Surfactant homeostasis is not corrected by additional NOS2 genetic ablation.

<p>Phospholipid content of BAL as well as small and large aggregate fractions was determined by the method of Bartlett. Data shown are mean ± S.E.. Statistically significant differences between groups (p<0.05) are indicated as:</p>*<p><i>vs.</i> WT,</p>ξ<p><i>vs.</i> NOS2^−/−,</p>#<p><i>vs.</i> Sftpd^−/− mice.</p

NOS2 ablation improves emphyatous phenotype resulting from the absence of SP-D.

<p>The number of alveoli per lung N(alv, lung) (<b>A</b>) is increased whereas the number-weighted mean volume of alveoli (alv) (<b>B</b>) is decreased in the DiNOS mice compared to Sftpd^−/− mice, indicating an attenuation of the pulmonary emphysema due to the additional ablation of the iNOS-gene in Sftpd deficient mice. Data shown are mean and individual data, n = 5–6 animals per genotype. Statistically significant differences between groups are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085722#pone-0085722-t002" target="_blank">Table 2</a>.</p

Ablation of NOS2 reduces NO metabolite production in DiNOS mice.

<p>BAL nitrates were measured by NOA as a marker of NOS activity. Values are mean ± S.E. (n = 6 to 15). Statistically significant differences between groups (p<0.05) are indicated as: * <i>vs</i> WT; ξ <i>vs</i> NOS2^−/−; # <i>vs</i> Sftpd^−/− mice.</p

Stereological data: AE2 cells and intracellular surfactant.

<p>Values are given as mean ± S.E. of n = 5–6 mice per genotype. Abbreviations: V = volume, V_V = volume fraction,  = number-weighted mean volume,  = volume-weighted mean volume, lb = lamellar body, type II = AE2. Statistically significant differences between groups (p<0.05) are indicated as:</p>*<p><i>vs.</i> WT,</p>ξ<p><i>vs.</i> NOS2^−/−,</p>#<p><i>vs.</i> Sftpd^−/−.</p

Stereological data of lung architecture.

<p>Values are given as mean ± S.E. of n = 5–6 mice per genotype. Abbreviations: V = volume, S = surface area, S_V = surface area density, N = number, N_V = numerical density,  = number-weighted mean volume,  = volume-weighted mean volume, par = parenchyma, alvepi = alveolar epithelium, alv = alveoli. Statistically significant differences between groups (p<0.05) are indicated as:</p>*<p><i>vs.</i> WT,</p>ξ<p><i>vs.</i> NOS2^−/−,</p>#<p><i>vs.</i> Sftpd^−/−.</p

DiNOS mice exhibit partial normalization of BAL cell counts.

<p>Lungs were lavaged with 0.5-ml aliquots of sterile saline to a total of 5 ml. Recovered BALF samples were centrifuged (300 g for 10 min) and the cell pellet was gently resuspended in 1 ml of PBS (with Ca²⁺ and Mg²⁺). (<b>A</b>) Total cell count was determinated using a Z1 Counter particle counter (Beckman Coulter). (<b>B</b>) Aliquots of cells were spun on a Thermo Shandon Cytospin-3 at 750 rpm for 3 min and stained with standard Diff-Quik for manual determination of cell differentials. Cells were identified as macrophages, eosinophils, neutrophils, and lymphocytes by standard morphology. Data shown are mean ±S.E., n = 7 animals per genotype. Statistically significant differences between groups (p<0.05) are indicated as: * <i>vs</i> WT; ξ<i>vs</i> NOS2^−/−; # <i>vs</i> Sftpd^−/− mice. (<b>C</b>) Representative Diff-Quik staining of cytospins from WT and Sftpd^−/−, DiNOS and NOS2^−/− mice.</p

Lungs of DiNOS and Sftpd^−/− mice exhibit AE2 hyperplasia and hypertrophy with increased numbers of lamellar bodies.

<p>Representative electron micrographs of AE2 cells of WT (<b>a</b>), NOS2^−/− (<b>b</b>), Sftpd^−/− (<b>c</b>) and DiNOS (<b>d</b>) mice. The profiles of AE2 cells in Sftpd^−/− and DiNOS are bigger and seem to contain more lamellar bodies compared to WT and iNOS^−/− mice.</p

NOS2 ablation alters the inflammatory phenotype of BAL in DiNOS mice.

