Kirsi Vaali

Effects of a COX-2 preferential agent nimesulide on TNBS-induced acute inflammation in the gut.

In inflammatory bowel disease, increased production of prostaglandins by cyclooxy- genase-2 (COX-2) contributes to bowel dysfunction, inflammatory edema, and hyperemia suggesting that inhibitors of COX-2 may have beneficial effect in gut inflammation. We compared the effects of nimesulide, a preferential COX-2 inhibitor, with those of indomethacin, acetylsalicylic acid (ASA), and dexamethasone in a 24-h model of 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced colitis in the rat. TNBS-induced colitis was associated with enhanced COX-2 expression in the gut and increased circulating concentrations of PGE2 metabolite (PGEM). Treatment with nimesulide (10 mg/kg), indomethacin (10 mg/kg), or dexamethasone (1 mg/kg) reduced plasma PGEM concentrations and edema in the inflamed bowel. In addition, nimesulide and dexamethasone treatments decreased neutrophil infiltration into the inflamed colon mucosa. ASA (10 mg/kg) did not have a significant effect on any of these measures of inflammation. None of the studied drugs reduced the size of inflammatory mucosal lesions in the colon. In TNBS-induced acute inflammation of the colon, nimesulide reduced the formation of inflammatory edema, probably by a mechanism related to inhibition of PGE2 production by COX-2 pathway. In addition, nimesulide inhibited neutrophil infiltration into inflamed mucosa mimicking the action of dexamethasone.

Murine model of food allergy after epicutaneous sensitization: role of mucosal mast cell protease-1.

Objective. Studies of the pathological mechanisms of food allergy have been impeded by the lack of relevant animal models. The purpose of this study was to develop a physiological model of food allergy that was not dependent on immunostimulatory adjuvants. Material and methods. Balb/c mice were epicutaneously sensitized four times at varying intervals over a 22-day period, and challenged orally from day 40, 6 times every 1–3 days with either saline or ovalbumin. Results. After sensitization (day 35) but before the oral challenges, the ovalbumin-sensitized groups showed increased specific IgE and IgG1 production when compared with the sham-sensitized groups. Mucosal mast cell protease-1 (MMCP-1) was undetectable in serum before the intragastric challenge. MMCP-1 concentrations were increased after the first ovalbumin dose, solely in the ovalbumin-sensitized and -challenged group. After the challenge period, the mean serum MMCP-1 concentration increased from an undetectable level in controls to an over 44-fold level in the ovalbumin-sensitized and -challenged mice. In this group, MMCP-1-positive cells were present in the small intestine and expressions of IFN-γ and CXCL-9 mRNA were decreased in the ileum, suggesting an impaired Th-1-type response. Within one hour of the last ovalbumin challenge, 5 out of 6 mice developed diarrhea in the ovalbumin-sensitized and -challenged group, but there was no diarrhea in the other groups. Conclusions. A murine model of food allergy based on sensitization via epicutaneous exposure to allergen without immunostimulatory adjuvants was developed. Effective production of MMCP-1 together with specific IgE and IgG1 suggests a breakdown in oral tolerance to the allergen. Intragastric challenges were accompanied by mast cell-dependent immunopathological changes and diarrhea.

Involvement of bradykinin B1 and B2 receptors in relaxation of mouse isolated trachea.

The aim of the present study was to investigate the effects of bradykinin and [des‐Arg9]‐bradykinin and their relaxant mechanisms in the mouse isolated trachea. In the resting tracheal preparations with intact epithelium, bradykinin and [des‐Arg9]‐bradykinin (each drug, 0.01–10 μm) induced neither contraction nor relaxation. In contrast, bradykinin (0.01–10 μm) induced concentration‐dependent relaxation when the tracheal preparations were precontracted with methacholine (1 μm). The relaxation induced by bradykinin was inhibited by the B2 receptor antagonist, d‐Arg0‐[Hyp3,Thi5,d‐Tic7,Oic8]‐bradykinin (Hoe 140, 0.01–1 μm) in a concentration‐dependent manner whereas the B1 receptor antagonist, [des‐Arg9,Leu8]‐bradykinin (0.01–1 μm), had no inhibitory effect on bradykinin‐induced relaxation. [des‐Arg9]‐bradykinin (0.01–10 μm) also caused concentration‐dependent relaxation after precontraction with methacholine. The relaxation induced by [des‐Arg9]‐bradykinin was concentration‐dependently inhibited by the B1 receptor antagonist, [des‐Arg9,Leu8]‐bradykinin (0.01–1 μm), whereas the B2 receptor antagonist, Hoe 140 (0.01–1 μm) was without effect. In the presence of the cyclo‐oxygenase inhibitor, indomethacin (0.01–1 μm), the relaxations induced by bradykinin and [des‐Arg9]‐bradykinin were inhibited concentration‐dependently. Two nitric oxide (NO) biosynthesis inhibitors NG‐nitro‐l‐arginine methyl ester (l‐NAME, 100 μm) and NG‐nitro‐l‐arginine (l‐NOARG, 100 μm) had no inhibitory effects on the relaxations induced by bradykinin and [des‐Arg9]‐bradykinin. Neither did the selective inhibitor of the soluble guanylate cyclase, 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ, 10 μm) inhibit the relaxations induced by bradykinin and [des‐Arg9]‐bradykinin. Prostaglandin E2 (PGE2, 0.01–33 μm) caused concentration‐dependent relaxation of the tracheal preparations precontracted with methacholine. Indomethacin (1 μm) and ODQ (10 μm) exerted no inhibitory effects on the relaxation induced by PGE2. The NO‐donor, sodium nitroprusside (SNP; 0.01–100 μm) also caused concentration‐dependent relaxation of the tracheal preparations precontracted with methacholine. ODQ (0.1–1 μm) concentration‐dependently inhibited the relaxation induced by SNP. These data demonstrate that bradykinin and [des‐Arg9]‐bradykinin relax the mouse trachea precontracted with methacholine by the activation of bradykinin B2‐receptors and B1‐receptors, respectively. The stimulation of bradykinin receptors induces activation of the cyclo‐oxygenase pathway, leading to the production of relaxing prostaglandins. The NO pathway is not involved in the bradykinin‐induced relaxation. The relaxation caused by NO‐donors in the mouse trachea is likely to be mediated via activation of soluble guanylate cyclase.

