Allergic Sheep Research Articles

Effect of novel adenosine A3 receptor antagonist SSR161421 in allergic sheep models

Effect of novel adenosine A3 receptor antagonist SSR161421 in allergic sheep models

Induction of Allergic Responses to Peanut Allergen in Sheep

Peanut allergy is the leading cause of deaths due to food-induced anaphylaxis but despite continued research, there are currently no specific treatments available. Challenge testing is limited in patients due to the high risk of adverse reactions, emphasising the need for an appropriate animal model. In the present study we examine the induction of allergic responses in a sheep model for peanut allergy. Sheep were sensitised with peanut (PN) extract and in separate injections with ovalbumin (OVA) or house dust mite (HDM) extract. Serum PN-specific IgE responses were detected in 40–50% of immunised sheep, while only 10% (1 of 10 sheep) showed detectable OVA-specific IgE. All PN-allergic sheep tested showed an Ara h 1-specific IgE response, while four out of five allergic sheep showed an Ara h 2-specific IgE response. Animals with high serum IgE levels to HDM were also PN IgE-positive. Of the PN-sensitised animals with high PN-specific IgE, 80% also showed an immediate hypersensitivity reaction following an intradermal PN injection. This new large animal model of peanut allergy may provide a useful tool for future investigations of allergen-associated immune mechanisms and specific immunotherapy.

Effect of oral and intravenous heparin tetrasaccharide on allergic airway responses: Critical role of N-sulfation

We have shown that inhaled heparin (hep) oligosaccharides attenuate allergic airway responses in sheep and that this anti-allergic activity resides in a tetrasaccharide sequence. Here we determined: (a) the anti-allergic activity of oral and intravenous hep-tetrasaccharide on allergic airway responses in the sheep model of asthma; and (b) the role of N-sulfation in mediating this anti-allergic activity. Ascaris suum-induced early (EAR) and Late (LAR) airway responses and airway hyperresponsiveness (AHR) to carbachol were measured in allergic sheep without and after treatment with different doses of oral or intravenous hep-tetrasaccharide. At doses of 0.06 mg/kg, 0.125 mg/kg, and 0.25 mg/kg, oral hep-tetrasaccharide caused a dose-dependent inhibition of EAR and LAR. Post-antigen AHR was also inhibited dose dependently. The same doses of intravenous hep-tetrasaccharide yielded comparable inhibition of EAR, LAR and AHR, confirming that orally delivered hep-tetrasaccharide has good bioavailability. The protection by hep-tetrasaccharide on EAR and LAR was dependent on N-sulfation, as N-desulfated/N-acetylated tetrasaccharide had a markedly reduced effect. However, inhibition of the post-antigen AHR was independent of N-sulfation. These results demonstrate that orally administered hep-tetrasaccharide inhibits allergic airway responses in the sheep model of asthma. Hep-tetrasaccharide has good oral bioavailability and its anti-allergic activity is critically dependent on N-sulfation of the glucosamine ring.

Increased Mast Cell Density and Airway Responses to Allergic and Non-Allergic Stimuli in a Sheep Model of Chronic Asthma

BackgroundIncreased mast cell (MC) density and changes in their distribution in airway tissues is thought to contribute significantly to the pathophysiology of asthma. However, the time sequence for these changes and how they impact small airway function in asthma is not fully understood. The aim of the current study was to characterise temporal changes in airway MC density and correlate these changes with functional airway responses in sheep chronically challenged with house dust mite (HDM) allergen.Methodology/Principal FindingsMC density was examined on lung tissue from four spatially separate lung segments of allergic sheep which received weekly challenges with HDM allergen for 0, 8, 16 or 24 weeks. Lung tissue was collected from each segment 7 days following the final challenge. The density of tryptase-positive and chymase-positive MCs (MCT and MCTC respectively) was assessed by morphometric analysis of airway sections immunohistochemically stained with antibodies against MC tryptase and chymase.MCT and MCTC density was increased in small bronchi following 24 weeks of HDM challenges compared with controls (P<0.05). The MCTC/MCT ratio was significantly increased in HDM challenged sheep compared to controls (P<0.05). MCT and MCTC density was inversely correlated with allergen-induced increases in peripheral airway resistance after 24 weeks of allergen exposure (P<0.05). MCT density was also negatively correlated with airway responsiveness after 24 challenges (P<0.01).ConclusionsMCT and MCTC density in the small airways correlates with better lung function in this sheep model of chronic asthma. Whether this finding indicates that under some conditions mast cells have protective activities in asthma, or that other explanations are to be considered requires further investigation.

