Cigarette smoking is the major risk factor for chronic obstructive pulmonary disease (COPD), a syndrome characterized by pulmonary neutrophil infiltration, chronic inflammation, and progressive tissue destruction. We examined here the acute effect of aqueous cigarette smoke extract (CSE) and of two α,β-unsaturated aldehydes (acrolein and crotonaldehyde) contained in CSE in cultured normal human lung fibroblasts and small airway epithelial cells. By examining a panel of 19 cytokines and chemokines, we found that IL-8 release was elevated by CSE as well as by acrolein, whereas other inflammatory mediators were mostly unaffected. CSE-evoked IL-8 release was mimicked by acrolein and crotonaldehyde at concentrations (3–60 μM each) found in CSE and fully prevented by 1 mM α,β-unsaturated aldehydes scavengers N-acetylcysteine (NAC) or sodium 2-mercaptoethanesulfonate. Neither the saturated aldehyde acetaldehyde nor H₂O₂evoked IL-8 release. In addition, CSE or crotonaldehyde upregulated the release of IL-8 from alveolar macrophages from both COPD patients and healthy nonsmokers, indicating that this is a response common to cells involved in lung inflammation. CSE-evoked IL-8 release was accompanied by increased phosphorylation of p38 MAPK and ERK1/2. CSE-evoked p38 and ERK1/2 phosphorylation was mimicked by acrolein and inhibited by NAC. IL-8 release elicited by both acrolein and CSE was blocked by pharmacological inhibition of p38 and ERK1/2 phosphorylation. In summary, our data show that α,β-unsaturated aldehydes-evoked phosphorylation of p38 and ERK1/2 underlies IL-8 release elicited by CSE, thus shedding light on the mechanisms through which cigarette smoke can initiate inflammation in the lung.

Type 2 (T2) inflammation in asthma encompasses the presence of eosinophilic inflammation and / or selected cytokines including interleukin (IL)‐13. Some chronic obstructive pulmonary disease (COPD) patients have increased airway eosinophil numbers, which is associated with greater effects of inhaled corticosteroids (ICS). Studies of T2 inflammation in COPD have used hypothesis free, open platform approaches.

BACKGROUND AND PURPOSE IFN‐γ levels are increased in chronic obstructive airway disease (COPD) patients compared with healthy subjects and are further elevated during viral exacerbations. IFN‐γ can ‘prime’ macrophages to enhance the response to toll‐like receptor (TLR) ligands, such as LPS. The aim of this study was to examine the effect IFN‐γ on corticosteroid sensitivity in alveolar macrophages (AM).EXPERIMENTAL APPROACH AM from non‐smokers, smokers and COPD patients were stimulated with IFN‐γ and/or LPS with or without dexamethasone. IL‐6, TNF‐α and IFN‐γ‐induced protein 10 kDa (IP‐10) levels were measured by elisa, and Western blots were used to investigate the IFN‐γ‐stimulated Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signalling pathway. Real‐time PCR and flow cytometry were used to investigate TLR levels following IFN‐γ treatment.KEY RESULTS In all three subject groups, IFN‐γ alone had no effect on IL‐6 and TNF‐α production but enhanced the effects of LPS on these cytokines. In contrast, IFN‐γ alone increased the production of IP‐10. IFN‐γ increased TLR2 and TLR4 expression in AM. Cytokine induction and STAT1 activation by IFN‐γ were insensitive to dexamethasone for all groups. The inhibition of JAK and STAT1 repressed all these IFN‐γ effects.CONCLUSIONS AND IMPLICATIONS Our results demonstrate that IFN‐γ–induced STAT‐1 signalling is corticosteroid resistant in AMs, and that targeting IFN‐γ signalling by JAK inhibitors is a potentially novel anti‐inflammatory strategy in COPD.

