A Single-Cell Atlas of the Human Healthy Airways.

Rationale: The respiratory tract constitutes an elaborate line of defense that is based on a unique cellular ecosystem.Objectives: We aimed to investigate cell population distributions and transcriptional changes along the airways by using single-cell RNA profiling.Methods: We have explored the cellular heterogeneity of the human airway epithelium in 10 healthy living volunteers by single-cell RNA profiling. A total of 77,969 cells were collected at 35 distinct locations, from the nose to the 12th division of the airway tree.Measurements and Main Results: The resulting atlas is composed of a high percentage of epithelial cells (89.1%) but also immune (6.2%) and stromal (4.7%) cells with distinct cellular proportions in different regions of the airways. It reveals differential gene expression between identical cell types (suprabasal, secretory, and multiciliated cells) from the nose (MUC4, PI3, SIX3) and tracheobronchial (SCGB1A1, TFF3) airways. By contrast, cell-type-specific gene expression is stable across all tracheobronchial samples. Our atlas improves the description of ionocytes, pulmonary neuroendocrine cells, and brush cells and identifies a related population of NREP-positive cells. We also report the association of KRT13 with dividing cells that are reminiscent of previously described mouse "hillock" cells and with squamous cells expressing SCEL and SPRR1A/B.Conclusions: Robust characterization of a single-cell cohort in healthy airways establishes a valuable resource for future investigations. The precise description of the continuum existing from the nasal epithelium to successive divisions of the airways and the stable gene expression profile of these regions better defines conditions under which relevant tracheobronchial proxies of human respiratory diseases can be developed.

Factors related to oxygen desaturation during flexible bronchoscopy and endobronchial ultrasound.

INTRODUCTION: Oxygen desaturation is a significant event during bronchoscopy. In this study, it was aimed to identify factors related to oxygen desaturation during flexible bronchoscopy (FB) and Endobronchial ultrasound (EBUS). MATERIALS AND METHODS: From 16 April 2019 to 14 February 2020, 196 consecutive patients (146 FB and 50 EBUS) undergoing bronchoscopy were evaluated retrospectively. The patients' oxygen saturations were monitored on admission and during the procedure by finger pulse oximetry. Desaturation was defined as saturation below 90%. Demographic characteristics, comorbidities, types of interventions, vitals before and during the procedure, amount of saturation decline, and sedative agents used were recorded. The data obtained were compared between the desaturated and non-desaturated groups in both FB and EBUS. We evaluated the risk factors for desaturation during bronchoscopic procedures. RESULT: The mean age of those who underwent FB was higher (62 [52-68] years vs. 55 [44-65] years, p= 0.05), and males were more frequent (54%, vs 19.2%, p<0.001) in the desaturated group. In FB, short lavage was more frequent in the non-desaturated group (28.8% vs. 9.5%, p<0.001). In EBUS, hypertension, diabetes mellitus and thyroid diseases were higher, and duration of procedure was longer (p= 0.02, p= 0.04, p= 0.01 and p<0.001 and p= 0.01, respectively), and SpO2 decline during procedures was higher (11% vs. 1% in FB, 18% vs. 3% in EBUS, p<0.001, each) in the desaturated group. CONCLUSIONS: This study suggested that baseline SpO2 and SpO2 decline during procedures as well as sex, hypertension, and concomitant endocrine - metabolic diseases, duration of procedure were factors associated with desaturation in patients who had undergone FB and EBUS.

Th1 cytokines TNF-α and IFN-γ promote corticosteroid resistance in developing human airway smooth muscle.

