brazilian lichen over the release of hydrogen peroxide and nitric oxide was evaluated in the culture of peritoneal macrophage cells from mice. Salazinic, secalonic A and fumarprotocetraric acids were the compounds that induced the greatest release of H2O2, whereas 12R-usnic and diffractaic acids induced the release of NO. These results indicate that lichen products have potential immunological modulating activities. (C) 2004 Elsevier B.V. All rights reserved.

Effect of the essential oil of Achillea millefolium L. in the production of hydrogen peroxide and tumor necrosis factor-alpha in murine macrophages

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Emprego de compostos organometálicos mononucleares de paládio(II) na ativação de macrófagos peritoneais de camundongos

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Structure and biological activities of eumenine mastoparan-AF (EMP-AF), a new mast cell degranulating peptide in the venom of the solitary wasp (Anterhynchium flavomarginatum micado)

A new mast cell degranulating peptide, eumenine mastoparan-AF (EMP-AF), was isolated from the venom of the solitary wasp Anterhynchium flavomarginatum micado, the most common eumenine wasp found in Japan. The structure was analyzed by FAB-MS/MS together with Edman degradation, which was corroborated by solid-phase synthesis. The sequence of EMP-AF, Ile-Asn-Leu-Leu-Lys-Ile-Ala-Lys-Gly-Ile-lle-Lys-Ser-Leu-NH(2), was similar to that of mastoparan, a mast cell degranulating peptide from a hornet venom; tetradecapeptide with C-terminus amidated and rich in hydrophobic and basic amino acids. In fact, EMP-AF exhibited similar activity to mastoparan in stimulating degranulation from rat peritoneal mast cells and RBL-2H3 cells. It also showed significant hemolytic activity in human erythrocytes. Therefore, this is the first example that a mast cell degranulating peptide is found in the solitary wasp venom. Besides the degranulation and hemolytic activity, EMP-AF also affects on neuromuscular transmission in the lobster walking leg preparation. Three analogs EMP-AF-1 similar to 3 were snythesized and biologically tested together with EMP-AF, resulting in the importance of the C-terminal amide structure for biological activities. (C) 2000 Elsevier B.V. Ltd. All rights reserved.