125 The Association between Mast Cells and Remodelling of the Small Airways in Chronic Asthma

BackgroundRepeated airway challenges with House Dust Mite (HDM) allergen results in marked remodelling and mast cell hyperplasia in the small airways of allergic sheep. We now examine mast cell activation and its association with small airway function and remodelling in these sheep using a novel segmental allergen challenge approach.MethodsEight allergic sheep received weekly intra-lung challenges of HDM to the left caudal lung for 24 weeks. Eight separate sheep were used as controls. Baseline lung function was assessed in the left caudal segments of all sheep throughout the challenge regime using a wedged-bronchoscope technique. Airway tissue was collected from challenged segments from all sheep, 7 days following the final intra-lung challenge. The airway tissues were immunohistochemically labelled for chymase-mast cells and eosinophils. Collagen and airway smooth muscle content were assessed on Masson's Trichrome stained sections.ResultsResting lung function in the left caudal segment is elevated in 4 out of 8 sheep at the end of the repeated allergen challenge regime. Chymase mast cell density was significantly increased in the small bronchial walls of the HDM-challenged group compared to the control group (52 ± 8 vs 8 ± 4; P < 0.01). There were significant increases in bronchial collagen deposition in HDM-exposed segments compared to control segments (0.17 ± 0.02 vs 0.11 ± 0.02 mm2/BM, P < 0.05). A correlation analysis of individual sheep data showed that there was a trend for a direct association between the increases in bronchial collagen deposition and the density of chymase-labelled mast cells (rs = 0.71, P = 0.088). Eosinophil density in the small bronchial walls of HDM-challenged segments was also significantly increased compared to controls (65 ± 19 vs 11 ± 3 cells/mm2, P < 0.001), but not associated with collagen content. The bronchial smooth muscle content was not different between HDM-challenged and unexposed control segments.ConclusionsThe results show that repeated exposure to allergen results in significant increases in density of chymase-labelled mast cells, together with increased levels of collagen content in the small airways. The segmental challenge protocol allows for a novel approach to characterise the progressive remodelling events occurring in the small airways in chronic asthma.

Inhibition of allergic airway responses by heparin derived oligosaccharides: identification of a tetrasaccharide sequence

BackgroundPrevious studies showed that heparin's anti-allergic activity is molecular weight dependent and resides in oligosaccharide fractions of <2500 daltons.ObjectiveTo investigate the structural sequence of heparin's anti-allergic domain, we used nitrous acid depolymerization of porcine heparin to prepare an oligosaccharide, and then fractionated it into disaccharide, tetrasaccharide, hexasaccharide, and octasaccharide fractions. The anti-allergic activity of each oligosaccharide fraction was tested in allergic sheep.MethodsAllergic sheep without (acute responder) and with late airway responses (LAR; dual responder) were challenged with Ascaris suum antigen with and without inhaled oligosaccharide pretreatment and the effects on specific lung resistance and airway hyperresponsiveness (AHR) to carbachol determined. Additional inflammatory cell recruitment studies were performed in immunized ovalbumin-challenged BALB/C mice with and without treatment.ResultsThe inhaled tetrasaccharide fraction was the minimal effective chain length to show anti-allergic activity. This fraction showed activity in both groups of sheep; it was also effective in inhibiting LAR and AHR, when administered after the antigen challenge. Tetrasaccharide failed to modify the bronchoconstrictor responses to airway smooth muscle agonists (histamine, carbachol and LTD4), and had no effect on antigen-induced histamine release in bronchoalveolar lavage fluid in sheep. In mice, inhaled tetrasaccharide also attenuated the ovalbumin-induced peribronchial inflammatory response and eosinophil influx in the bronchoalveolar lavage fluid. Chemical analysis identified the active structure to be a pentasulfated tetrasaccharide ([IdoU2S (1→4)GlcNS6S (1→4) IdoU2S (1→4) AMan-6S]) which lacked anti-coagulant activity.ConclusionsThese results demonstrate that heparin tetrasaccharide possesses potent anti-allergic and anti-inflammatory properties, and that the domains responsible for anti-allergic and anti-coagulant activity are distinctly different.