Corticosteroids (CSs) are commonly used to manage wheezing and asthma in pediatric populations. Although corticosteroids are effective in alleviating airway diseases, some children with more moderate-severe asthma phenotypes show CS resistance and exhibit significant airflow obstruction, persistent inflammation, and more frequent exacerbations. Previous studies have demonstrated that Th1 cytokines, such as TNF-α and IFN-γ, promote CS resistance in adult human airway smooth muscle (ASM). In the present study, using a human fetal ASM cell model, we tested the hypothesis that TNF-α/IFN-γ induces CS resistance. In contrast to TNF-α or IFN-γ alone, the combination of TNF-α/IFN-γ blunted the ability of fluticasone propionate (FP) to reduce expression of the chemokines CCL5 and CXCL10 despite expression of key anti-inflammatory glucocorticoid receptor target genes being largely unaffected by TNF-α/IFN-γ. Expression of the NF-κB subunit p65 and phosphorylation of Stat1 were elevated in cells treated with TNF-α/IFN-γ, an effect that remained in the presence of FP. siRNA knockdown studies demonstrated the effects of TNF-α/IFN-γ on increased p65 are mediated by Stat1, a transcription factor activated by IFN-γ. Expression of TNFAIP3, a negative regulator of NF-κB activity, was not altered by TNF-α/IFN-γ. However, the effects of TNF-α/IFN-γ were partially reduced by overexpression of TNFAIP3 but did not influence p65 expression. Together, these data suggest that IFN-γ augments the effects of TNF-α on chemokines by enhancing expression of key inflammatory pathways in the presence of CS. Interactions between TNF-α- and IFN-γ-mediated pathways may promote inflammation in asthmatic children resistant to CSs.

Parabronchial remodeling in chicks in response to embryonic hypoxia.

The embryonic development of parabronchi occurs mainly during the second half of incubation in precocious birds, which makes this phase sensitive to possible morphological modifications induced by O2 supply limitation. Thus, we hypothesized that hypoxia during the embryonic phase of parabronchial development induces morphological changes that remain after hatching. To test this hypothesis, chicken embryos were incubated entirely (21 days) under normoxia or partially under hypoxia (15% O2 during days 12 to 18). Lung structures, including air capillaries, blood capillaries, infundibula, atria, parabronchial lumen, bronchi, blood vessels larger than capillaries and interparabronchial tissue, in 1- and 10-day-old chicks were analyzed using light microscopy-assisted stereology. Tissue barrier and surface area of air capillaries were measured using electron microscopy-assisted stereology, allowing for calculation of the anatomical diffusion factor. Hypoxia increased the relative volumes of air and blood capillaries, structures directly involved in gas exchange, but decreased the relative volumes of atria in both groups of chicks, and the parabronchial lumen in older chicks. Accordingly, the surface area of the air capillaries and the anatomical diffusion factor were increased under hypoxic incubation. Treatment did not alter total lung volume, relative volumes of infundibula, bronchi, blood vessels larger than capillaries, interparabronchial tissue or the tissue barrier of any group. We conclude that hypoxia during the embryonic phase of parabronchial development leads to a morphological remodeling, characterized by increased volume density and respiratory surface area of structures involved in gas exchange at the expense of structures responsible for air conduction in chicks up to 10 days old.

Inhibitory effects of openers of large-conductance Ca2+-activated K+ channels on agonist-induced phasic contractions in rabbit and mouse bronchial smooth muscle.

Airway smooth muscle expresses abundant BKCa channels, but their role in regulating contractions remains controversial. This study examines the effects of two potent BKCa channel openers on agonist-induced phasic contractions in rabbit and mouse bronchi. First, we demonstrated the ability of 10 µM GoSlo-SR5-130 to activate BKCa channels in inside-out patches from rabbit bronchial myocytes, where it shifted the activation V1/2 by -88 ± 11 mV (100 nM Ca2+, n = 7). In mouse airway smooth muscle cells, GoSlo-SR5-130 dose dependently shifted V1/2 by 12-83 mV over a concentration range of 1-30 µM. Compound X, a racemic mixture of two enantiomers, reported to be potent BKCa channel openers, shifted V1/2 by 20-79 mV over a concentration range of 0.3-3 µM. In rabbit bronchial rings, exposure to histamine (1 µM) induced phasic contractions after a delay of ~35 min. These were abolished by GoSlo-SR5-130 (30 µM). Nifedipine (100 nM) and CaCCinhA01 (10 µM), a TMEM16A blocker, also abolished histamine-induced phasic contractions. In mouse bronchi, similar phasic contractions were evoked by exposure to U46619 (100 nM) and carbachol (100 nM). In each case, these were inhibited by concentrations of GoSlo-SR5-130 and compound X that shifted the activation V1/2 of BKCa channels in the order of -80 mV. In conclusion, membrane potential-dependent regulation of L-type Ca2+ channels appears to be important for histamine-, U46619-, and carbachol-induced phasic contractions in airway smooth muscle. Contractions can be abolished by BKCa channel openers, suggesting that these channels are potential targets for treating some causes of airway obstruction.

