Search

BACKGROUND: Double lumen endobronchial tubes (DLTs) are frequently used to employ single lung ventilation strategies during thoracic surgical procedures. Placement of these tubes can be challenging even for experienced clinicians. We hypothesized that airway anatomy, particularly of the glottis and proximal trachea, significantly impacts the ease or difficulty in placement of these tubes. METHODS: Images from 24 randomly selected Positron Emission Tomography - Computed Tomography (PET-CT) scans were evaluated for several anatomic aspects of the upper airway, including size and angulation of the glottis and proximal tracheal using calibrated CT measurements and an online digital protractor. The anatomic issues identified were confirmed in cadaveric anatomic models. RESULTS: Proximal tracheal diameter measurements in PET-CT scans demonstrated a mean ± standard deviation of 20.4 ± 2.5 mm in 12 males and 15.5 ± 0.98 mm in 12 females (p

Understanding viral tropism is an essential step toward reducing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) transmission, decreasing mortality from coronavirus disease 2019 (COVID-19) and limiting opportunities for mutant strains to arise. Currently, little is known about the extent to which distinct tissue sites in the human head and neck region and proximal respiratory tract selectively permit SARS-CoV-2 infection and replication. In this translational study, we discover key variabilities in expression of angiotensin-converting enzyme 2 (ACE2) and transmembrane serine protease 2 (TMPRSS2), essential SARS-CoV-2 entry factors, among the mucosal tissues of the human proximal airways. We show that SARS-CoV-2 infection is present in all examined head and neck tissues, with a notable tropism for the nasal cavity and tracheal mucosa. Finally, we uncover an association between smoking and higher SARS-CoV-2 viral infection in the human proximal airway, which may explain the increased susceptibility of smokers to developing severe COVID-19. This is at least partially explained by differences in interferon (IFN)-β1 levels between smokers and non-smokers.

The immunological features that distinguish COVID-19-associated acute respiratory distress syndrome (ARDS) from other causes of ARDS are incompletely understood. Here, we report the results of comparative lower respiratory tract transcriptional profiling of tracheal aspirate from 52 critically ill patients with ARDS from COVID-19 or from other etiologies, as well as controls without ARDS. In contrast to a "cytokine storm," we observe reduced proinflammatory gene expression in COVID-19 ARDS when compared to ARDS due to other causes. COVID-19 ARDS is characterized by a dysregulated host response with increased PTEN signaling and elevated expression of genes with non-canonical roles in inflammation and immunity. In silico analysis of gene expression identifies several candidate drugs that may modulate gene expression in COVID-19 ARDS, including dexamethasone and granulocyte colony stimulating factor. Compared to ARDS due to other types of viral pneumonia, COVID-19 is characterized by impaired interferon-stimulated gene (ISG) expression. The relationship between SARS-CoV-2 viral load and expression of ISGs is decoupled in patients with COVID-19 ARDS when compared to patients with mild COVID-19. In summary, assessment of host gene expression in the lower airways of patients reveals distinct immunological features of COVID-19 ARDS.

Rapid and accurate assessment of endotracheal tube (ETT) location is essential in the intensive care unit (ICU) setting, where timely identification of a mispositioned support device may prevent significant patient morbidity and mortality. This study proposes a series of deep learning-based algorithms which together iteratively identify and localize the position of an ETT relative to the carina on chest radiographs. Using the open-source MIMIC Chest X-Ray (MIMIC-CXR) dataset, a total of 16,000 patients were identified (8000 patients with an ETT and 8000 patients without an ETT). Three different convolutional neural network (CNN) algorithms were created. First, a regression loss function CNN was trained to estimate the coordinate location of the carina, which was then used to crop the original radiograph to the distal trachea and proximal bronchi. Second, a classifier CNN was trained using the cropped inputs to determine the presence or absence of an ETT. Finally, for radiographs containing an ETT, a third regression CNN was trained to both refine the coordinate location of the carina and identify the location of the distal ETT tip. Model accuracy was assessed by comparing the absolute distance of prediction and ground-truth coordinates as well as CNN predictions relative to measurements documented in original radiologic reports. Upon five-fold cross validation, binary classification for the presence or absence of ETT demonstrated an accuracy, sensitivity, specificity, PPV, NPV, and AUC of 97.14%, 97.37%, 96.89%, 97.12%, 97.15%, and 99.58% respectively. CNN predicted coordinate location of the carina, and distal ETT tip was estimated within a median error of 0.46 cm and 0.60 cm from ground-truth annotations respectively. Overall final CNN assessment of distance between the carina and distal ETT tip was predicted within a median error of 0.60 cm from manual ground-truth annotations, and a median error of 0.66 cm from measurements documented in the original radiology reports. A serial cascaded CNN approach demonstrates high accuracy for both identification and localization of ETT tip and carina on chest radiographs. High performance of the proposed multi-step strategy is in part related to iterative refinement of coordinate localization as well as explicit image cropping which focuses algorithm attention to key anatomic regions of interest.

