Ventilator-induced lung injury results in oxidative stress response and mitochondrial swelling in a mouse model

© 2022. The Author(s).BACKGROUND: Mechanical ventilation is a life-saving therapy for critically ill patients, providing rest to the respiratory muscles and facilitating gas exchange in the lungs. Ventilator-induced lung injury (VILI) is an unfortunate side effect of mechanical ventilation that may lead to serious consequences for the patient and increase mortality. The four main injury mechanisms associated with VILI are: baro/volutrauma caused by overstretching the lung tissues; atelectrauma, caused by repeated opening and closing of the alveoli resulting in shear stress; oxygen toxicity due to use of high ratio of oxygen in inspired air, causing formation of free radicals; and biotrauma, the resulting biological response to tissue injury, that leads to a cascade of events due to excessive inflammatory reactions and may cause multi-organ failure. An often-overlooked part of the inflammatory reaction is oxidative stress. In this research, a mouse model of VILI was set up with three tidal volume settings (10, 20 and 30 mL/kg) at atmospheric oxygen level. Airway pressures and heart rate were monitored and bronchoalveolar lavage fluid (BALF) and lung tissue samples were taken. RESULTS: We show a correlation between increased inflammation and barrier failure, and higher tidal volumes, evidenced by increased IL-6 expression, high concentration of proteins in BALF along with changes in expression of adhesion molecules. Furthermore, swelling of mitochondria in alveolar type II cells was seen indicating their dysfunction and senescence-like state. RNA sequencing data present clear increases in inflammation, mitochondrial biogenesis and oxidative stress as tidal volume is increased, supported by degradation of Keap1, a redox-regulated substrate adaptor protein. CONCLUSIONS: Oxidative stress seems to be a more prominent mechanism of VILI than previously considered, indicating that possible treatment methods against VILI might be identified by impeding oxidative pathways.Peer reviewe

Azithromycin ameliorates sulfur dioxide-induced airway epithelial damage and inflammatory responses

Publisher's version (útgefin grein)Background: The airway epithelium (AE) forms the first line of defence against harmful particles and pathogens. Barrier failure of the airway epithelium contributes to exacerbations of a range of lung diseases that are commonly treated with Azithromycin (AZM). In addition to its anti-bacterial function, AZM has immunomodulatory effects which are proposed to contribute to its clinical effectiveness. In vitro studies have shown the AE barrier-enhancing effects of AZM. The aim of this study was to analyze whether AE damage caused by inhalation of sulfur dioxide (SO2) in a murine model could be reduced by pre-treatment with AZM. Methods: The leakiness of the AE barrier was evaluated after SO2 exposure by measuring levels of human serum albumin (HSA) in bronchoalveolar lavage fluid (BALF). Protein composition in BALF was also assessed and lung tissues were evaluated across treatments using histology and gene expression analysis. Results: AZM pre-treatment (2 mg/kg p.o. 5 times/week for 2 weeks) resulted in reduced glutathione-S-transferases in BALF of SO2 injured mice compared to control (without AZM treatment). AZM treated mice had increased intracellular vacuolization including lamellar bodies and a reduction in epithelial shedding after injury in addition to a dampened SO2-induced inflammatory response. Conclusions: Using a mouse model of AE barrier dysfunction we provide evidence for the protective effects of AZM in vivo, possibly through stabilizing the intracellular microenvironment and reducing inflammatory responses. Our data provide insight into the mechanisms contributing to the efficacy of AZM in the treatment of airway diseases.This work was supported by the Icelandic Research Council Technical Fund (Rannís Grant numbers: 186943–0611) and EpiEndo Pharmaceuticals.Peer Reviewe

Melflufen, a peptide‐conjugated alkylator, is an efficient anti‐neo‐plastic drug in breast cancer cell lines

Publisher's version (útgefin grein)Melphalan flufenamide (hereinafter referred to as “melflufen”) is a peptide-conjugated drug currently in phase 3 trials for the treatment of relapsed or refractory multiple myeloma. Due to its lipophilic nature, it readily enters cells, where it is converted to the known alkylator melphalan leading to enrichment of hydrophilic alkylator payloads. Here, we have analysed in vitro and in vivo the efficacy of melflufen on normal and cancerous breast epithelial lines. D492 is a normal-derived nontumorigenic epithelial progenitor cell line whereas D492HER2 is a tumorigenic version of D492, overexpressing the HER2 oncogene. In addition we used triple negative breast cancer cell line MDA-MB231. The tumorigenic D492HER2 and MDA-MB231 cells were more sensitive than normal-derived D492 cells when treated with melflufen. Compared to the commonly used anti-cancer drug doxorubicin, melflufen was significantly more effective in reducing cell viability in vitro while it showed comparable effects in vivo. However, melflufen was more efficient in inhibiting metastasis of MDA-MB231 cells. Melflufen induced DNA damage was confirmed by the expression of the DNA damage proteins ƴH2Ax and 53BP1. The effect of melflufen on D492HER2 was attenuated if cells were pretreated with the aminopeptidase inhibitor bestatin, which is consistent with previous reports demonstrating the importance of aminopeptidase CD13 in facilitating melflufen cleavage. Moreover, analysis of CD13high and CD13low subpopulations of D492HER2 cells and knockdown of CD13 showed that melflufen efficacy is mediated at least in part by CD13. Knockdown of LAP3 and DPP7 aminopeptidases led to similar efficacy reduction, suggesting that also other aminopeptidases may facilitate melflufen conversion. In summary, we have shown that melflufen is a highly efficient anti-neoplastic agent in breast cancer cell lines and its efficacy is facilitated by aminopeptidases.We thank Inovotion for their work and support in the CAM assays.Peer Reviewe

Rannsóknir á lakkasagenum

Lakkasar eru ensím sem hafa það hlutverk í náttúrunni að brjóta niður lignín til þess að komast að öðrum kolvetnum í trjávið (sellulósa og hemisellulósa). Þar til nýlega hafa lakkasar einungis fundist í heilkjörnungum eins og sveppum, plöntum og skordýrum. Nú hafa lakkasar hinsvegar einnig fundist í dreifkjörnungum. Lakkasar hafa verið nýttir í ýmsum iðnaði, t.d. líflitun pappírskvoðu, afeitrun úrgangs, sem aukefni í mat og fleira. Munur á heilkjörnunga- og dreifkjörnungalakkasa hefur verið rannsakaður en nýjar uppgötvanir eru stöðugt að koma fram. Tilgangur þessa verkefnis var að raðgreina og staðfesta þrjú lakkasa gen, CotA, LaccSi og LaccCu. Einnig að mæla ensím virkni með þremur hvarfefnum, ABTS, guaiacol og cathecol. Lykilorð: Kuldavirk ensím, lakkasi, PCR, raðgreining, ensím mælingar.Laccases are enzymes that have the role in nature to break down lignin in order to gain access to other carbohydrates in wood (cellulose and hemicellulose). Until recently, laccases were only found in eukaryotes such as fungi, plants and insects. Now laccases have been discovered in prokaryotes as well. Laccase has been used for various industrial purposes, such as bio-bleaching of pulps, detoxification of waste, additives in food and beverage processing to name a few. The difference between eukaryotic and prokaryotic laccases has been studied but new discoveries are constantly surfacing. The purpose of this project was to sequence and identify three laccase specific genes, CotA, LaccSi and LaccCu. Also measure the enzymes activity by three different assaying methods using Cathecol, ABTS and Guaiacol as substrates. Keywords: Cold active enzymes, laccase, PCR, sequencing, enzyme assays