Calcification of the lower respiratory tract in relation to flight development in Jamaican fruit bats (Phyllostomidae, Artibeus jamaicensis).

The production of echolocation calls in bats along with forces produced by contraction of thoracic musculature used in flight presumably puts relatively high mechanical loads on the lower respiratory tract (LRT). Thus, there are likely adaptations to prevent collapse or distortion of the bronchial tree and trachea during flight in echolocating bats. By clearing and staining (Alcian blue and Alizarin red) LRTs removed from nonvolant neonates, semivolant juveniles, volant subadults, and adult Jamaican fruit bats (Artibeus jamaicensis), I found that calcification of the tracheal, primary bronchial, and secondary bronchial (lobar) cartilage rings occurs over the span of about 3 days and coincides with later developmental stages of flight and the increased production of echolocation calls. Tracheal rings that are immediately adjacent to the larynx calcified first, followed by more caudal tracheal rings and then the rings of the primary and secondary bronchi. I suggest that calcification of LRT cartilage rings in echolocating bats provides increased rigidity to counter the thoracic compressions incurred during flight. Calcification of the LRT rings is an adaptation to support the emission of laryngeally produced echolocation calls during flight in bats.

Functional characterization of a novel 3D model of the epithelial-mesenchymal trophic unit.

BACKGROUND/AIM: Epithelial-mesenchymal communication plays a key role in tissue homeostasis and abnormal signaling contributes to chronic airways disease such as COPD. Most in vitro models are limited in complexity and poorly represent this epithelial-mesenchymal trophic unit. We postulated that cellular outgrowth from bronchial tissue would enable development of a mucosal structure that recapitulates better in vivo tissue architecture. MATERIALS AND METHODS: Bronchial tissue was embedded in Matrigel and outgrowth cultures monitored using time-lapse microscopy, electrical resistance, light and electron microscopy. Cultures were challenged repetitively with cigarette smoke extract (CSE). RESULTS: The outgrowths formed as a multicellular sheet with motile cilia becoming evident as the Matrigel was remodeled to provide an air interface; cultures were viable for more than one year. Immunofluorescence and electron microscopy (EM) identified an upper layer of mucociliary epithelium and a lower layer of highly organized extracellular matrix (ECM) interspersed with fibroblastic cells separated by a basement membrane. EM analysis of the mucosal construct after repetitive exposure to CSE revealed epithelial damage, loss of cilia, and ECM remodeling, as occurs in vivo. CONCLUSIONS: We have developed a robust bronchial mucosal model. The structural changes observed following CSE exposure suggest the model should have utility for drug discovery and preclinical testing, especially those targeting airway remodeling.

Branching morphogenesis of immortalized human bronchial epithelial cells in three-dimensional culture.

While mouse models have contributed in our understanding of lung development, repair and regeneration, inherent differences between the murine and human airways requires the development of new models using human airway epithelial cells. In this study, we describe a three-dimensional model system using human bronchial epithelial cells (HBECs) cultured on reconstituted basement membrane. HBECs form complex budding and branching structures on reconstituted basement membrane when co-cultured with human lung fetal fibroblasts. These structures are reminiscent of the branching epithelia during lung development. The HBECs also retain markers indicative of epithelial cell types from both the central and distal airways suggesting their multipotent potential. In addition, we illustrate how the model can be utilized to understand respiratory diseases such as lung cancer. The 3D novel cell culture system recapitulates stromal-epithelial interactions in vitro that can be utilized to understand important aspects of lung development and diseases.

