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.

Imatinib mesylate alleviates diarrhea in a mouse model of intestinal allergy.

BACKGROUND: When sensitized epicutaneously and challenged orally with ovalbumin, Balb/c mice develop allergen-induced diarrhea. As mast cells play important roles in diarrhea, we studied whether allergic diarrhea could be alleviated with imatinib mesylate. METHODS: Balb/c mice were sensitized and challenged with ovalbumin and treated orally with imatinib. Cytokine mRNA expressions were determined with quantitative RT-PCR and numbers of small intestinal mast cells determined by staining for chloroacetate esterase and mucosal mast cell protease-1. Immunofluorescence staining was used to assess the intestinal CCL1 expression. KEY RESULTS: Ovalbumin-sensitized and challenged Balb/c mice developed diarrhea, which was associated with increased number of mast cells and expression of interleukin (IL)-4 and -13, and chemokines CCL1 and CCL17 in the small intestine. Treatment with imatinib reduced the incidence of diarrhea, inhibited the development of mastocytosis and jejunal mRNA expression of IL-13, CCL1, CCL17 and CCL22. Mast cell-deficient W/W(-V) mice, and surprisingly, also their mast cell-competent control (+/+) littermates failed to develop diarrhea as a response to ovalbumin. This strain-dependent difference was associated with the inability of +/+ and W/W(-V) mice to increase the number of intestinal mast cells and expression of IL-4, IL-13, CCL1 and CCL17 after ovalbumin challenge. CONCLUSIONS & INFERENCES: Development of allergic diarrhea is associated with the ability of mice to develop intestinal mastocytosis. Imatinib inhibited the development of intestinal mastocytosis, reduced the incidence of diarrhea, and reduced the expression of IL-13, CCL1, and CCL17. Targeting intestinal mast cells could be a feasible approach to treat allergic diarrhea.

IgE sensitization to the fish parasite Anisakis simplex in a Norwegian population: a pilot study.

The reports on fish parasite Anisakis simplex allergy have increased in countries with high fish consumption in the last decade. In Norway, a high consumption country, the prevalence of immunoglobulin E (IgE) sensitization to A. simplex was still unknown. Thus, our objective was to investigate the sensitization prevalence in this country. At the Haukeland University Hospital, Bergen, Norway, two main groups of surplus serum samples were collected: one from newly recruited blood donors (BDO) and the other from the Allergy laboratory (ALL) after analysing IgE and IgE antibodies. The latter was divided into three series: one containing unsorted sera and two sorted by either Phadiatop(®) ≥0.35 kU(A)/l or total IgE ≥1000 kU/l. The sera were analysed for total IgE and IgE antibodies against A. simplex, shrimp, house dust mite (HDM), cod and cross-reactive carbohydrates (CCDs). The prevalence of IgE sensitization to A. simplex was 2.0%, 2.2% and 6.6% in BDO, the unsorted and Phadiatop(®) positive serum groups, respectively. A considerable degree of cross-sensitization to shrimp and HDM is further suggested. Unspecific binding because of high total IgE or by binding to CCDs seemed to play a minor role. The prevalence of IgE sensitization to A. simplex appears to be lower in a Norwegian population than in other high fish-consuming countries, but might still be overestimated owing to cross-sensitization.

Comparison between ovalbumin and ovalbumin peptide 323-339 responses in allergic mice: humoral and cellular aspects.