<p>RNA was extracted from BAL cells isolated from WT, Sftpd^−/−, NOS2^−/− and DiNOS mice. Gene markers were quantified by RT-qPCR as described. Ct values obtained were normalized to β-actin signals and further analyzed using the relative quantization (ΔΔCt) method. Data are expressed as fold change (means ± S.E.M, n = 5–8 in each group). Statistically significant differences between groups (p<0.05) are indicated as: * <i>vs</i> WT, ξ <i>vs</i> NOS2^−/−, # <i>vs</i> Sftpd^−/−.</p

Image_1_Use of Submicron Vaterite Particles Serves as an Effective Delivery Vehicle to the Respiratory Portion of the Lung.jpg

<p>Nano- and microencapsulation has proven to be a useful technique for the construction of drug delivery vehicles for use in vascular medicine. However, the possibility of using these techniques within the lung as an inhalation delivery mechanism has not been previously considered. A critical element of particle delivery to the lung is the degree of penetrance that can be achieved with respect to the airway tree. In this study we examined the effectiveness of near infrared (NIR) dye (Cy7) labeled calcium carbonate (vaterite) particles of 3.15, 1.35, and 0.65 μm diameter in reaching the respiratory portion of the lung. First of all, it was shown that, interaction vaterite particles and the components of the pulmonary surfactant occurs a very strong retardation of the recrystallization and dissolution of the particles, which can subsequently be used to create systems with a prolonging release of bioactive substances after the particles penetrate the distal sections of the lungs. Submicro- and microparticles, coated with Cy7 labeled albumin as a model compound, were delivered to mouse lungs via tracheostomy with subsequent imaging performed 24, 48, and 72 h after delivery by in vivo fluorescence. 20 min post administration particles of all three sizes were visible in the lung, with the deepest penetrance observed with 0.65 μm particles. In vivo biodistribution was confirmed by fluorescence tomography imaging of excised organs post 72 h. Laser scanning confocal microscopy shows 0.65 μm particles reaching the alveolar space. The delivery of fluorophore to the blood was assessed using Cy7 labeled 0.65 μm particles. Cy7 labeled 0.65 μm particles efficiently delivered fluorescent material to the blood with a peak 3 h after particle administration. The pharmacokinetics of NIR fluorescence dye will be shown. These studies establish that by using 0.65 μm particles loaded with Cy7 we can efficiently access the respiratory portion of the lung, which represents a potentially efficient delivery mechanism for both the lung and the vasculature.</p

Image_2_Use of Submicron Vaterite Particles Serves as an Effective Delivery Vehicle to the Respiratory Portion of the Lung.jpg

<p>Nano- and microencapsulation has proven to be a useful technique for the construction of drug delivery vehicles for use in vascular medicine. However, the possibility of using these techniques within the lung as an inhalation delivery mechanism has not been previously considered. A critical element of particle delivery to the lung is the degree of penetrance that can be achieved with respect to the airway tree. In this study we examined the effectiveness of near infrared (NIR) dye (Cy7) labeled calcium carbonate (vaterite) particles of 3.15, 1.35, and 0.65 μm diameter in reaching the respiratory portion of the lung. First of all, it was shown that, interaction vaterite particles and the components of the pulmonary surfactant occurs a very strong retardation of the recrystallization and dissolution of the particles, which can subsequently be used to create systems with a prolonging release of bioactive substances after the particles penetrate the distal sections of the lungs. Submicro- and microparticles, coated with Cy7 labeled albumin as a model compound, were delivered to mouse lungs via tracheostomy with subsequent imaging performed 24, 48, and 72 h after delivery by in vivo fluorescence. 20 min post administration particles of all three sizes were visible in the lung, with the deepest penetrance observed with 0.65 μm particles. In vivo biodistribution was confirmed by fluorescence tomography imaging of excised organs post 72 h. Laser scanning confocal microscopy shows 0.65 μm particles reaching the alveolar space. The delivery of fluorophore to the blood was assessed using Cy7 labeled 0.65 μm particles. Cy7 labeled 0.65 μm particles efficiently delivered fluorescent material to the blood with a peak 3 h after particle administration. The pharmacokinetics of NIR fluorescence dye will be shown. These studies establish that by using 0.65 μm particles loaded with Cy7 we can efficiently access the respiratory portion of the lung, which represents a potentially efficient delivery mechanism for both the lung and the vasculature.</p