Lung progenitors from lambs can differentiate into specialized alveolar or bronchiolar epithelial cells.

BACKGROUND: Airways progenitors may be involved in embryogenesis and lung repair. The characterization of these important populations may enable development of new therapeutics to treat acute or chronic lung disease. In this study, we aimed to establish the presence of bronchioloalveolar progenitors in ovine lungs and to characterize their potential to differentiate into specialized cells. RESULTS: Lung cells were studied using immunohistochemistry on frozen sections of the lung. Immunocytochemistry and flow cytometry were conducted on ex-vivo derived pulmonary cells. The bronchioloalveolar progenitors were identified by their co-expression of CCSP, SP-C and CD34. A minor population of CD34(pos)/SP-C(pos)/CCSP(pos) cells (0.33% ± 0.31) was present ex vivo in cell suspensions from dissociated lungs. Using CD34 magnetic positive-cell sorting, undifferentiated SP-C(pos)/CCSP(pos) cells were purified (>80%) and maintained in culture. Using synthetic media and various extracellular matrices, SP-C(pos)/CCSP(pos) cells differentiated into either club cells (formerly named Clara cells) or alveolar epithelial type-II cells. Furthermore, these ex vivo and in vitro derived bronchioloalveolar progenitors expressed NANOG, OCT4 and BMI1, specifically described in progenitors or stem cells, and during lung development. CONCLUSIONS: We report for the first time in a large animal the existence of bronchioloalveolar progenitors with dual differentiation potential and the expression of specialized genes. These newly described cell population in sheep could be implicated in regeneration of the lung following lesions or in development of diseases such as cancers.

Development of the bronchial epithelial reticular basement membrane: relationship to epithelial height and age.

BACKGROUND: The bronchial epithelium and underlying reticular basement membrane (RBM) have a close spatial and functional inter-relationship and are considered an epithelial-mesenchymal trophic unit (EMTU). An understanding of RBM development is critical to understanding the extent and time of appearance of its abnormal thickening that is characteristic of asthma. METHODS: RBM thickness and epithelial height were determined in histological sections of cartilaginous bronchi obtained postmortem from 47 preterm babies and infants (median age 40 weeks gestation (22 weeks gestation-8 months)), 40 children (2 years (1 month-17 years)) and 23 adults (44 (17-90) years) who had died from non-respiratory causes, and had no history of asthma. RESULTS: The RBM was visible by light microscopy at 30 weeks gestation. RBM thickness increased in successive age groups in childhood; in infants (r=0.63, p<0.001) and in children between 1 month and 17 years (r=0.82, p<0.001). After 18 years, RBM thickness decreased with increasing age (r=-0.42, p<0.05). Epithelial height showed a similar relationship with age, a positive relationship from preterm to 17 years (r=0.50, p<0.001) and a negative relationship in adulthood (r=-0.84, p<0.0001). There was a direct relationship between epithelial height and RBM thickness (r=0.6, p<0.001). CONCLUSIONS: The RBM in these subjects was microscopically identifiable by 30 weeks gestation. It thickened during childhood and adolescence. In adults, there was either no relationship with age, or a slow reduction in thickness in older age. Developmental changes of RBM thickness were accompanied by similar changes in epithelial height, supporting the close relationship between RBM and epithelium within the EMTU.

Postnatal growth of tracheobronchial airways of Sprague-Dawley rats.