Repeated exposure to aerosolized brevetoxin-3 induces prolonged airway hyperresponsiveness and lung inflammation in sheep

Context: During a Florida red tide, brevetoxins (PbTxs) produced by Karenia brevis become aerosolized and can cause both immediate and prolonged airway symptoms in humans, especially in those with preexisting airway disease (e.g., asthma). Although environmental monitoring indicates that toxins remain airborne for up to 4 consecutive days, there is little information on airway responses after multiple-day exposures.Objectives: To delineate putative mechanisms leading to pulmonary dysfunction after PbTx exposure, we studied airway responses before and after multiple exposures to aerosol PbTx-3, the most potent PbTx produced, in nonallergic (healthy) and in allergic sheep, which serve as a surrogate for patients with compromised airways.Methods: Both groups were exposed to 20 breaths of increasing concentrations of PbTx-3 (30–300 pg/mL) for 4 consecutive days. Airway responsiveness to carbachol (1 and 8 days after) and airway inflammation as assessed by bronchoalveolar lavage (0 and 7 days after) were measured.Results: Both groups developed airway hyperresponsiveness (AHR) 1 day after challenge; the severity was concentration dependent and more severe in the allergic group. AHR remained after 8 days, but the difference in the severity between the groups was lost. Both groups developed an inflammatory response after exposure to 300 pg/mL PbTx-3. Immediately after exposure, lung neutrophilia was prominent. This neutrophilia persisted for 7 days in addition to increases in total cells and macrophages.Conclusion: Repeated exposures to PbTx-3 result in prolonged AHR and lung inflammation. These pathophysiologic responses could be underlying contributors to the prolonged respiratory symptoms in humans after red tides.

An ovine tracheal explant culture model for allergic airway inflammation

BackgroundThe airway epithelium is thought to play an important role in the pathogenesis of asthmatic disease. However, much of our understanding of airway epithelial cell function in asthma has been derived from in vitro studies that may not accurately reflect the interactive cellular and molecular pathways active between different tissue constituents in vivo.MethodsUsing a sheep model of allergic asthma, tracheal explants from normal sheep and allergic sheep exposed to house dust mite (HDM) allergen were established to investigate airway mucosal responses ex vivo. Explants were cultured for up to 48 h and tissues were stained to identify apoptotic cells, goblet cells, mast cells and eosinophils. The release of cytokines (IL-1α, IL-6 and TNF-α) by cultured tracheal explants, was assessed by ELISA.ResultsThe general morphology and epithelial structure of the tracheal explants was well maintained in culture although evidence of advanced apoptosis within the mucosal layer was noted after culture for 48 h. The number of alcian blue/PAS positive mucus-secreting cells within the epithelial layer was reduced in all cultured explants compared with pre-cultured (0 h) explants, but the loss of staining was most evident in allergic tissues. Mast cell and eosinophil numbers were elevated in the allergic tracheal tissues compared to naïve controls, and in the allergic tissues there was a significant decline in mast cells after 24 h culture in the presence or absence of HDM allergen. IL-6 was released by allergic tracheal explants in culture but was undetected in cultured control explants.ConclusionsSheep tracheal explants maintain characteristics of the airway mucosa that may not be replicated when studying isolated cell populations in vitro. There were key differences identified in explants from allergic compared to control airways and in their responses in culture for 24 h. Importantly, this study establishes the potential for the application of tracheal explant cultures in relevant ex vivo investigations on the therapeutic and mechanistic modalities of asthmatic disease.

Discovery of an Oral Potent Selective Inhibitor of Hematopoietic Prostaglandin D Synthase (HPGDS)

Hematopoietic prostaglandin D synthase (HPGDS) is primarly expressed in mast cells, antigen-presenting cells, and Th-2 cells. HPGDS converts PGH2 into PGD2, a mediator thought to play a pivotal role in airway allergy and inflammatory processes. In this letter, we report the discovery of an orally potent and selective inhibitor of HPGDS that reduces the antigen-induced response in allergic sheep.

