Capture of 0.1-µm aerosol particles containing viable H1N1 influenza virus by N95 filtering facepiece respirators.

Nosocomial infections pose an escalating threat to both patients and healthcare workers (HCWs). A widely recommended device for individual respiratory protection, the N95 filtering facepiece respirator (FFR) has been shown to provide efficient filtration of inert particles larger and smaller than the nominal most-penetrating particle size (MPPS) range, 0.03-0.3 µm. Humans generate respiratory aerosols in the MPPS range, suggesting that short-range disease transmission could occur via small infectious particles. Data presented here show that the N95 FFR will afford a significant measure of protection against infectious particles as small as a bare H1N1 influenza virion, and that the capture mechanism does not discriminate in favor of, or against, biological particles.

Correlation of respirator fit measured on human subjects and a static advanced headform.

This study assessed the correlation of N95 filtering facepiece respirator (FFR) fit between a Static Advanced Headform (StAH) and 10 human test subjects. Quantitative fit evaluations were performed on test subjects who made three visits to the laboratory. On each visit, one fit evaluation was performed on eight different FFRs of various model/size variations. Additionally, subject breathing patterns were recorded. Each fit evaluation comprised three two-minute exercises: "Normal Breathing," "Deep Breathing," and again "Normal Breathing." The overall test fit factors (FF) for human tests were recorded. The same respirator samples were later mounted on the StAH and the overall test manikin fit factors (MFF) were assessed utilizing the recorded human breathing patterns. Linear regression was performed on the mean log10-transformed FF and MFF values to assess the relationship between the values obtained from humans and the StAH. This is the first study to report a positive correlation of respirator fit between a headform and test subjects. The linear regression by respirator resulted in R(2) = 0.95, indicating a strong linear correlation between FF and MFF. For all respirators the geometric mean (GM) FF values were consistently higher than those of the GM MFF. For 50% of respirators, GM FF and GM MFF values were significantly different between humans and the StAH. For data grouped by subject/respirator combinations, the linear regression resulted in R(2) = 0.49. A weaker correlation (R(2) = 0.11) was found using only data paired by subject/respirator combination where both the test subject and StAH had passed a real-time leak check before performing the fit evaluation. For six respirators, the difference in passing rates between the StAH and humans was < 20%, while two respirators showed a difference of 29% and 43%. For data by test subject, GM FF and GM MFF values were significantly different for 40% of the subjects. Overall, the advanced headform system has potential for assessing fit for some N95 FFR model/sizes.

Effects of dynamic compression on lentiviral transduction in an in vitro airway wall model.

Asthmatic patients are more susceptible to viral infection, and we asked whether dynamic strain on the airway wall (such as that associated with bronchoconstriction) would influence the rate of viral infection of the epithelial and subepithelial cells. To address this, we characterized the barrier function of a three-dimensional culture model of the bronchial airway wall mucosa, modified the culture conditions for optimization of ciliogenesis, and compared epithelial and subepithelial green fluorescent protein (GFP) transduction by a pWpts-GFP lentivirus, pseudotyped with VSV-G, under static vs. dynamic conditions. The model consisted of human lung fibroblasts, bronchial epithelial cells, and a type I collagen matrix, and after 21 days of culture at air liquid interface, it exhibited a pseudostratified epithelium comprised of basal cells, mucus-secreting cells, and ciliated columnar cells with beating cilia. Microparticle tracking revealed partial coordination of mucociliary transport among groups of cells. Slow dynamic compression of the airway wall model (15% strain at 0.1 Hz over 3 days) substantially enhanced GFP transduction of epithelial cells and underlying fibroblasts. Fibroblast-only controls showed a similar degree of transduction enhancement when undergoing dynamic strain, suggesting enhanced transport through the matrix. Tight junction loss in the epithelium after mechanical stress was observed by immunostaining. We conclude that dynamic compressive strain such as that associated with bronchoconstriction may promote transepithelial transport and enhance viral transgene delivery to epithelial and subepithelial cells. This finding has significance for asthma pathophysiology as well as for designing delivery strategies of viral gene therapies to the airways.

