Background: Asthma is a chronic airway inflammatory disease characterized by an imbalance in both Th1 and Th2 cytokines. Exhaled nitric oxide (NO) is elevated in asthma, and is a potentially useful non-invasive marker of airway inflammation. However, the origin and underlying mechanisms of intersubject variability of exhaled NO are not yet fully understood. We have previously described NO gas phase release from normal human bronchial epithelial cells (NHBEs, tracheal origin). However, smaller airways are the major site of morbidity in asthma. We hypothesized that IL-13 or cytomix (IL-1 beta, TNF-alpha, and IFN-gamma) stimulation of differentiated small airway epithelial cells (SAECs, generation 10-12) and A549 cells (model cell line of alveolar type II cells) in culture would enhance NO gas phase release. Methods: Confluent monolayers of SAECs and A549 cells were cultured in Transwell plates and SAECs were allowed to differentiate into ciliated and mucus producing cells at an air-liquid interface. The cells were then stimulated with IL-13 (10 ng/mL) or cytomix (10 ng/mL for each cytokine). Gas phase NO release in the headspace air over the cells was measured for 48 hours using a chemiluminescence analyzer. Results: In contrast to our previous result in NHBE, baseline NO release from SAECs and A549 is negligible. However, NO release is significantly increased by cytomix (0.51 +/- 0.18 and 0.29 +/- 0.20 pl.s(-1).cm(-2), respectively) reaching a peak at approximately 10 hours. iNOS protein expression increases in a consistent pattern both temporally and in magnitude. In contrast, IL-13 only modestly increases NO release in SAECs reaching a peak (0.06 +/- 0.03 pl.s-1.cm(-2)) more slowly (30 to 48 hours), and does not alter NO release in A549 cells. Conclusion: We conclude that the airway epithelium is a probable source of NO in the exhaled breath, and intersubject variability may be due, in part, to variability in the type (Th1 vs Th2) and location (large vs small airway) of inflammation.

Multiphoton microscopy (MPM) holds promise as a noninvasive imaging technique for characterizing collagen structure, and thus mechanical properties, through imaging second harmonic generation (SHG) and two-photon fluorescence in engineered and real connective tissues. Controlling polymerization pH to manipulate collagen gel microstructure, we quantified pore and fiber dimensions using both standard methods and image correlation spectroscopy (ICS) on MPM, scanning electron, and darkfield microscopy images. The latter two techniques are used to confirm microstructural measurements made from MPM images. As polymerization pH increased from 5.5 to 8.5, mean fiber diameter decreased from 3.7 +/- 0.7 microm to 1.6 +/- 0.3 microm, the average pore size decreased from 81.7 +/- 3.7 microm(2) to 7.8 +/- 0.4 microm(2), and the pore area fraction decreased from 56.8% +/- 0.8% to 18.0% +/- 1.3% (measured from SHG images), whereas the storage modulus G' and loss modulus G'', components of the shear modulus, increased approximately 33-fold and approximately 16-fold, respectively. A characteristic length scale measured using ICS, W(ICS), correlates well with the mean fiber diameter from SHG images (R(2) = 0.95). Semiflexible network theory predicts a scaling relationship of the collagen gel storage modulus (G') depending upon mesh size and fiber diameter, which are estimated from SHG images using ICS. We conclude that MPM and ICS are an effective combination to assess bulk mechanical properties of collagen hydrogels in a noninvasive, objective, and systematic fashion and may be useful for specific in vivo applications.

Multiphoton microscopy of collagen hydrogels produces second harmonic generation (SHG) and two-photon fluorescence (TPF) images, which can be used to noninvasively study gel microstructure at depth ( approximately 1 mm). The microstructure is also a primary determinate of the mechanical properties of the gel; thus, we hypothesized that bulk optical properties (i.e., SHG and TPF) could be used to predict bulk mechanical properties of collagen hydrogels. We utilized polymerization temperature (4-37 degrees C) and glutaraldehyde to manipulate collagen hydrogel fiber diameter, space-filling properties, and cross-link density. Multiphoton microscopy and scanning electron microscopy reveal that as polymerization temperature decreases (37-4 degrees C) fiber diameter and pore size increase, whereas hydrogel storage modulus (G', from 23 +/- 3 Pa to 0.28 +/- 0.16 Pa, respectively, mean +/- SE) and mean SHG decrease (minimal change in TPF). In contrast, glutaraldehyde significantly increases the mean TPF signal (without impacting the SHG signal) and the storage modulus (16 +/- 3.5 Pa before to 138 +/- 40 Pa after cross-linking, mean +/- SD). We conclude that SHG and TPF can characterize differential microscopic features of the collagen hydrogel that are strongly correlated with bulk mechanical properties. Thus, optical imaging may be a useful noninvasive tool to assess tissue mechanics.