The discovery of potent, selective, and reversible inhibitors of the house dust mite peptidase allergen Der p 1: an innovative approach to the treatment of allergic asthma.

Blocking the bioactivity of allergens is conceptually attractive as a small-molecule therapy for allergic diseases but has not been attempted previously. Group 1 allergens of house dust mites (HDM) are meaningful targets in this quest because they are globally prevalent and clinically important triggers of allergic asthma. Group 1 HDM allergens are cysteine peptidases whose proteolytic activity triggers essential steps in the allergy cascade. Using the HDM allergen Der p 1 as an archetype for structure-based drug discovery, we have identified a series of novel, reversible inhibitors. Potency and selectivity were manipulated by optimizing drug interactions with enzyme binding pockets, while variation of terminal groups conferred the physicochemical and pharmacokinetic attributes required for inhaled delivery. Studies in animals challenged with the gamut of HDM allergens showed an attenuation of allergic responses by targeting just a single component, namely, Der p 1. Our findings suggest that these inhibitors may be used as novel therapies for allergic asthma

Mucosal sensitization to German cockroach involves protease-activated receptor-2

<p>Abstract</p> <p>Background</p> <p>Allergic asthma is on the rise in developed countries. A common characteristic of allergens is that they contain intrinsic protease activity, and many have been shown to activate protease-activated receptor (PAR)-2 <it>in vitro</it>. The role for PAR-2 in mediating allergic airway inflammation has not been assessed using a real world allergen.</p> <p>Methods</p> <p>Mice (wild type or PAR-2-deficient) were sensitized to German cockroach (GC) feces (frass) or protease-depleted GC frass by either mucosal exposure or intraperitoneal injection and measurements of airway inflammation (IL-5, IL-13, IL-17A, and IFNγ levels in the lung, serum IgE levels, cellular infiltration, mucin production) and airway hyperresponsiveness were performed.</p> <p>Results</p> <p>Following systemic sensitization, GC frass increased airway hyperresponsiveness, Th2 cytokine release, serum IgE levels, cellular infiltration and mucin production in wild type mice. Interestingly, PAR-2-deficient mice had similar responses as wild type mice. Since these data were in direct contrast to our finding that mucosal sensitization with GC frass proteases regulated airway hyperresponsiveness and mucin production in BALB/c mice (Page et. al. 2007 Resp Res 8:91), we backcrossed the PAR-2-deficient mice into the BALB/c strain. Sensitization to GC frass could now occur via the more physiologically relevant method of intratracheal inhalation. PAR-2-deficient mice had significantly reduced airway hyperresponsiveness, Th2 and Th17 cytokine release, serum IgE levels, and cellular infiltration compared to wild type mice when sensitization to GC frass occurred through the mucosa. To confirm the importance of mucosal exposure, mice were systemically sensitized to GC frass or protease-depleted GC frass via intraperitoneal injection. We found that removal of proteases from GC frass had no effect on airway inflammation when administered systemically.</p> <p>Conclusions</p> <p>We showed for the first time that allergen-derived proteases in GC frass elicit allergic airway inflammation via PAR-2, but only when allergen was administered through the mucosa. Importantly, our data suggest the importance of resident airway cells in the initiation of allergic airway disease, and could make allergen-derived proteases attractive therapeutic targets.</p

Clara Cell 10-kDa Protein Gene Transfection Inhibits NF-κB Activity in Airway Epithelial Cells