The outermost layer of spores of the Bacillus cereus family is a loose structure known as the exosporium. Spores of a library of Tn 917 -LTV1 transposon insertion mutants of B. cereus ATCC 10876 were partitioned into hexadecane; a less hydrophobic mutant that was isolated contained an insertion in the exsA promoter region. ExsA is the equivalent of SafA (YrbA) of Bacillus subtilis , which is also implicated in spore coat assembly; the gene organizations around both are identical, and both proteins contain a very conserved N-terminal cortex-binding domain of ca. 50 residues, although the rest of the sequence is much less conserved. In particular, unlike SafA, the ExsA protein contains multiple tandem oligopeptide repeats and is therefore likely to have an extended structure. The exsA gene is expressed in the mother cell during sporulation. Spores of an exsA mutant are extremely permeable to lysozyme and are blocked in late stages of germination, which require coat-associated functions. Two mutants expressing differently truncated versions of ExsA were constructed, and they showed the same gross defects in the attachment of exosporium and spore coat layers. The protein profile of the residual exosporium harvested from spores of the three mutants—two expressing truncated proteins and the mutant with the original transposon insertion in the promoter region—showed some differences from the wild type and from each other, but the major exosporium glycoproteins were retained. The exsA gene is extremely important for the normal assembly and anchoring of both the spore coat and exosporium layers in spores of B. cereus .

Tripartite ATP-independent periplasmic (TRAP) transporters are relatively common prokaryotic secondary transporters which comprise an extracytoplasmic solute receptor (ESR) protein and two dissimilar membrane proteins or domains, yet the substrates and physiological functions of only a few of these systems are so far known. In this study, a biophysical approach was used to identify the ligands for the purified Rhodobacter capsulatus RRC01191 and Escherichia coli YiaO proteins, which are members of two phylogenetically distinct families of TRAP-ESRs found in diverse bacteria. In contrast to previous indirect evidence pointing to RRC01191 orthologues being involved in polyol uptake, it was shown that RRC01191 binds pyruvate, 2-oxobutyrate and a broad range of aliphatic monocarboxylic 2-oxoacid anions with varying affinities (K_d values 0·08–3 μM), consistent with a predicted role in monocarboxylate transport related to branched-chain amino-acid biosynthesis. The E. coli YiaMNO TRAP transporter has previously been proposed to be an l-xylulose uptake system [Plantinga et al. (2004) Mol Membr Biol 21, 51–57], but purified YiaO did not bind l- or d-xylulose as judged by fluorescence spectroscopy, circular dichroism or mass spectrometry. Instead, these techniques showed that a breakdown product of l-ascorbate, 2,3-diketo-l-gulonate (2,3-DKG), binds by a simple one-step mechanism with sub-micromolar affinity. The data provide the first evidence for the existence of ESR-dependent transporters for 2-oxoacids and 2,3-DKG, homologues of which appear to be widespread amongst prokaryotes. The results also underline the utility of direct ESR ligand-binding studies for TRAP transporter characterization

GerN, a Bacillus cereus spore germination protein, exhibits homology to a widely distributed group of putative cation transporters or channel proteins. GerN complemented the Na ⁺ -sensitive phenotype of an Escherichia coli mutant that is deficient in Na ⁺ /H ⁺ antiport activity (strain KNabc). GerN also reduced the concentration of K ⁺ required to support growth of an E. coli mutant deficient in K ⁺ uptake (strain TK2420). In a fluorescence-based assay of everted E. coli KNabc membrane vesicles, GerN exhibited robust Na ⁺ /H ⁺ antiport activity, with a K _m for Na ⁺ estimated at 1.5 mM at pH 8.0 and 25 mM at pH 7.0. Li ⁺ , but not K ⁺ , served as a substrate. GerN-mediated Na ⁺ /H ⁺ antiport was further demonstrated in everted vesicles as energy-dependent accumulation of ²² Na ⁺ . GerN also used K ⁺ as a coupling ion without completely replacing H ⁺ , as indicated by partial inhibition by K ⁺ of H ⁺ uptake into right-side-out vesicles loaded with Na ⁺ . K ⁺ translocation as part of the antiport was supported by the stimulatory effect of intravesicular K ⁺ on ²² Na ⁺ uptake by everted vesicles and the dependence of GerN-mediated ⁸⁶ Rb ⁺ efflux on the presence of Na ⁺ in trans . The inhibitory patterns of protonophore and thiocyanate were most consistent with an electrogenic Na ⁺ /H ⁺ -K ⁺ antiport. GerN-mediated Na ⁺ /H ⁺ -K ⁺ antiport was much more rapid than GerN-mediated Na ⁺ /H ⁺ antiport.