Ovalbumin (OVA) is widely used in allergy research. OVA peptide 323-339 has been reported to be responsible for 25-35% of isolated BALB/c mouse T-cell response to intact OVA. An investigation of whether OVA and OVA 323-339 molecules can induce equivalent in vivo and in vitro immune responses was conducted. Eight-week-old BALB/c mice were randomly divided into three groups: OVA, OVA 323-339 and saline. On days 0, 7, 14, mice were intraperitoneally injected with 25 microg OVA or OVA 323-339 absorbed on 300 microg Alum, or saline; on days 21-23, all groups were challenged intranasally with either 20 microl of 1% OVA, 1% OVA 323-339 or saline. On day 28, after killing, splenocytes were isolated and cultured under the stimulus of each allergen or medium. Evaluated by hematoxylin/eosin and major basic protein immunohistochemical stainings, OVA and OVA 323-339 induced similar lung inflammation. Interestingly, significant serum total IgE and OVA-specific IgE were observed in OVA mice when compared to saline control. OVA 323-339 mice showed higher serum OVA-specific IgE, OVA 323-339-specific IgE, IL-4 and lower IFN-gamma similar to OVA mice. The proliferative response to OVA was found in cultured splenocytes of both OVA and OVA 323-339 mice, while the similar proliferative response to OVA 323-339 was only observed in the splenocytes of OVA 323-339-sensitized and challenged mice. Although OVA 323-339 induced a Th2-like response in the mouse model as did OVA, OVA 323-339 has clearly limited immunogenic potency to activate OVA-sensitized and challenged mice splenocytes, unlike OVA.

Abnormal small-intestinal endocrine cells in patients with irritable bowel syndrome.

BACKGROUND: General disturbances in gastrointestinal motility have been reported in patients with irritable bowel syndrome (IBS). The gastrointestinal tract hormones play an important role in regulating gastrointestinal motility. AIMS: To investigate a possible abnormality in the small intestinal endocrine cells of IBS patients. METHODS: Included in the study were 41 patients with irritable bowel syndrome according to Rome Criteria III and 42 healthy controls. Duodenal biopsies were obtained from both patients and controls during gastroscopy. The biopsies were immunostained by avidin-biotin-complex method for secretin, CCK, GIP, somatostatin, and serotonin cells. The cell densities were quantified by computerized image analysis. RESULTS: The density of secretin- and CCK-immunoreactive cells in patients with IBS was significantly reduced. The reduction in secretin and CCK cells occurred only in IBS-diarrhea patients, but not in IBS-constipation subtype. Both GIP and somatostatin cell densities were reduced in the duodenum of IBS patients. There was no statistical difference between the subtypes of IBS patients, regarding secretin, CCK, GIP, or somatostatin cell densities. Serotonin cell density was not affected in patients with IBS. CONCLUSIONS: The low densities of secretin and CCK cells in IBS-diarrhea patients may cause a functional pancreatic insufficiency as well as inadequate gall emptying, as these hormones stimulate pancreatic bicarbonate and enzyme secretion and CCK stimulates as well gall bladder contraction. Low densities of secretin, GIP, and somatostatin cells in IBS patients might result in a high secretion of gastric acid, as secretin, GIP, and somatostatin inhibit gastric acid secretion.

Suppression of acute experimental colitis by a highly selective inducible nitric-oxide synthase inhibitor, N-[3-(aminomethyl)benzyl]acetamidine.

High concentrations of nitric oxide (NO) produced by the inducible nitric-oxide synthase (iNOS) are associated with ulcerative inflammation and disease activity in colitis. Therefore, inhibition of iNOS serves as a novel experimental approach to treat gut inflammation. The aim of the present study was to investigate the effects of a novel highly selective iNOS inhibitor, N-[3-(aminomethyl)benzyl]acetamidine (1400W), as compared with a nonselective NOS inhibitor, N(G)-nitro-L-arginine-methyl-ester (L-NAME), in 2,4,6-trinitrobenzenesulfonic acid (TNBS)-induced acute colitis in the rat. Increased expression of iNOS protein and mRNA was found in acute TNBS-induced colitis along with neutrophil infiltration, inflammatory edema, and tissue damage. In a 24-h model of acute colitis, subcutaneous injections of 1400W (5 or 10 mg/kg t.i.d.) produced a 56 and 95% reduction in inflammatory edema formation, a 68 and 63% reduction in neutrophil infiltration (measured as myeloperoxidase activity), and a 19 and 26% decrease in the size of mucosal lesions as compared with vehicle treatment. Administration of L-NAME (35 mg/kg) failed to produce any significant beneficial effects as compared with vehicle treatment in this experimental model of acute colitis. Treatment with 1400W, a highly selective inhibitor of iNOS, reduced formation of edema, neutrophil infiltration, and macroscopic inflammatory damage in experimentally induced acute colitis in the rat. In contrast, nonselective nitric-oxide synthase inhibition with L-NAME provided no benefit. These results support the idea that selective iNOS inhibitors have a promise in the treatment of colitis.