Rats are widely used for the studies of pulmonary toxicology in both juveniles and adults. To facilitate such studies, investigators have developed models of lung architecture based on manual or computerized airway measurements. However, postnatal growth of conducting airways of rat lungs has never been reported. In this paper, we present conducting airway architecture statistics for male Sprague-Dawley rat lungs at ages 15, 28, 40, and 81 days by analyzing CT images from airway silicon casts. Detailed branching characteristics and intersubject variance are presented. This study shows that (i) airway growth in diameter and length is not linear with age, (ii) growth of airway length is faster than that of diameter during the 15-81-day postnatal period, and (iii) asymmetry in airway diameter (ratio of major to minor daughter diameter) increases with age.

Disruption of tracheobronchial airway growth following postnatal exposure to ozone and ultrafine particles.

This study examined airway structure changes in adult rats after a long recovery period due to sub-chronic juvenile exposure to ozone and ultrafine particles that have a high organic fraction. Neonatal male Sprague-Dawley rats were exposed during lung development to 3 cycles of 0.5 ppm ozone from postnatal day 7 through 25. Two different exposure patterns were used: 5-day exposure per week (Ozone52) or 2-day exposure per week (Ozone25) with or without co-exposure to ultrafine particles (OPFP5252, OPFP5225). Airway architecture was evaluated at 81 days of age, after 56 days of continued development beyond the exposure period in filtered air (FA). By analyzing CT images from lung airway casts, we determined airway diameter, length, branching angle, and rotation angle for most conducting airways. Compared with the FA control group, the Ozone52 group showed significant decreases in airway diameter in generations larger than 10 especially in the right diaphragmatic lobe and in airway length in distal generations, while changes in airway structure due to the Ozone25 exposure were not appreciable. Interaction effects of ozone and ultrafine particle exposures were not significant. These results suggest that airway alterations due to postnatal ozone exposure are not limited to the distal region but occur extensively from the middle to distal conducting airways. Further, alterations due to early ozone exposure do not recover nearly 2 months after exposure has ceased demonstrating a persistent airway structural change following an early life exposure to ozone.

Pten controls lung morphogenesis, bronchioalveolar stem cells, and onset of lung adenocarcinomas in mice.

PTEN is a tumor suppressor gene mutated in many human cancers. We generated a bronchioalveolar epithelium-specific null mutation of Pten in mice [SP-C-rtTA/(tetO)(7)-Cre/Pten(flox/flox) (SOPten(flox/flox)) mice] that was under the control of doxycycline. Ninety percent of SOPten(flox/flox) mice that received doxycycline in utero [SOPten(flox/flox)(E10-16) mice] died of hypoxia soon after birth. Surviving SOPten(flox/flox)(E10-16) mice and mice that received doxycycline postnatally [SOPten(flox/flox)(P21-27) mice] developed spontaneous lung adenocarcinomas. Urethane treatment accelerated number and size of lung tumors developing in SOPten(flox/flox) mice of both ages. Histological and biochemical examinations of the lungs of SOPten(flox/flox)(E10-16) mice revealed hyperplasia of bronchioalveolar epithelial cells and myofibroblast precursors, enlarged alveolar epithelial cells, and impaired production of surfactant proteins. Numbers of bronchioalveolar stem cells (BASCs), putative initiators of lung adenocarcinomas, were increased. Lungs of SOPten(flox/flox)(E10-16) mice showed increased expression of Spry2, which inhibits the maturation of alveolar epithelial cells. Levels of Akt, c-Myc, Bcl-2, and Shh were also elevated in SOPten(flox/flox)(E10-16) and SOPten(flox/flox)(P21-27) lungs. Furthermore, K-ras was frequently mutated in adenocarcinomas observed in SOPten(flox/flox)(P21-27) lungs. These results indicate that Pten is essential for both normal lung morphogenesis and the prevention of lung carcinogenesis, possibly because this tumor suppressor is required for BASC homeostasis.

In vitro 3D culture lung model from expanded primary cystic fibrosis human airway cells.