Dual Inhibition of Cathepsin G and Chymase Is Effective in Animal Models of Pulmonary Inflammation

Mast cells and neutrophils are key contributors to the pathophysiological inflammatory processes that underpin asthma and chronic obstructive pulmonary disease, partly through the release of noxious serine proteases, including cathepsin G (Cat G) and chymase. From this standpoint, a dual inhibitor of neutrophil Cat G and mast cell chymase could protect against these disease-related inflammatory responses. We examined the antiinflammatory pharmacology of RWJ-355871, a dual inhibitor of Cat G and chymase, in animal models of inflammation that evince pathophysiological pathways relevant to asthma and chronic obstructive pulmonary disease to determine the therapeutic potential of this compound. In an ovalbumin (OVA)-sensitized rat model, RWJ-355871 was administered to block the mast-cell-mediated increase in paw volume caused by OVA injection. In a sheep asthma model, antigen-induced airway responses were assessed with and without aerosol treatment with RWJ-355871. In a murine tobacco-smoke model of airway inflammation, the effect of RWJ-355871 on smoke-induced neutrophilia was determined. Intravenous treatment of OVA-sensitized rats with RWJ-355871 provided dose-dependent reduction in the increase in rat paw volume. In allergic sheep, aerosol pretreatment with RWJ-355871 showed dose-dependent inhibition of the antigen-induced early response, late response, and post-antigen-induced airway hyperreponsiveness. In tobacco-smoke-exposed mice, nebulized RWJ-355871 significantly reduced the smoke-induced neutrophilia from the levels observed in untreated mice. The preclinical antiinflammatory effects of RWJ-355871 in these animal models of inflammation indicate that this dual inhibitor may have therapeutic utility for treating airway inflammatory diseases involving mechanisms that depend on Cat G and/or chymase.

Specific inhibition of tissue kallikrein 1 with a human monoclonal antibody reveals a potential role in airway diseases

KLK1 (tissue kallikrein 1) is a member of the tissue kallikrein family of serine proteases and is the primary kinin-generating enzyme in human airways. DX-2300 is a fully human antibody that inhibits KLK1 via a competitive inhibition mechanism (Ki=0.13 nM). No binding of DX-2300 to KLK1 was observed in a surface-plasmon-resonance biosensor assay when KLK1 was complexed to known active-site inhibitors, suggesting that DX-2300 recognizes the KLK1 active site. DX-2300 did not inhibit any of the 21 serine proteases that were each tested at a concentration of 1 microM. We validated the use of DX-2300 for specific KLK1 inhibition by measuring the inhibition of KLK1-like activity in human urine, saliva and bronchoalveolar lavage fluid, which are known to contain active KLK1. In human tracheobronchial epithelial cells grown at the air/liquid interface, DX-2300 blocked oxidative-stress-induced epidermal-growth-factor receptor activation and downstream mucus cell proliferation and hypersecretion, which have been previously shown to be mediated by KLK1. In an allergic sheep model of asthma, DX-2300 inhibited both allergen-induced late-phase bronchoconstriction and airway hyper-responsiveness to carbachol. These studies demonstrate that DX-2300 is a potent and specific inhibitor of KLK1 that is efficacious in in vitro and in vivo models of airway disease.

Hyaluronan blocks porcine pancreatic elastase-induced mucociliary dysfunction in allergic sheep

Neutrophil elastase is a mediator common to asthma, chronic obstructive pulmonary disease, and cystic fibrosis and thought to contribute to the pathophysiology of these diseases. Previously, we found that inhaled hyaluronan blocked elastase-induced bronchoconstriction in allergic sheep through its control of tissue kallikrein. Here, we extend those studies by determining if inhaled hyaluronan can protect against the elastase-induced depression in tracheal mucus velocity, a surrogate marker of whole lung mucociliary clearance. We measured tracheal mucus velocity in allergic sheep before, and sequentially for 6 h after, aerosol challenge with porcine pancreatic elastase alone and after pretreatment with 1.5 or 6 mg aerosolized hyaluronan. Elastase (2.55 U) decreased tracheal mucus velocity. Pretreatment with 6 mg, but not 1.5 mg, hyaluronan inhibited the elastase-induced decrease in tracheal mucus velocity. Hyaluronan (6 mg) given 1 h after elastase challenge was ineffective, suggesting the involvement of secondary mediators. The elastase-induced depression in mucus transport appeared to be mediated, in part, by reactive oxygen species and bradykinin because pretreatment with either aerosolized catalase (38 mg/3 ml) or the bradykinin B2-receptor antagonist HOE140 (400 nM/kg) was also effective in blocking the response. These latter two findings are consistent with oxygen radical-induced degradation of hyaluronan with concomitant loss of its regulatory effect on tissue kallikrein, resulting in kinin generation. This hypothesis is supported by the demonstration that hyaluronan failed to block the oxygen radical-induced fall in tracheal mucus velocity resulting from xanthine-xanthine oxidase challenge and that inhaled bradykinin itself can slow mucociliary transport.

