BackgroundIL-6 trans-signalling (IL-6TS) is emerging as a pathogenic mechanism in chronic respiratory diseases, however the drivers of IL-6TS in the airways and the phenotypic characteristic of patients with increased IL-6TS pathway activation remain poorly understood.ObjectiveOur aim was to identify and characterize COPD patients with increased airway IL-6TS and to elucidate the biological drivers of IL-6TS pathway activation.MethodsWe used an IL-6TS-specific sputum biomarker profile (sIL-6R, IL-6, IL-1β, IL-8, MIP-1β) to stratify sputum data from patients with COPD (n=74; BEAT-COPD) by hierarchical clustering. The IL-6TS signature was related to clinical characteristics and sputum microbiome profiles. The induction of neutrophil extracellular trap formation (NETosis) and IL-6TS by Haemophilus influenzae were studied in human neutrophils.ResultsHierarchical clustering revealed an IL-6TS-high subset (n=24) of COPD patients, which shared phenotypic traits with an IL-6TS-high subset previously identified in asthma. The subset was characterized by increased sputum cell counts (p=0.0001), persistent sputum neutrophilia (p=0.0004), reduced quality of life (CRQ total score; p=0.008), and increased levels of pro-inflammatory mediators and MMPs in sputum. IL-6TS-high COPD patients showed an increase in Proteobacteria, with Haemophilus as the dominating genus. NETosis induced by H. influenzae was identified as a potential mechanism for increased soluble IL-6 receptor (sIL-6R) levels. This was supported by a significant positive correlation between sIL-6R and NETosis markers in bronchoalveolar lavage fluid from COPD patients.ConclusionIL-6TS pathway activation due to chronic colonization with Haemophilus may be an important disease driver in a subset of COPD patients.
Increased plasma concentrations of lipoprotein(a) (Lp(a)) are associated with an increased risk for cardiovascular disease. Lp(a) is composed of apolipoprotein(a) (apo(a)) covalently bound to apolipoprotein B of low-density lipoprotein (LDL). Many of apo(a)'s potential pathological properties, such as inhibition of plasmin generation, have been attributed to its main structural domains, the kringles, and have been proposed to be mediated by their lysine-binding sites. However, available small-molecule inhibitors, such as lysine analogs, bind unselectively to kringle domains and are therefore unsuitable for functional characterization of specific kringle domains. Here, we discovered small molecules that specifically bind to the apo(a) kringle domains KIV-7, KIV-10, and KV. Chemical synthesis yielded compound AZ-05, which bound to KIV-10 with a Kd of 0.8 μm and exhibited more than 100-fold selectivity for KIV-10, compared with the other kringle domains tested, including plasminogen kringle 1. To better understand and further improve ligand selectivity, we determined the crystal structures of KIV-7, KIV-10, and KV in complex with small-molecule ligands at 1.6–2.1 Å resolutions. Furthermore, we used these small molecules as chemical probes to characterize the roles of the different apo(a) kringle domains in in vitro assays. These assays revealed the assembly of Lp(a) from apo(a) and LDL, as well as potential pathophysiological mechanisms of Lp(a), including (i) binding to fibrin, (ii) stimulation of smooth-muscle cell proliferation, and (iii) stimulation of LDL uptake into differentiated monocytes. Our results indicate that a small-molecule inhibitor targeting the lysine-binding site of KIV-10 can combat the pathophysiological effects of Lp(a).
Little is known about factors affecting anthocyanin biosynthesis in red-fleshed apples (Malus × domestica Borkh.). The objective was to establish the effects of orchard management factors on flesh anthocyanin content of dark-colored (DC) and light-colored (LC) apple clones. Flesh color was assessed by measuring color in the L, a, b mode using a spectrophotometer and predicting the anthocyanin content based on relationships between the absorption of a flesh extract at 530 nm and the L-value determined using a spectrophotometer (r2 = 0.99 ***). Fruit from the DC clone were red by 86 days after full bloom (DAFB), whereas the LC clone began to color at 136 DAFB. Color intensity in both clones decreased from the top of the tree to the base. Further, the intensity of the flesh color of the DC clone decreased with shading (94% absorption of incident photosynthetic active radiation). Covering a fruit with a UV absorbing film (100% UV absorption) had no effect on flesh color in the DC clone but decreased color in the LC clone. Fruit thinning increased color in DC and LC fruit. There was little change in flesh color during storage. However, the DC clone developed severe flesh browning as storage progressed beyond 30 days. The results demonstrated that light (visible and UV wavelength) stimulated, whereas shade inhibited, anthocyanin biosynthesis in the flesh under orchard conditions.
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