BACKGROUND: In vitro cystic fibrosis (CF) models are crucial for understanding the mechanisms and consequences of the disease. They are also the gold standard for pre-clinical efficacy studies of current and novel CF drugs. However, few studies have investigated expansion and differentiation of primary CF human bronchial epithelial (CF-HBE) cells. Here we describe culture conditions to expand primary CF airway cells while preserving their ability to differentiate into 3D epithelial cultures expressing functional cystic fibrosis transmembrane conductance regulator (CFTR) ion channels that responds to CFTR modulators. METHODS: Primary CF airway cells were expanded using PneumaCultTM-Ex Plus (StemCell Technologies) medium with no feeder cells or added Rho kinase (ROCK) inhibitor. Differentially passaged CF-HBE cells at the air-liquid interface (ALI) were characterized phenotypically and functionally in response to the CFTR corrector drug VX-661 (Tezacaftor). RESULTS: CF-HBE primary cells, expanded up to six passages (~25 population doublings), differentiated into 3D epithelial cultures as evidenced by trans-epithelial electrical resistance (TEER) of >400 Ohms∙cm2 and presence of pseudostratified columnar ciliated epithelium with goblet cells. However, up to passage five cells from most donors showed increased CFTR-mediated short-circuit currents when treated with the corrector drug, VX-661. Ciliary beat frequency (CBF) also increased with the corrector VX-661. CONCLUSIONS: CF donor-derived airway cells can be expanded without the use of feeder cells or additional ROCK inhibitor, and still achieve optimal 3D epithelial cultures that respond to CFTR modulators. The study of rare CF mutations could benefit from cell expansion and could lead to the design of personalized medicine/treatments.

Nicotine activates and up-regulates nicotinic acetylcholine receptors in bronchial epithelial cells.

Prenatal nicotine exposure impairs normal lung development and leads to diminished pulmonary function after birth. Previous work from our laboratory has demonstrated that nicotine alters lung development by affecting a nonneuronal cholinergic autocrine loop that is expressed in lung. Bronchial epithelial cells (BECs) express choline acetyltransferase, the choline high-affinity transporter and nicotinic acetylcholine (ACh) receptor (nAChR) subunits. We now demonstrate through a combination of morphological and electrophysiological techniques that nicotine affects this autocrine loop by up-regulating and activating cholinergic signaling. RT-PCR showed the expression of alpha 3, alpha 4, alpha 7, alpha 9, alpha 10, beta2, and beta 4 nAChR mRNAs in rhesus monkey lung and cultured BECs. The expression of alpha 7, alpha 4, and beta2 nAChR was confirmed by immunofluorescence in the cultured BECs and lung. The electrophysiological characteristics of nAChR in BECs were determined using whole-cell patch-clamp on cultured BECs. Both ACh and nicotine evoked an inward current, with a rapid desensitizing current. Nicotine induced inward currents in a concentration-dependent manner, with an EC(50) of 26.7 microM. Nicotine-induced currents were reversibly blocked by the nicotinic antagonists, mecamylamine, dihydro-beta-erythroidine, and methyllcaconitine. Incubation of BECs with 1 microM nicotine for 48 hours enhanced nicotine-induced currents by roughly 26%. The protein tyrosine phosphorylation inhibitor, genistein, increased nicotine-induced currents by 58% and enhanced methyllcaconitine-sensitive currents (alpha 7 nAChR activities) 2.3-fold, whereas the protein tyrosine phosphatase inhibitor, pervanadate, decreased the effects of nicotine. These results demonstrate that chronic nicotine exposure up-regulates nAChR activity in developing lung, and that nAChR activity can be further modified by tyrosine phosphorylation.

Effect of perinatal secondhand tobacco smoke exposure on in vivo and intrinsic airway structure/function in non-human primates.