Whole-Body Periodic Acceleration Modifies Experimental Asthma in Sheep

Nitric oxide is released from vascular endothelium in response to increased pulsatile shear stress. Nitric oxide inhibits mast cell activation and is antiinflammatory and therefore might be protective in asthma. We determined if a noninvasive motion platform that imparts periodic sinusoidal inertial forces to the whole body along the spinal axis (pGz) causing release of endothelial nitric oxide modulates experimental asthma in sheep. Allergic sheep were untreated (control) or were treated with pGz alone or after receiving intravenously the nitric oxide synthase inhibitor N(w)-nitro-L-arginine methyl ester (L-NAME) before aerosol challenge with Ascaris suum, and the effect on antigen-induced airway responses was determined. Bronchoalveolar lavage cells obtained 6 h after antigen challenge were analyzed for nuclear factor-kappaB (NF-kappaB) activity in the respective groups. pGz treatment for 1 h before antigen challenge reduced the early airway response and blocked the late airway response but did not prevent the antigen-induced airway hyperresponsiveness 24 h after challenge. Administration of L-NAME before pGz completely reversed this protection, whereas L-NAME alone did not affect the antigen-induced responses. NF-kappaB activity was 1.9- and 1.8-fold higher in the control and L-NAME + pGz groups, respectively, compared with pGz-treated animals. Extending the pGz treatment to twice daily for 3 d and then 1 h before antigen challenge blocked the early and late airway responses, the 24-h airway hyperresponsiveness, and the airway inflammatory cell response. Whole-body pGz modulates allergen-induced airway responses in allergic sheep.

Nafamostat Mesilate (FUT) and Gabexate Mesilate (FOY) Attenuates Allergic Airway Inflammation and Eosinophilia in Dermatophagoides pteronyssinus - Sensitized Mice

RATIONALE: Serine and tryptase inhibitors have been shown to reduce antigen-induced airway hyper-reactivity in allergic guinea pigs and sheep. But the precise modes of anti-inflammatory mechanisms of these tryptase inhibitors are still unclear.We sought to investigate the effects of protease inhibitors (FUT, FOY, UTI) on airway inflammation in a mouse model of allergic asthma.

Seasick Lungs: How Airborne Algal Toxins Trigger Asthma Symptoms

Adverse health effects from harmful algal blooms have most frequently been linked to eating fish or shellfish that have accumulated algal toxins. However, people have also suffered asthma-like symptoms after inhaling minute amounts of algal toxins that were aerosolized by waves. Now a research team uses an animal model to gain a better understanding of how exposure to airborne algal toxins causes these symptoms and whether available drugs can be used to prevent or relieve them [EHP 113:632–637]. The researchers focused on toxins produced by a subtropical species of dinoflagellate (Karenia brevis) that is a major component of a bloom known as Florida red tide. K. brevis produces at least nine types of brevetoxins. When ingested, brevetoxins cause neurotoxic shellfish poisoning, with symptoms that can include numbness, tingling, and gastrointestinal distress. Persons exposed to aerosolized brevetoxins may suffer shortness of breath, sneezing, and other allergy- and asthma-like symptoms. Persons with preexisting airway disease appear most likely to be affected. To study airborne toxin exposure in a more controlled setting, the research team used a sheep model of asthma. The sheep model used is naturally sensitive to an antigen derived from the roundworm Ascaris suum, developing asthma-like symptoms (such as airway constriction) when exposed to this antigen. The sheep therefore can serve as surrogates for persons with asthma. To simulate environmental brevetoxin exposures, these allergic sheep were exposed to crude brevetoxins. These samples contained a variety of brevetoxin species (because multiple types are usually found in Florida red tide) as well as other parts of algal cells (because the toxin is usually released as the algae die and begin to decompose). In addition, the animals were also exposed to two types of purified brevetoxin, presumed to be the primary agent causing respiratory symptoms. In some studies the animals were treated before or after exposure with one of several clinically available medications. Exposure to crude brevetoxins caused immediate bronchoconstriction in the sheep as evidenced by a twofold increase in airway constriction. This immediate bronchoconstriction was inhibited by 49% (compared with untreated animals) in animals that were pre-treated with budesonide, by 71% in animals pretreated with albuterol, by 58% in animals pretreated with atropine, and by 47% in animals pretreated with diphenhydramine. In addition, bronchoconstriction was quickly reversed in animals that had not been premedicated if they were dosed with albuterol immediately after exposure. The fact that diphenhydramine, a histamine antagonist, reduced airway symptoms indicates that brevetoxins activate histamine-producing cells, such as mast cells and basophils. Further proof of the involvement of these cells was gained from studies where toxin was injected into the animals’ skin. In these skin tests, the reaction was up to 75% smaller if animals were pretreated with diphenhydramine. The effectiveness of atropine, an anticholinergic agent, indicates that brevetoxins also activate neural pathways. The cholinergic pathway is involved in the regulation of the neurotransmitter acetylcholine, and is also activated by exposure to organophosphate pesticides. The researchers also found that bronchoconstriction was reduced by 34% when animals were treated with HOE-140, a bradykinin β2 receptor antagonist. This response indicates that, in addition to raising histamine levels, exposure to brevetoxins also increases the level of bradykinin, a protein with effects similar to histamine that has also been linked to asthma symptoms. Asthmatics are more sensitive to bradykinin than are individuals with normal airways, and kinin levels are increased in inflamed airways. This may explain why the researchers found that animals whose airways were already inflamed responded more strongly to brevetoxins. Thus, the increased responsiveness to toxin in persons with preexisting airway disease may be linked to their underlying airway inflammation at the time of toxin exposure. This research shows that brevetoxins are potent airway constrictors, triggering several physiological pathways. The research has also determined drugs that could mitigate symptoms and serve as rescue medications for persons with severe reactions to brevetoxins.