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 cyclooxygenase-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.

Role of BK(Ca) 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.

Relaxing effects of NO donors on guinea pig trachea in vitro are mediated by calcium-sensitive potassium channels.

The relaxing effects of the nitric oxide (NO) donors 1,2,3,4-oxatriazolium,3-(3-chloro-2-methylphenyl-5-[[(4-methoxyphe nyl) sulfonyl]amino]-,hydroxide inner salt (GEA 3268) 1,2,3,4-oxatriazolium,3-(3-chloro-2-methyphenyl-5-[methys ulfonyl)amino]- hydroxide inner salt (GEA 5145), 3-morpholinosydnonimine (SIN-1) and S-nitroso-N-acetylpenicillamine (SNAP) were inhibited in vitro by iberiotoxin (IbTX) and charybdotoxin (ChTX), the two selective inhibitors of Ca(++)-activated K+ channels (KCa) in guinea pig trachea. When studied in cumulative concentrations in metacholine constriction, the relaxing effects of the NO donors were inhibited by at least 70% in the presence of the toxins, with the exception of SIN-1 in the presence of ChTX. The inhibitory effect of ChTX was less marked than that of IbTX. This suggests that the relaxing effects of the structurally different NO donors are mediated through KCa channels and that IbTX is more potent than ChTX. A selective inhibitor of soluble guanylate cyclase, 1H-[1,2,4]oxadiazolo[4,3-a]quinozalin-1-one (ODQ), significantly inhibited the relaxing effects of GEA 3268 and GEA 5145 on metacholine and KCl constriction and almost totally inhibited the relaxing effects of SIN-1 and SNAP. The inhibitor of the delayed rectifier K+ channel current 4-aminopyridine did not influence the relaxations of the NO donors, and under the experimental conditions of this study, the ATP-sensitive K+ channel inhibitor glibenclamide had no effect. In conclusion, the relaxing effects of the structurally different NO-releasing compounds are mediated via KCa channels. However, the significance of some other possible mechanisms unrelated to K+ channels cannot be excluded.

Flufenamic and tolfenamic acids and lemakalim relax guinea-pig isolated trachea by different mechanisms.

The effects of K+ channel inhibitors on the relaxations induced by flufenamic and tolfenamic acids and lemakalim were examined in guinea-pig isolated trachea precontracted with prostaglandin F2alpha (PGF2alpha, 1 microM). Flufenamic and tolfenamic acids (0.1-33 microM) and lemakalim (0.01-33 microM) relaxed guinea-pig trachea in a concentration-dependent manner. Tetraethylammonium (0.5-2 mM), a nonspecific inhibitor of K+ channels, inhibited the relaxations induced by flufenamic and tolfenamic acids without affecting lemakalim-induced relaxation. Charybdotoxin (ChTX, 33-100 nM), an inhibitor of the large Ca2+-activated K+ channels (BK(Ca)), also inhibited the relaxations induced by flufenamic and tolfenamic acids without affecting lemakalim-induced relaxation. Glipizide (3.3-33 microM), an inhibitor of the ATP-sensitive K+ channels (K(ATP)) inhibited lemakalim-induced relaxation without affecting those induced by flufenamic and tolfenamic acids. Our results indicate that the relaxations of guinea-pig isolated trachea by flufenamic and tolfenamic acids are due to activation of BK(Ca). The relaxant mechanism of flufenamic and tolfenamic acids thus differs from that of lemakalim, an activator of K(ATP).