Infants exposed to second hand smoke (SHS) experience more problems with wheezing. This study was designed to determine if perinatal SHS exposure increases intrinsic and/or in vivo airway responsiveness to methacholine and whether potential structural/cellular alterations in the airway might explain the change in responsiveness. Pregnant rhesus monkeys were exposed to filtered air (FA) or SHS (1 mg/m(3) total suspended particulates) for 6 h/day, 5 days/week starting at 50 days gestational age. The mother/infant pairs continued the SHS exposures postnatally. At 3 months of age each infant: 1) had in vivo lung function measurements in response to inhaled methacholine, or 2) the right accessory lobe filled with agarose, precision-cut to 600 mum slices, and bathed in increasing concentrations of methacholine. The lumenal area of the central airway was determined using videomicrometry followed by fixation and histology with morphometry. In vivo tests showed that perinatal SHS increases baseline respiratory rate and decreases responsiveness to methacholine. Perinatal SHS did not alter intrinsic airway responsiveness in the bronchi. However in respiratory bronchioles, SHS exposure increased airway responsiveness at lower methacholine concentrations but decreased it at higher concentrations. Perinatal SHS did not change eosinophil profiles, epithelial volume, smooth muscle volume, or mucin volume. However it did increase the number of alveolar attachments in bronchi and respiratory bronchioles. In general, as mucin increased, airway responsiveness decreased. We conclude that perinatal SHS exposure alters in vivo and intrinsic airway responsiveness, and alveolar attachments.

Presence of N-acetylgalactosamine/galactose residues on bronchioloalveolar cells during rat postnatal development.

In mammals, the alveolarization process develops predominantly after birth. Airway cells display a complex assemblage of glycans on their surface. These glycans, particularly terminal glycan extensions, are important effective carriers of information that change during the differentiation process. Nevertheless, few systematic data are reported about the cell surface sugar residue content during post-natal lung development. In the present work, we aimed to identify and semi-quantify N-acetylgalactosamine (GalNAc)/galactose (Gal) residues on the bronchioloalveolar cell surface in rat lung sections from 1-, 4-, 8- day old and adult animals and link these data with the lung glycocalyx composition. Horseradish peroxidase-conjugated lectin from Glycine max (soybean agglutinin, SBA) was used, and light microscopy methodologies were performed. SBA labelling intensity was studied before and after sialidase pre-treatment, at one-, four- and eight-day-old animals and adult animals. For semi-quantitative evaluation of SBA binding intensity, two investigators performed the analysis independently, blinded to the type of experiment. Reactivity of the lectin was assessed in bronchiolar and respiratory portion/alveolar epithelial cell surfaces. We evidenced a stronger positive reaction when lung sections were pre-treated with neuraminidase before incubation with the lectin in one- and four-day-old animals and adult animals. These results were not so manifest in eight-day-old animals. This binding pattern, generally points towards the presence of terminal but mainly sub-terminal GalNAc/Gal residues probably capped by sialic acids on the rat bronchiolar/respiratory tract epithelial cells. As this glycan extension is common in O- and N-glycans, our results suggest that these glycan classes can be present in bronchioloalveolar cells immediately after birth and exist during the postnatal period. The results observed in eight-day-old rat lung sections may be due to the dramatic lung morphologic changes and the possible underlying biological mechanisms that occur during this age-moment.

Concomitant deletion of HRAS and NRAS leads to pulmonary immaturity, respiratory failure and neonatal death in mice.