Therapeutic Targeting of Endothelial Ligands for L-selectin (PNAd) in a Sheep Model of Asthma

The homing of lymphocytes to peripheral lymph nodes is initiated by an adhesive interaction between L-selectin on lymphocytes and PNAd, a set of sialomucins that are constitutively displayed on high endothelial venules of lymph nodes. PNAd is defined by monoclonal antibody MECA-79 that recognizes a sulfated oligosaccharide carried by the sialomucins. This epitope overlaps with 6-sulfo sialyl Lewis x, a recognition determinant for L-selectin. Previous work has shown that administration of a L-selectin monoclonal antibody blocks both late-phase airway responses and airway hyperresponsiveness in a sheep model of asthma. We show here that airway-associated lymphoid collections from lungs of allergic sheep exhibited PNAd(+) venules as detected by immunostaining with MECA-79. The same vessels also expressed a GlcNAc-6-O-sulfotransferase known as HEC-GlcNAc6ST, which is known to contribute to the formation of the MECA-79 epitope in high endothelial venules of mouse lymph nodes. Intravenous administration of MECA-79 to allergic sheep significantly blunted both the late-phase airway response and airway hyperresponsiveness induced by airway allergen challenge. Furthermore, MECA-79 inhibited the accumulation of all classes of leukocytes in bronchoalveolar lavage fluid. These findings represent the first demonstration that targeting of PNAd has therapeutic efficacy in an inflammatory disease.

Synthesis and Biological Evaluation of 1,2,4-Triazinylphenylalkylthiazolecarboxylic Acid Esters as Cytokine-Inhibiting Antedrugs with Strong Bronchodilating Effects in an Animal Model of Asthma

The influx of leukocytes (eosinophils, lymphocytes, and monocytes) into the airways and their production of proinflammatory cytokines contribute to the severity of allergic asthma. We describe here the synthesis and pharmacological evaluation of a series of triazinylphenylalkylthiazolecarboxylic acid esters that were designed to act as lung-specific antedrugs and inhibitors of the production of interleukin (IL)-5, a primary eosinophil-activating and proinflammatory cytokine. Closer examination of the hydroxypropyl ester, 15, indicated its high metabolic stability (t(1/2) > 240 min) in human lung S9 fraction but rapid conversion (t(1/2) = 15 min) into the pharmacologically inactive carboxylic acid by human liver preparations. In stimulated human whole blood cultures, 15 reduced not only the production of IL-5 (IC(50) = 78 nM) but also the biosynthesis of the monocyte chemotactic proteins MCP-1 (IC(50) = 220 nM), MCP-2 (IC(50) = 580 nM), and MCP-3 (IC(50) = 80 nM). In vivo, intratracheal administration of 15 (6 mg/animal) to allergic sheep, either before (-4 h) or after (+1.5 h) the pulmonary allergen challenge, completely abrogated the late-phase airway response and reduced the bronchial hyperreactivity to inhaled carbachol.