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

1. 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. 2. In the resting tracheal preparations with intact epithelium, bradykinin and [des-Arg9]-bradykinin (each drug, 0.01-10 microM) induced neither contraction nor relaxation. In contrast, bradykinin (0.01-10 microM) induced concentration-dependent relaxation when the tracheal preparations were precontracted with methacholine (1 microM). 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 microM) in a concentration-dependent manner whereas the B1 receptor antagonist, [des-Arg9,Leu8]-bradykinin (0.01-1 microM), had no inhibitory effect on bradykinin-induced relaxation. [des-Arg9]-bradykinin (0.01-10 microM) 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 microM), whereas the B2 receptor antagonist, Hoe 140 (0.01-1 microM) was without effect. 3. In the presence of the cyclo-oxygenase inhibitor, indomethacin (0.01-1 microM), the relaxations induced by bradykinin and [des-Arg9]-bradykinin were inhibited concentration-dependently. 4. Two nitric oxide (NO) biosynthesis inhibitors NG-nitro-L-arginine methyl ester (L-NAME, 100 microM) and NG-nitro-L-arginine (L-NOARG, 100 microM) 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 microM) inhibit the relaxations induced by bradykinin and [des-Arg9]-bradykinin. 5. Prostaglandin E2 (PGE2, 0.01-33 microM) caused concentration-dependent relaxation of the tracheal preparations precontracted with methacholine. Indomethacin (1 microM) and ODQ (10 microM) exerted no inhibitory effects on the relaxation induced by PGE2. 6. The NO-donor, sodium nitroprusside (SNP; 0.01-100 microM) also caused concentration-dependent relaxation of the tracheal preparations precontracted with methacholine. ODQ (0.1-1 microM) concentration-dependently inhibited the relaxation induced by SNP. 7. 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.

Interactions between beta2-adrenoreceptor agonist and NO donors in the relaxation of guinea pig trachea in vitro.

The effects of interaction between the beta2-adrenoreceptor agonist, salbutamol, and the nitric oxide donors, sodium nitroprusside and 3-morpholinosydnonimine (SIN-1), on guinea pig trachea contraction were studied. Cumulatively increased concentrations (0.1-10 nM) of salbutamol together with a single concentration of SNP (0.33 microM) or with SIN-1 (1 microM) showed significant (p < 0.001) synergy for the inhibition of 1 microM metacholine-induced contraction. Significant synergy (p < 0.05) was also found for the inhibition of this contraction by cumulatively increased concentrations (0.1-33 microM) of SNP and a single concentration (1 nM) of salbutamol. No synergistic effect was found on the 40 mM KC1-induced contraction. We suggest that the combination of NO donors with salbutamol has a synergistic effect on metacholine contraction.

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.

Bradykinin, lemakalim and sodium nitroprusside relax the mouse trachea in vitro by different mechanisms.

The role of K+ channels in the relaxations induced by bradykinin, lemakalim, an activator of ATP-sensitive K+ channels and sodium nitroprusside (SNP), a nitric oxide (NO) donor was examined in the isolated mouse trachea precontracted by methacholine (1 microM). 4-aminopyridine (4-AP, 0.1-2 mM), an inhibitor of 4-AP sensitive delayed rectifier channels, did not alter relaxations induced by bradykinin, lemakalim or SNP. Glibenclamide and glipizide (10-33 microM), inhibitors of ATP-sensitive K+ channels, inhibited relaxation to lemakalim without affecting responses to bradykinin and SNP. Charybdotoxin (10-100 nM) and iberiotoxin (10-100 nM), inhibitors of large conductance Ca2+-activated K+ channels, failed to inhibit relaxation to bradykinin, lemakalim or SNP. Apamin (0.1-1 microM), an inhibitor of small conductance Ca2+-activated K+ channels, did not alter responses to bradykinin, lemakalim and SNP. The results implicate that the mechanism of relaxation induced by bradykinin and SNP is different from that of lemakalim. Relaxation of the isolated mouse trachea by lemakalim appears to be mediated by ATP-sensitive K+ channels. Bradykinin and SNP induced relaxations are not mediated via Ca2+-activated K+ channels.

Role of nitric oxide in experimental obliterative bronchiolitis (chronic rejection) in the rat.

