Oxidative Stress and Lung Fibrosis: Towards an Adverse Outcome Pathway.

Lung fibrosis is a progressive fatal disease in which deregulated wound healing of lung epithelial cells drives progressive fibrotic changes. Persistent lung injury due to oxidative stress and chronic inflammation are central features of lung fibrosis. Chronic cigarette smoking causes oxidative stress and is a major risk factor for lung fibrosis. The objective of this manuscript is to develop an adverse outcome pathway (AOP) that serves as a framework for investigation of the mechanisms of lung fibrosis due to lung injury caused by inhaled toxicants, including cigarette smoke. Based on the weight of evidence, oxidative stress is proposed as a molecular initiating event (MIE) which leads to increased secretion of proinflammatory and profibrotic mediators (key event 1 (KE1)). At the cellular level, these proinflammatory signals induce the recruitment of inflammatory cells (KE2), which in turn, increase fibroblast proliferation and myofibroblast differentiation (KE3). At the tissue level, an increase in extracellular matrix deposition (KE4) subsequently culminates in lung fibrosis, the adverse outcome. We have also defined a new KE relationship between the MIE and KE3. This AOP provides a mechanistic platform to understand and evaluate how persistent oxidative stress from lung injury may develop into lung fibrosis.

Designing Studies to Inform Tobacco Harm Reduction: Learnings From an Oral Nicotine Pouch Actual Use Pilot Study.

BACKGROUND: Introduction of new tobacco products in the United States, including those that may be lower on the risk continuum than traditional combustible cigarettes, requires premarket authorization by the US Food and Drug Administration and information on the potential impact of the products on consumer behaviors. Efficient recruitment and data capture processes are needed to collect relevant information in a near-to-real-world environment. OBJECTIVE: The aim of this pilot study was to develop and test a protocol for an actual use study of a new tobacco product. The product included in this study was a commercially available oral nicotine pouch. Through the process of study design and execution, learnings were garnered to inform the design, execution, analysis, and report writing of future full-scale actual use studies with tobacco products. METHODS: A small sample (n=100) of healthy adult daily smokers of 7 or more cigarettes per day were recruited to participate in an 8-week prospective observational study conducted at 4 geographically dispersed sites in the United States. A smartphone-based customized electronic diary (eDiary) was employed to capture daily tobacco product use, including 1 week of baseline smoking and 6 weeks during which participants were provided with oral nicotine pouches for use as desired. RESULTS: Online screening procedures with follow-up telephone interviews and on-site enrollment were successfully implemented. Of 100 participants, 97 completed the study, with more than half (59/99, 60%) identifying as dual- or poly-users of cigarettes and other types of tobacco products at baseline. There was more than 90% (91-93/99, 92%-94%) compliance with daily eDiary reporting, and the majority (92/99, 93%) of participants expressed satisfaction with the study processes. Product use data from the eDiary indicated that after an initial period of trial use, pouches per day increased among those continuing to use the products, while per day average cigarette consumption decreased for 82% (79/97) of all study participants. At the end of the week 6, 16% (15/97) of participants had reduced their cigarette consumption by more than half. CONCLUSIONS: The design of this study, including recruiting, enrollment, eDiary use, and oversight, was successfully implemented through the application of a detailed protocol, a user-friendly eDiary, electronically administered questionnaires, and remote monitoring procedures. High-resolution information was obtained on prospective changes in tobacco product use patterns in the context of availability of a new tobacco product. Future, larger actual use studies will provide important evidence supporting the role that alternatives to combustible cigarettes may play in smoking reduction and/or cessation and lowering the population health burden of tobacco and nicotine-containing products.

Leucine Rich α-2 Glycoprotein: A Novel Neutrophil Granule Protein and Modulator of Myelopoiesis.

Leucine-rich α2 glycoprotein (LRG1), a serum protein produced by hepatocytes, has been implicated in angiogenesis and tumor promotion. Our laboratory previously reported the expression of LRG1 in murine myeloid cell lines undergoing neutrophilic granulocyte differentiation. However, the presence of LRG1 in primary human neutrophils and a role for LRG1 in regulation of hematopoiesis have not been previously described. Here we show that LRG1 is packaged into the granule compartment of human neutrophils and secreted upon neutrophil activation to modulate the microenvironment. Using immunofluorescence microscopy and direct biochemical measurements, we demonstrate that LRG1 is present in the peroxidase-negative granules of human neutrophils. Exocytosis assays indicate that LRG1 is differentially glycosylated in neutrophils, and co-released with the secondary granule protein lactoferrin. Like LRG1 purified from human serum, LRG1 secreted from activated neutrophils also binds cytochrome c. We also show that LRG1 antagonizes the inhibitory effects of TGFß1 on colony growth of human CD34+ cells and myeloid progenitors. Collectively, these data invoke an additional role for neutrophils in innate immunity that has not previously been reported, and suggest a novel mechanism whereby neutrophils may modulate the microenvironment via extracellular release of LRG1.

Bioavailability of polyphenols from peanut skin extract associated with plasma lipid lowering function.

Peanut skin is a rich source of polyphenols including procyanidins and is shown to have hypolipidemic properties. This study investigated the bioavailability of peanut skin polyphenols using a rat model. First, the bioavailability of peanut skin polyphenols in rat plasma was evaluated. Our results showed procyanidin A2 levels in plasma peaked within 30 min of ingestion. The results of a second study show that peanut skin extract supplemented in addition to oil gavage resulted in significant decrease in plasma triglyceride and VLDL within 5h. In the third study, rats were given a Western type diet for 5 weeks with peanut skin extract at a dose of 150 and 300 mg/kg body weight. The main effects observed were lowering of total blood lipid and reduction of the plasma fatty acids profile. Our results suggest that procyanidin A may impart a key role of hypolipidemic effect seen in peanut skin polyphenols.

The heterodimerization of platelet-derived chemokines.

Chemokines encompass a large family of proteins that act as chemoattractants and are involved in many biological processes. In particular, chemokines guide the migration of leukocytes during normal and inflammatory conditions. Recent studies reveal that the heterophilic interactions between chemokines significantly affect their biological activity, possibly representing a novel regulatory mechanism of the chemokine activities. The co-localization of platelet-derived chemokines in vivo allows them to interact. Here, we used nano-spray ionization mass spectrometry to screen eleven different CXC and CC platelet-derived chemokines for possible interactions with the two most abundant chemokines present in platelets, CXCL4 and CXCL7. Results indicate that many screened chemokines, although not all of them, form heterodimers with CXCL4 and/or CXCL7. In particular, a strong heterodimerization was observed between CXCL12 and CXCL4 or CXCL7. Compared to other chemokines, the main structural difference of CXCL12 is in the orientation and packing of the C-terminal alpha-helix in relation to the beta-sheet. The analysis of one possible structure of the CXCL4/CXCL12 heterodimer, CXC-type structure, using molecular dynamics (MD) trajectory reveals that CXCL4 may undergo a conformational transition to alter the alpha helix orientation. In this new orientation, the alpha-helix of CXCL4 aligns in parallel with the CXCL12 alpha-helix, an energetically more favorable conformation. Further, we determined that CXCL4 and CXCL12 physically interact to form heterodimers by co-immunoprecipitations from human platelets. Overall, our results highlight that many platelet-derived chemokines are capable of heterophilic interactions and strongly support future studies of the biological impact of these interactions.