We reported previously that adult (HRAS-/-; NRAS-/-) double knockout (DKO) mice showed no obvious external phenotype although lower-than-expected numbers of weaned DKO animals were consistently tallied after crossing NRAS-KO and HRAS-KO mice kept on mixed genetic backgrounds. Using mouse strains kept on pure C57Bl/6 background, here we performed an extensive analysis of the offspring from crosses between HRAS-KO and NRAS-KO mice and uncovered the occurrence of very high rates of perinatal mortality of the resulting DKO littermates due to respiratory failure during the first postnatal 24-48 h. The lungs of newborn DKO mice showed normal organ structure and branching but displayed marked defects of maturation including much-reduced alveolar space with thick separating septa and significant alterations of differentiation of alveolar (AT1, AT2 pneumocytes) and bronchiolar (ciliated, Clara cells) cell lineages. We also observed the retention of significantly increased numbers of undifferentiated progenitor precursor cells in distal lung epithelia and the presence of substantial accumulations of periodic acid-Schiff-positive (PAS+) material and ceramide in the lung airways of newborn DKO mice. Interestingly, antenatal dexamethasone treatment partially mitigated the defective lung maturation phenotypes and extended the lifespan of the DKO animals up to 6 days, but was not sufficient to abrogate lethality in these mice. RNA microarray hybridization analyses of the lungs of dexamethasone-treated and untreated mice uncovered transcriptional changes pointing to functional and metabolic alterations that may be mechanistically relevant for the defective lung phenotypes observed in DKO mice. Our data suggest that delayed alveolar differentiation, altered sphingolipid metabolism and ceramide accumulation are primary contributors to the respiratory stress and neonatal lethality shown by DKO mice and uncover specific, critical roles of HRAS and NRAS for correct lung differentiation that are essential for neonatal survival and cannot be substituted by the remaining KRAS function in this organ.

Airway geometry models of children's lungs for use in dosimetry modeling.

Single-path whole-lung and lobar models of the lungs of 11 children between 3 mo and 21 yr of age were developed based on a combination of cast data and published information on distal airway dimensions. The cast data used to generate these models were taken from one of the largest databases of actual measurements in children. The methods used to develop the children's models were based on techniques that have been used to develop adult single-path airway geometry models. Model dimensions for the conducting airways, as well as the estimated dead space, for all children fell within the range of the limited published information. Thus, the method for estimating airway dimensions in adults may be successfully applied to develop estimates of airway dimensions in children. The predicted total lung capacity (TLC) for the older children (aged 8 to 21 yr) fell within or near the range arising from published scaling equations. The assumptions used to generate the gas exchange region for children 8 yr and older produced results that were reasonably consistent with available physiological data. However, these assumptions do not result in a physiologically consistent gas exchange region for children 3 yr of age and younger; also, to maintain physiologically reasonable relationships between dead space and alveolar volume, the models for children 3 yr of age and younger resulted in predicted TLCs well below those predicted using published scaling equations. These discrepancies may be reflective of dysanaptic growth, in which the alveolar region is growing more rapidly than the airways. The results for children 3 yr of age and under suggest the need for a greater understanding of lung development during this critical period. This is particularly important considering the increasing evidence that exposure to pollutants and other toxicants and allergens during the first 2 yr of life may have long-term consequences on respiratory disease outcomes. Our results suggest that the geometry model airway dimensions for all ages are appropriate for use with dosimetry models, but dosimetry modelers need to assess carefully the reasonableness of TLC and functional residual capacity volumes to which airway dimensions are scaled for children 3 yr of age and under.

High repeatability from 3D experimental platform for quantitative analysis of cellular branch pattern formations.

Three-dimensional (3D) cell and tissue cultures more closely mimic biological environments than two-dimensional (2D) cultures and are therefore highly desirable in culture experiments. However, 3D cultures often fail to yield repeatable experimental results because of variation in the initial culture conditions, such as cell density and distribution in the extracellular matrix, and therefore reducing such variation is a paramount concern. Here, we present a 3D culture platform that demonstrates highly repeatable experimental results, obtained by controlling the initial cell cluster shape in the gel cube culture device. A micro-mould with the desired shape was fabricated by photolithography or machining, creating a 3D pocket in the extracellular matrix contained in the device. Highly concentrated human bronchial epithelial cells were then injected in the pocket so that the cell cluster shape matched the fabricated mould shape. Subsequently, the cubic device supplied multi-directional scanning, enabling high-resolution capture of the whole tissue structure with only a low-magnification lens. The proposed device significantly improved the repeatability of the developed branch pattern, and multi-directional scanning enabled quantitative analysis of the developed branch pattern formations. A mathematical simulation was also conducted to reveal the mechanisms of branch pattern formation. The proposed platform offers the potential to accelerate any research field that conducts 3D culture experiments, including tissue regeneration and drug development.