Clara cell 10-kDa protein (CC10) is a multifunctional protein with anti-inflammatory and immunomodulatory effects. Induction of CC10 expression by gene transfection may possess potential therapeutic effect. Nuclear factor κB (NF-κB) plays a key role in the inflammatory processes of airway diseases.To investigate potential therapeutic effect of CC10 gene transfection in controlling airway inflammation and the underlying intracellular mechanisms, in this study, we constructed CC10 plasmid and transfected it into bronchial epithelial cell line BEAS-2B cells and CC10 knockout mice. In BEAS-2B cells, CC10's effect on interleukin (IL)-1β induced IL-8 expression was explored by means of RT-PCR and ELISA and its effect on NF-κB classical signaling pathway was studied by luciferase reporter, western blot, and immunoprecipitation assay. The effect of endogenous CC10 on IL-1β evoked IL-8 expression was studied by means of nasal explant culture. In mice, CC10's effect on IL-1β induced IL-8 and nuclear p65 expression was examined by immunohistochemistry. First, we found that the CC10 gene transfer could inhibit IL-1β induced IL-8 expression in BEAS-2B cells. Furthermore, we found that CC10 repressed IL-1β induced NF-κB activation by inhibiting the phosphorylation of IκB-α but not IκB kinase-α/β in BEAS-2B cells. Nevertheless, we did not observe a direct interaction between CC10 and p65 subunit in BEAS-2B cells. In nasal explant culture, we found that IL-1β induced IL-8 expression was inversely correlated with CC10 levels in human sinonasal mucosa. In vivo study revealed that CC10 gene transfer could attenuate the increase of IL-8 and nuclear p65 staining in nasal epithelial cells in CC10 knockout mice evoked by IL-1β administration.These results indicate that CC10 gene transfer may inhibit airway inflammation through suppressing the activation of NF-κB, which may provide us a new consideration in the therapy of airway inflammation

Stimulation of surfactant lipid secretion from fetal type II pneumocytes by gastrin-releasing peptide

Gastrin-releasing peptide (GRP) and bombesin apparently enhance the rate of secretion of surfactant lipids from cultured fetal rat type II pneumocytes. This effect, evident within 1h of addition of the peptide, is concentration-dependent, with a maximal response at 3.0 nM. When the effect of GRP was assessed in comparison with other known secretagogues, it was found that, whereas GRP and isoproterenol were additive in their effect, there was no response to GRP in the presence of saturating concentrations of A23187 or phorbol 12-myristate 13-acetate. This suggests that the secretory response to GRP is via activation of Ca2+/calmodulin-dependent protein kinase and/or protein kinase C and is independent of adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase. This conclusion is supported by the observation that the GRP-induced secretion is inhibited by calphostin C, an inhibitor of protein kinase C, but not by H-89, an inhibitor of cAMP-dependent protein kinase. The fact that GRP regulates surfactant secretion from type II pneumocytes suggests that it and/or related peptides may play a significant role in the physiological maturation of the lung

Proteases and Protease-activated receptors signalling: at the crossroads of acquired and innate immunity

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/71652/1/j.1365-2222.2007.02738.x.pd

Comparison of expression profiles induced by dust mite in airway epithelia reveals a common pathway

Background: Airway epithelial cells have shown to be active participants in the defense against pathogens by producing signaling and other regulatory molecules in response to the encounter. Methods: In previous manuscripts, we have studied the effect of house dust mite (HDM) extract on both an epithelial cell-line (H292) and primary nasal epithelial cell. When we compare these responses we conclude that the H292 cells more closely resemble nasal epithelium of healthy controls (share 107 probe-sets) than of allergic individuals (share 17 probe-sets). Results: Interestingly, probably because of an absent intraindividual variation between samples, more probe-sets (8280) change expression significantly in H292 than in either healthy (555) or allergic (401) epithelium. Conclusions: A direct comparison of all the responses in these epithelial cells reveals a core-response to HDM of just 29 genes. These genes (CCL20, IL-8, CXCL2, CXCL1, IL-1B, AREG, TNFAIP3, HBEGF, PTGS2, BMP2, LDLR, PLAUR, PLAU, NFKB2, NFKB1, JUN, ATF3, EGR1, NPC1, TICAM1, EPHA2, CTGF, DUSP1, SPRY1, TLR-3, complement factor C3, IVNS1ABP, SerpinB3, and PSAT1) have described links with allergy or inflammation and may even describe the well-established relationship between viral infections and allergic exacerbations or allergy development