FENAMATES INHIBIT CONTRACTION OF GUINEA-PIG ISOLATED BRONCHUS IN VITRO INDEPENDENT OF PROSTANOID SYNTHESIS INHIBITION

The inhibitory and relaxant effects of flufenamic and tolfenamic acids on guinea-pig isolated bronchus were compared with those of verapamil and indomethacin. Flufenamic and tolfenamic acids (each drug, 20 microM) and verapamil (1 microM) inhibited bronchial contraction induced by Ca2+, KCl or PGF2alpha whereas indomethacin (20 microM) had no inhibitory effect. Only verapamil, but not flufenamic and tolfenamic acids and indomethacin, inhibited methacholine-induced contraction. Flufenamic and tolfenamic acids and verapamil (each drug, 0.1-33 microM) relaxed the bronchus precontracted by KCl or PGF2alpha. In contrast, indomethacin (0.1-33 microM) did not relax KCl- or PGF2alpha-precontracted bronchus. Verapamil, but not flufenamic and tolfenamic acids and indomethacin, relaxed methacholine precontracted bronchus. In conclusion, fenamates inhibit Ca2+-, KCl- and PGF2alpha-induced contractions in guinea-pig isolated bronchus in a manner involving inhibition of Ca2+ influx but not inhibition of prostanoid synthesis.

In-vitro bronchorelaxing effects of novel nitric oxide donors GEA 3268 and GEA 5145 in guinea-pigs and rats

Endogenously released nitric oxide (NO) in airways might contribute to physiological bronchodilation; induced production of NO might play a role in the pathogenesis of asthma, although it could also be a compensatory mechanism to other factors that cause bronchoconstriction or inflammation. To investigate the efficacy of NO donors on bronchial tone, the bronchorelaxing efficacies of NO donors, new experimental GEA compounds 3268 and 5145 (oxatriazole sulphonylamides) were compared with those of sodium nitroprusside and SIN‐1 (3‐morpholinosydnonimine) and to the standard β2‐adrenergic agonist, salbutamol, in bronchi of guinea‐pigs and rats in‐vitro. Their relaxing effects were also studied in rat mesentery arteries to compare the selectivity for airways. The capacity of the NO donors to produce nitrites and nitrates was assayed by the Griess reaction.

Hemin and Cobalt Protoporphyrin Inhibit NLRP3 Inflammasome Activation by Enhancing Autophagy: A Novel Mechanism of Inflammasome Regulation

Inflammasomes are intracellular protein platforms, which, upon activation, produce the highly proinflammatory cytokines interleukin (IL)-1β and IL-18. Heme, hemin and their degradation products possess significant immunomodulatory functions. Here, we studied whether hemin regulates inflammasome function in macrophages. Both hemin and its derivative, cobalt protoporphyrin (CoPP), significantly reduced IL-1β secretion by cultured human primary macrophages, the human monocytic leukemia cell line and also mouse bone marrow-derived and peritoneal macrophages. Intraperitoneal administration of CoPP to mice prior to urate crystal-induced peritonitis alleviated IL-1β secretion to the peritoneal cavity. In cultured macrophages, hemin and CoPP inhibited NLRP3 inflammasome assembly by reducing the amount of intracellular apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC). The reduction of ASC was associated with enhanced autophagosome formation and autophagic flux. Inhibition of autophagy prevented the CoPP-induced depletion of ASC, implying that the depletion was caused by increased autophagy. Our data indicate that hemin functions as an endogenous negative regulator of the NLRP3 inflammasome. The inhibition is mediated via enhanced autophagy that results in increased degradation of ASC. This regulatory mechanism may provide a novel approach for the treatment of inflammasome-related diseases.