The role of nitric oxide in obliterative bronchiolitis development, i.e., chronic rejection, was investigated in the heterotopic rat tracheal allograft model. An increase in the intragraft inducible nitric oxide synthase (iNOS) mRNA and mononuclear inflammatory cell iNOS immunoreactivity was demonstrated during progressive loss of respiratory epithelium and airway occlusion in nontreated allografts compared to syngeneic grafts. In nontreated allografts, however, intragraft nitric oxide production was decreased, most likely because of loss of iNOS epithelial expression. Treatment with aminoguanidine, a preferential inhibitor of inducible nitric oxide synthase, was associated with enhanced proliferation of alpha-smooth muscle actin immunoreactive cells and the intensity of obliterative bronchiolitis early after transplantation. Aminoguanidine treatment did not affect iNOS mRNA synthesis or intragraft nitric oxide production, but decreased iNOS immunoreactivity in smooth muscle cells. Treatment with L-arginine, a precursor of nitric oxide, significantly reduced obliterative changes. L-arginine supplementation enhanced intragraft iNOS mRNA synthesis and iNOS immunoreactivity in capillary endothelial and smooth muscle cells as well as intragraft nitric oxide production. Immunohistochemical analysis of allografts showed that neither iNOS inhibition nor supplementation of the nitric oxide pathway affected the number of graft-infiltrating CD4+ and CD8+ T cells, ED1+ and ED3+ macrophages, immune activation with expression of IL-2R or MHC class II, or production of macrophage or Th1 cytokines. In contrast, L-arginine treatment was associated with increased staining for Th2 cytokines IL-4 and IL-10. In conclusion, this study demonstrates that nitric oxide has a protective role in obliterative bronchiolitis development in this model, and suggests that nitric oxide either directly or indirectly inhibits smooth muscle cell proliferation and modulates immune response towards Th2 cytokines.

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 beta 2-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. The novel NO donors GEA 3268 and GEA 5145 were more potent bronchorelaxing agents than the old NO donors sodium nitroprusside and SIN-1. In guinea-pig bronchi, however, salbutamol was most potent. In rat bronchi the GEA compounds induced the strongest relaxation effect when compared with the old NO donors or with salbutamol. The airway selectivity of the drugs studied decreased in the order of salbutamol, SIN-1, GEA 5145, GEA 3268, sodium nitroprusside. The nitrites and nitrates produced spontaneously did not correlate with the efficacy of the relaxants. The results obtained suggest that NO is only partly responsible for the relaxation and the potency is dependent on the animal species and constricting agents used.

Effects of potassium-channel opener HOE 234 in guinea-pig airways.

The mechanism of the bronchospasmolytic effect of HOE 234 ((3S,4R)-3-hydroxy-2,2-dimethyl-4-(2-oxo-1-pyrrolidinyl) 6-phenylsulphonylchromane hemihydrate), a novel opener of ATP-sensitive potassium channels, has been studied by in-vitro testing in ring preparations of trachea and different parts of the principle bronchus of guinea-pigs, using methacholine, histamine and KCl as preconstrictors. The contribution of prostanoids was estimated in the presence and absence of indomethacin. Regional differences in the bronchodilatory effect of HOE 234 were compared with that of salbutamol. HOE 234 had a concentration-dependent relaxing effect on the smooth muscle preparations. In the absence of indomethacin no regional differences were seen for the effect of HOE 234 against the metacholine or KCl preconstriction, whereas with histamine a particularly strong relaxation was detectable in trachea. In the presence of indomethacin, distal bronchus after methacholine and trachea after KCl preconstriction were relaxed significantly more strongly than the other parts. The relaxation of the histamine-constricted trachea rings was not increased in comparison with that without indomethacin pretreatment. Thus the bronchospasmolytic effect of HOE 234 varied depending on the pretreatment, method of preconstriction and part of the airways examined. It was strongest in trachea rings with histamine preconstriction but its potency was clearly less than that of salbutamol. The latter had regionally different effects, being stronger in trachea than in the bronchi for all methods of preconstriction. The results suggest that the formation of bronchoconstricting prostanoids can attenuate parts of the relaxing effect of HOE 234. The possible advantages of HOE 234 as an anti-asthma drug might be related to an effect on bronchial hyper-reactivity and mucus secretion, because as a direct bronchodilator it is inferior to salbutamol.