1 Endogenous nitric oxide (NO) can be detected in exhaled air and accumulates in in¯amed airways. However its physiological role has not been fully elucidated. In this study, we investigated a role for endogenous NO in allergen-induced airway responses. Sensitised guinea-pigs were treated with N G -nitro-L-arginine methyl ester L-NAME (2.0 mM) or aminoguanidine (AG) (2.0 mM) 30 min before the allergen challenge, and 3 and 4 h after the challenge. Alternatively, L-arginine (2.4 mM) treatment was performed 30 min before, and 2 and 3 h after the challenge. In all groups, ovalbumin (OVA) challenge (2 mg ml 71 for 2 min) was performed, and airway responses, NO production, in®ltration of in¯ammatory cells, plasma exudation and histological details were examined. 2 Allergen-challenged animals showed an immediate airway response (IAR) and a late airway response (LAR), which synchronised with an increase in exhaled NO. Treatment with L-NAME and AG did not aect IAR while they signi®cantly blocked LAR (72% and 80% inhibition compared to vehicle) and production of NO (35% and 40% inhibition). On the other hand, treatment with L-arginine did not aect IAR but potentiated LAR (74% augmentation). 3 In bronchoalveolar lavage (BAL)¯uid, allergen-induced increases in eosinophils were reduced by 48% for L-NAME treatment compared to vehicle, and increased by 56% for L-arginine treatment. 4 Treatment with L-NAME signi®cantly decreased airway microvascular permeability to both Monastral blue (MB) and Evans blue (EB) dye (50.6% and 44% inhibition). 5 We conclude that allergen-induced LAR is closely associated with NO production, and that NO plays a critical role in in¯ammatory cell in®ltration and plasma exudation in the allergic condition.

Inflammatory bowel disease (IBD), including ulcerative colitis and Crohn’s disease, is a chronic intestinal inflammatory condition initiated by integrins-mediated leukocyte adhesion to the activated colonic microvascular endothelium. Calreticulin (CRT), a calcium-binding chaperone, is known as a partner in the activation of integrin α subunits (ITGAs). The relationship between their interaction and the pathogenesis of IBD is largely unknown. Here we show that a small molecule, orally active ER-464195-01, inhibits the CRT binding to ITGAs, which suppresses the adhesiveness of both T cells and neutrophils. Transcriptome analysis on colon samples from dextran sodium sulfate-induced colitis mice reveals that the increased expression of pro-inflammatory genes is downregulated by ER-464195-01. Its prophylactic and therapeutic administration to IBD mouse models ameliorates the severity of their diseases. We propose that leukocytes infiltration via the binding of CRT to ITGAs is necessary for the onset and development of the colitis and the inhibition of this interaction may be a novel therapeutic strategy for the treatment of IBD.

Phosphodiesterase (PDE) 4 inhibition is a well-known anti-inflammatory mechanism, but the development of PDE4 inhibitors has been hampered by side effects such as nausea and emesis. Local delivery of a PDE4 inhibitor to the site of inflammation may overcome these issues. The purpose of this study was to assess the therapeutic potential of E6005 (methyl 4-[({3-[6,7-dimethoxy-2-(methylamino)quinazolin-4-yl]phenyl}amino)carbonyl]benzoate), a novel PDE4 inhibitor developed as a topical agent for atopic dermatitis (AD). E6005 potently and selectively inhibited human PDE4 activity with an IC 50 of 2.8 nM and suppressed the production of various cytokines from human lymphocytes and monocytes with IC 50 values ranging from 0.49 to 3.1 nM. In mice models, the topical application of E6005 produced an immediate antipruritic effect as well as an anti-inflammatory effect with reduced expression of cytokines/adhesion molecules. On the basis of these observed effects, topical E6005 ameliorated the appearance of atopic dermatitis-like skin lesions in two types of AD models, hapten-and mite-elicited models, exhibiting inhibitory effects comparable to that of tacrolimus. The use of 14 Clabeled E6005 showed rapid clearance from the blood and low distribution to the brain, contributing to the low emetic potential of this compound. These results suggest that E6005 may be a promising novel therapeutic agent with antipruritic activity for the treatment of AD.

We have isolated a full-length cDNA for an inducible nitric oxide synthase (iNOS) from guinea-pig lung. The cDNA has a 3447 bp open reading frame encoding 1149 amino acid residues. The deduced amino acid sequence is approx. 80% identical with iNOS of human epithelial cells and murine macrophages. Consensus recognition sites for cofactors are highly conserved. COS cell lysate transfected with the guinea-pig iNOS shows significant levels of nitric oxide synthase (NOS) activity, and this is inhibited by 79% by chelation of Ca2+ ions. The NOS activity is restored in a concentration-dependent manner by increasing the free Ca2+ level. The NOS activity is also inhibited by trifluoperazine, a calmodulin antagonist, which suggests that the Ca2+ dependence is due to Ca2+-dependent calmodulin binding to the enzyme. Northern blot analysis reveals that the cloned iNOS mRNA is expressed in the lung and the colon in normal guinea pigs. Stimulation in vivo by lipopolysaccharide induces the expression of iNOS in the kidney, the spleen and the colon, but in the lung the same stimulation decreases its expression. These results suggest that the cloned guinea-pig iNOS is distinct in characteristics and expression from previously described iNOS forms.

When stimulated with immune complex or C5a anaphylatoxin, human neutrophils undergo an increase in the concentration of intracellular CaZ+ ([Ca"],) that precedes the onset of superoxide (0;) production. The extracellular Ca*+ is required for the 0; production of neutrophils stimulated by C5a. but is only partially required for those by immune complex The addition of pertussis toxin to neutrophils does not inhibit the rise in [Ca*'], and 0; production induced by immune complex but does inhibit those induced by C5a. These results suggest that a different sequence of reaction is involved by different stimulants, the anaphylatoxin and immune complex.