Econazole, miconazole and SK&F 96365 inhibit depolarization-induced and receptor-operated contraction of guinea-pig isolated trachea in vitro

Econazole, miconazole, SK & F 96365 and nifedipine inhibited Ca2+- and depolarization-induced and receptor-operated contraction of guinea-pig isolated trachea. Econazole, miconazole and SK & F 96365 inhibited histamine- and methacholine-induced tracheal contraction more than nifedipine. Nifedipine was more potent in inhibiting KCl-induced contraction. Nifedipine, salbutamol and theophylline, but not econazole, miconazole or SK & F 96365, relaxed KCl, histamine-, and methacholine-precontracted trachea. It appears that in the guinea-pig tracheal smooth muscle, econazole, miconazole and SK & F 96365 behave differently from nifedipine, theophylline and salbutamol. Econazole, miconazole and SK & F 96365 are thus introduced as novel antagonists of receptor-operated airway smooth muscle contraction.

Proteomic identification of allergenic seed proteins, napin and cruciferin, from cold-pressed rapeseed oils.

In Finland and France atopic children commonly react to seeds of oilseed rape and turnip rape in skin prick tests (SPT) and open food challenges. These seeds are not as such in dietary use and therefore the routes of sensitization are unknown. Possible allergens were extracted from commercial cold-pressed and refined rapeseed oils and identified by gel-based tandem nanoflow liquid chromatography mass spectrometry (LC-MS/MS). Napin (a 2S albumin), earlier identified as a major allergen in the seeds of oilseed rape and turnip rape, and cruciferin (an 11S globulin), a new potential seed allergen, were detected in cold-pressed oils, but not in refined oils. Pooled sera from five children sensitized or allergic to oilseed rape and turnip rape seeds reacted to these proteins from cold-pressed oil preparations and individual sera from five children reacted to these proteins extracted from the seeds when examined with IgE immunoblotting. Hence cold-pressed rapeseed oil might be one possible route of sensitization for these allergens.

Role of BKCa channels and cyclic nucleotides in synergistic relaxation of trachea

beta-Adrenoceptor agonists, nitric oxide (NO), and NO donors have been shown to mediate their effects through large conductance Ca(2+)-activated K(+) (BK(Ca)) channels. The mechanism of the synergistic effect of the beta(2)-adrenoceptor agonist, salbutamol, and an NO donor, sodium nitroprusside, was studied in guinea pig tracheal preparations. Salbutamol (0.1 nM) and sodium nitroprusside (0.33 microM) alone relaxed the acetyl-beta-methylcholine chloride (methacholine)-contracted preparations only by 0.5% and 28%, respectively, but their combination caused a maximum of 60% relaxation (at 3 min), which stabilized to 40% (at 10 min). Iberiotoxin, a selective inhibitor of the BK(Ca) channels, did not abolish the synergistic effect. 3-isobutyl-1-methylxanthine (IBMX) did not modify relaxation evoked by the drugs. Concentrations of cyclic nucleotides did not correlate with relaxations as a function of time. The mechanism of synergy remains to be clarified. The results show that NO is an important modulator in the relaxation of guinea pig trachea induced by beta(2)-adrenoceptor agonists in vitro.

Effects of K+ channel inhibitors on relaxation induced by flufenamic and tolfenamic acids in guinea-pig trachea

The effects of different K(+) channel inhibitors on flufenamic- and tolfenamic-acids-induced relaxation were studied in prostaglandin F(2alpha) (1 microM) precontracted guinea-pig trachea. Flufenamic and tolfenamic acids (each 0.1-33 microM) and lemakalim (0.01-33 microM), but not indomethacin (0.1-33 microM), caused relaxation. Iberiotoxin (33 and 100 nM) inhibited flufenamic- and tolfenamic-acids-, but not lemakalim-, induced relaxation. Iberiotoxin (100 nM) inhibited nifedipine (10 nM-10 microM)-induced relaxation. Nifedipine (0.1 microM) inhibited the blockade of fenamate-induced relaxation by iberiotoxin. Apamin (0.1 and 1 microM) did not affect flufenamic- and tolfenamic-acids- and lemakalim-induced relaxation. Glibenclamide (10 and 33 microM) inhibited lemakalim-, but not flufenamic- and tolfenamic-acids-, induced relaxation. 4-Aminopyridine (0.5 and 2 mM) inhibited flufenamic- and tolfenamic- acids- and lemakalim-induced relaxation. Flufenamic- and tolfenamic-acids-induced relaxation is likely to be activation of Ca(2+)-activated K(+) channels and differs from that of lemakalim.