No abstract

Induction of intercellular adhesion molecule-1 (ICAM-1) by proinflammatory cytokines during inflammation plays an important role in regulating polymorphonuclear neutrophil (PMN) migration and localization. In this report, we examined the effects of tumor necrosis factor-alpha (TNF alpha) and interferon-gamma (IFN gamma) on specific lung cell expression of ICAM-1 in vivo and the accompanying morphological changes. Balb-c mice were treated with phosphate-buffered saline (PBS) alone or with PBS containing 5 micrograms TNF alpha or IFN gamma through intranasal instillation. Twenty-four hours after treatment, their lungs were processed for immunoblot analysis and electron microscope immunocytochemistry. In the normal lung, the ICAM-1 level is high on type I alveolar epithelial cells, medium on arterial and venous endothelial cells, low on type II epithelial cells and capillary endothelium, and not detectable on bronchial epithelium. Topical treatment of the lung with either TNF alpha or IFN gamma induced a 50-60% increase in total lung and alveolar ICAM-1. A dramatic increase of alveolar type II cell surface ICAM-1 was observed (> 20-fold). Both cytokines caused 2-3-fold higher ICAM-1 expression on capillary endothelial cells and a 40% increase of ICAM-1 on alveolar type I cells that was not uniform. However, due to the large total surface area of type I epithelium, type I cells contribute 70-86% of total alveolar septal ICAM-1 and > 90% of alveolar surface ICAM-1 in either treated or normal mouse lungs. Increased ICAM-1 expression was also observed on nonparenchymal endothelial and epithelial cells. Margination and sequestration of PMN in cytokine-treated lungs were observed by histologic examination, measurements of total lung myloperoxidase activity, and number of neutrophils recovered in bronchoalveolar lavage fluid. These results showed that TNF alpha and IFN gamma induce ICAM-1 expression and infiltration of neutrophils in the lung. The response of specific lung cells in terms of induction of ICAM-1 in response to cytokine stimulation varied significantly, particularly between type I and type II epithelial cells.

No abstract

In contrast to the processes controlling the complexation, targeting and uptake of polycationic gene delivery vectors, the molecular mechanisms regulating their cytoplasmic dissociation remains poorly understood. Upon cytosolic entry, vectors become exposed to a complex, concentrated mixture of molecules and biomacromolecules. In this report, we characterise the cytoplasmic interactome associated with polycationic vectors based on poly(dimethylaminoethyl methacrylate) (PDMAEMA) and poly(2-methacrylolyloxyethyltrimethylammonium chloride) (PMETAC) brushes. To quantify the contribution of different classes of low molar mass molecules and biomacromolecules to RNA release, we develop a kinetics model based on competitive binding. Our results identify the importance of competition from highly charged biomacromolecules, such as cytosolic RNA, as a primary regulator of RNA release. Importantly, our data indicate the presence of ribosome associated proteins, proteins associated with translation and transcription factors that may underly a broader impact of polycationic vectors on translation. In addition, we bring evidence that molecular crowding modulates competitive binding and demonstrate how the modulation of such interactions, for example via quaternisation or the design of charge-shifting moieties, impacts on the long-term transfection efficiency of polycationic vectors. Understanding the mechanism regulating cytosolic dissociation will enable the improved design of cationic vectors for long term gene release and therapeutic efficacy.

No abstract