Airway responses to aerosolized brevetoxins in an animal model of asthma.

Florida red tide brevetoxins are sodium channel neurotoxins produced by the dinoflagellate Karenia brevis. When aerosolized, the toxin causes airway symptoms in normal individuals and patients with airway disease, but systematic exposures to define the pulmonary consequences and putative mechanisms are lacking. Here we report the effects of airway challenges with lysed cultures of Karenia brevis (crude brevetoxin), pure brevetoxin-2, brevetoxin-3, and brevetoxin-tbm (brevetoxin-2 minus the side chain) on pulmonary resistance and tracheal mucus velocity, a marker of mucociliary clearance, in allergic and nonallergic sheep. Picogram concentrations of toxin caused bronchoconstriction in both groups of sheep. Brevetoxin-tbm was the least potent, indicating the importance of the side chain for maximum effect. Both histamine H(1)- and cholinergic-mediated pathways contributed to the bronchoconstriction. A synthetic antagonist, beta-naphthoyl-brevetoxin-3, and brevenal, a natural antagonist, inhibited the bronchoconstriction. Only crude brevetoxin and brevetoxin-3 decreased tracheal mucus velocity; both antagonists prevented this. More importantly, picomolar concentrations of the antagonists alone improved tracheal mucus velocity to the degree seen with mM concentrations of the sodium channel blocker amiloride. Thus, Karenia brevis, in addition to producing toxins that adversely affect the airways, may be a source of agents for treating mucociliary dysfunction.

Leukocytic cell sources of airway tissue kallikrein.

Lung tissue kallikrein (TK) is a serine proteinase that putatively plays a role in the pathophysiology of asthma by generating kallidin and bradykinin, mediators that contribute to airway hyperresponsiveness. In previous studies we observed biphasic increases in TK activity in bronchoalveolar lavage fluid following airway allergen challenge in allergic sheep. Although glandular TK is likely a major source of the initial increase in TK, the sources of the late increases in TK that are associated with the development of airway hyperresponsiveness may be dependent on activated resident and recruited inflammatory cells including alveolar macrophages (AMs) and neutrophils (PMNs). These cells increase concomitantly with the late increases in TK activity. To test this hypothesis, we obtained AMs from bronchoalveolar lavage fluid and PMNs and monocytes (precursors of AMs) from sheep blood and determined whether these cells contained TK and whether these same cells could release TK upon activation. Using confocal microscopy, immunocytochemical techniques, and enzyme activity assays, we found that all three cell types contained and secreted TK. All three cell types demonstrated basal release of TK, which could be increased after stimulation with zymosan. In addition, PMNs also released TK in the presence of phorbol ester, suggesting multiple secretory pathways in these cells. Furthermore, we showed that human monocytes also contain and secrete TK. We conclude that in the airways, monocytes, PMNs, and AMs may contribute to increased TK activity. Knowing the sources of TK in the airways could be important in understanding the mechanisms of inflammation that contribute to the pathophysiology of asthma and may help in the development of new therapies to control the disease.

Potent, small-molecule inhibitors of human mast cell tryptase. Antiasthmatic action of a dipeptide-based transition-state analogue containing a benzothiazole ketone.

Inhibitors of human mast cell tryptase (EC 3.4.21.59) have therapeutic potential for treating allergic or inflammatory disorders. We have investigated transition-state mimetics possessing a heterocycle-activated ketone group and identified in particular benzothiazole ketone (2S)-6 (RWJ-56423) as a potent, reversible, low-molecular-weight tryptase inhibitor with a K(i) value of 10 nM. A single-crystal X-ray analysis of the sulfate salt of (2S)-6 confirmed the stereochemistry. Analogues 12 and 15-17 are also potent tryptase inhibitors. Although RWJ-56423 potently inhibits trypsin (K(i) = 8.1 nM), it is selective vs other serine proteases, such as kallikrein, plasmin, and thrombin. We obtained an X-ray structure of (2S)-6 complexed with bovine trypsin (1.9-A resolution), which depicts inter alia a hemiketal involving Ser-189, and hydrogen bonds with His-57 and Gln-192. Aerosol administration of 6 (2R,2S; RWJ-58643) to allergic sheep effectively antagonized antigen-induced asthmatic responses, with 70-75% blockade of the early response and complete ablation of the late response and airway hyperresponsiveness.