Extracellular matrix remodeling by dynamic strain in a three-dimensional tissue-engineered human airway wall model.

Airway wall remodeling is a hallmark of asthma, characterized by subepithelial thickening and extracellular matrix (ECM) remodeling. Mechanical stress due to hyperresponsive smooth muscle cells may contribute to this remodeling, but its relevance in a three-dimensional environment (where the ECM plays an important role in modulating stresses felt by cells) is unclear. To characterize the effects of dynamic compression in ECM remodeling in a physiologically relevant three-dimensional environment, a tissue-engineered human airway wall model with differentiated bronchial epithelial cells atop a collagen gel containing lung fibroblasts was used. Lateral compressive strain of 10 or 30% at 1 or 60 cycles per hour was applied using a novel straining device. ECM remodeling was assessed by immunohistochemistry and zymography. Dynamic strain, particularly at the lower magnitude, induced airway wall remodeling, as indicated by increased deposition of types III and IV collagen and increased secretion of matrix metalloproteinase-2 and -9. These changes paralleled increased myofibroblast differentiation and were fibroblast-dependent. Furthermore, the spatial pattern of type III collagen deposition correlated with that of myofibroblasts; both were concentrated near the epithelium and decreased diffusely away from the surface, indicating some epithelial control of the remodeling response. Thus, in a physiologically relevant three-dimensional model of the bronchial wall, dynamic compressive strain induced tissue remodeling that mimics many features of remodeling seen in asthma, in the absence of inflammation and dependent on epithelial-fibroblast signaling.

Physiological 3D tissue model of the airway wall and mucosa.

This protocol describes the setup, maintenance and characteristics of a tissue-engineered model of the human bronchial mucosa that can be used for basic physiology and pathophysiology studies. The model includes a well-differentiated epithelium with functional cilia, mucus secretion and subepithelial fibroblasts within type I collagen. The tissue is created within porous polymeric wells to prevent gel contraction and allow culture at the air-liquid interface. It requires at least 2 wk to be established and can be maintained thereafter for over 4 wk, with tissue differentiation moving towards a more physiologically relevant phenotype with increasing time in culture. Over time, the extracellular matrix also remodels, depositing proteins such as types III and IV collagen and fibronectin. Because it recapitulates many key anatomical and functional features of the airway wall, this model is well suited for a wide range of studies, including those on airway remodeling, transepithelial transport and inflammatory cell interactions with the mucosa. The entire protocol takes 4-6 wk, including cell expansion, depending on the extent of ciliogenesis desired.

An in vitro airway wall model of remodeling.

Recent studies have shown that mechanical forces on airway epithelial cells can induce upregulation of genes involved in airway remodeling in diseases such as asthma. However, the relevance of these responses to airway wall remodeling is still unclear since 1). mechanotransduction is highly dependent on environment (e.g., matrix and other cell types) and 2). inflammatory mediators, which strongly affect remodeling, are also present in asthma. To assess the effects of mechanical forces on the airway wall in a relevant three-dimensional inflammatory context, we have established a tissue culture model of the human airway wall that can be induced to undergo matrix remodeling. Our model contains differentiated human bronchial epithelial cells characterized by tight junctions, cilia formation, and mucus secretion atop a collagen gel embedded with human lung fibroblasts. We found that addition of activated eosinophils and the application of 50% strain to the same system increased the epithelial thickness compared with either condition alone, suggesting that mechanical strain affects airway wall remodeling synergistically with inflammation. This integrated model more closely mimics airway wall remodeling than single-cell, conditioned media, or even two-dimensional coculture systems and is relevant for examining the importance of mechanical strain on airway wall remodeling in an inflammatory environment, which may be crucial for understanding and treating pathologies such as asthma.