Identification of targeting peptides for the diagnosis of myocarditis.

AIM: Current diagnostic tests for myocarditis are invasive and have low diagnostic value. Our aim was to identify potential targeting peptides to detect early myocarditis following intravenous delivery. MATERIALS & METHODS: We used an animal model of experimental autoimmune myocarditis and a phage display library to identify potential targeting peptides. After several steps, we selected two peptides, MyH-PhD-05 and MyH-PhD-120, for in vivo screening using fluorescent imaging. Immunofluorescence and proteonomic analysis was used to identify potential cellular and molecular targets of MyH-PhD-05. Echocardiography was used to assess functional changes. RESULTS: Peptide MyH-PhD-05 was able to detect animals with severe myocarditis even in the absence of functional changes. Immunofluorescence demonstrated that MyH-PhD-05 colocalizes with CD4+ T cells and monocytes (CD11b+) in cardiac infiltrates. CONCLUSION: We identified potential targeting peptides for the diagnosis of myocarditis. Future studies will focus on better identification of potential targets and translating this technology to clinically relevant imaging modalities.

Acute effects of cigarette smoke extract on alveolar epithelial sodium channel activity and lung fluid clearance.

Cigarette smoke contains high levels of reactive species. Moreover, cigarette smoke can induce cellular production of oxidants. The purpose of this study was to determine the effect of cigarette smoke extract (CSE)-derived oxidants on epithelial sodium channel (ENaC) activity in alveolar type 1 (T1) and type 2 (T2) cells and to measure corresponding rates of fluid clearance in mice receiving a tracheal instillation of CSE. Single-channel patch clamp analysis of T1 and T2 cells demonstrate that CSE exposure increases ENaC activity (NPo), measured as the product of the number of channels (N) and a channels open probability (Po), from 0.17 ± 0.07 to 0.34 ± 0.10 (n = 9; P = 0.04) in T1 cells. In T2 cells, CSE increased NPo from 0.08 ± 0.03 to 0.35 ± 0.10 (n = 9; P = 0.02). In both cell types, addition of tetramethylpiperidine and glutathione attenuated CSE-induced increases in ENaC NPo. Biotinylation and cycloheximide chase assays indicate that CSE-derived ROS increases channel activity, in part, by maintaining cell surface expression of the α-ENaC subunit. In vivo studies show that tracheal instillation of CSE promoted alveolar fluid clearance after 105 minutes compared with vehicle control (n = 10/group; P < 0.05).

Ethanol alters alveolar fluid balance via Nadph oxidase (NOX) signaling to epithelial sodium channels (ENaC) in the lung.

Chronic alcohol consumption is associated with increased incidence of ICU-related morbidity and mortality, primarily from acute respiratory distress syndrome (ARDS). However, the mechanisms involved are unknown. One explanation is that alcohol regulates epithelial sodium channels (ENaC) via oxidant signaling to promote a pro- injury environment. We used small rodent models to mimic acute and chronic alcohol consumption and tested the hypothesis that ethanol (EtOH) would affect lung fluid clearance by up-regulating ENaC activity in the lung. Fluorescence labeling of rat lung slices and in vivo mouse lung revealed an increase in ROS production in response to acute EtOH exposure. Using western blots and fluorescein-5-maleimide labeling, we conclude that EtOH exposure modifies cysteines of α-ENaC while data from single channel patch clamp analysis confirm that 0.16% EtOH increased ENaC activity in rat alveolar cells. In vivo lung fluid clearance demonstrated a latent increase in fluid clearance in mice receiving EtOH diet. Ethanol mice given a tracheal instillation of LPS demonstrated early lung fluid clearance compared to caloric control mice and C57Bl/6 mice. Standard biochemical techniques reveal that chronic EtOH consumption resulted in greater protein expression of the catalytic gp91(phox) subunit and the obligate Rac1 protein. Collectively these data suggest that chronic EtOH consumption may lead to altered regulation of ENaC, contributing to a 'pro-injury' environment in the alcohol lung.