Severe asthma remains challenging to manage and has limited treatment options. We have previously shown that targeting smooth muscle integrin α5β1 interaction with fibronectin can mitigate the effects of airway hyperresponsiveness by impairing force transmission. In this study, we show that another member of the integrin superfamily, integrin α2β1, is present in airway smooth muscle and capable of regulating force transmission via cellular tethering to the matrix protein collagen I and, to a lesser degree, laminin-111. The addition of an inhibitor of integrin α2β1 impaired IL-13-enhanced contraction in mouse tracheal rings and human bronchial rings and abrogated the exaggerated bronchoconstriction induced by allergen sensitization and challenge. We confirmed that this effect was not due to alterations in classic intracellular myosin light chain phosphorylation regulating muscle shortening. Although IL-13 did not affect surface expression of α2β1, it did increase α2β1-mediated adhesion and the level of expression of an activation-specific epitope on the β1 subunit. We developed a method to simultaneously quantify airway narrowing and muscle shortening using 2-photon microscopy and demonstrated that inhibition of α2β1 mitigated IL-13-enhanced airway narrowing without altering muscle shortening by impairing the tethering of muscle to the surrounding matrix. Our data identified cell matrix tethering as an attractive therapeutic target to mitigate the severity of airway contraction in asthma.

Huntington's disease (HD) is caused by an expansion of a poly glutamine (polyQ) stretch in the huntingtin protein (HTT) that is necessary to cause pathology and formation of HTT aggregates. Here we ask whether expanded polyQ is sufficient to cause pathology and aggregate formation. By addressing the sufficiency question, one can identify cellular processes and structural parameters that influence HD pathology and HTT subcellular behavior (i.e. aggregation state and subcellular location). Using Drosophila, we compare the effects of expressing mutant full-length human HTT (fl-mHTT) to the effects of mutant human HTTexon1 and to two commonly used synthetic fragments, HTT171 and shortstop (HTT118). Expanded polyQ alone is not sufficient to cause inclusion formation since full-length HTT and HTTex1 with expanded polyQ are both toxic although full-length HTT remains diffuse while HTTex1 forms inclusions. Further, inclusions are not sufficient to cause pathology since HTT171-120Q forms inclusions but is benign and co-expression of HTT171-120Q with non-aggregating pathogenic fl-mHTT recruits fl-mHTT to aggregates and rescues its pathogenicity. Additionally, the influence of sequences outside the expanded polyQ domain is revealed by finding that small modifications to the HTT118 or HTT171 fragments can dramatically alter their subcellular behavior and pathogenicity. Finally, mutant HTT subcellular behavior is strongly modified by different cell and tissue environments (e.g. fl-mHTT appears as diffuse nuclear in one tissue and diffuse cytoplasmic in another but toxic in both). These observations underscore the importance of cellular and structural context for the interpretation and comparison of experiments using different fragments and tissues to report the effects of expanded polyQ.

The conducting airway forms a protective mucosal barrier and is the primary target of airway disorders. The molecular events required for the formation and function of the airway mucosal barrier, as well as the mechanisms by which barrier dysfunction leads to early onset airway diseases, remain unclear. In this study, we systematically characterized the developmental landscape of the mouse airway using single-cell RNA sequencing and identified remarkably conserved cellular programs operating during human fetal development. We demonstrated that in mouse, genetic inactivation of chloride channel Ano1/Tmem16a compromises airway barrier function, results in early signs of inflammation, and alters the airway cellular landscape by depleting epithelial progenitors. Mouse Ano1-/-mutants exhibited mucus obstruction and abnormal mucociliary clearance that resemble the airway defects associated with cystic fibrosis. The data reveal critical and non-redundant roles for Ano1 in organogenesis, and show that chloride channels are essential for mammalian airway formation and function.

The combination of vertebral, anal, cardiac, tracheo-esophageal, renal and limb anomalies termed VACTERL association, also referred to as VATER, has been used as a clinical descriptor and more recently, a diagnosis of exclusion, for a specific group of phenotypic manifestations that have been observed to co-occur non-randomly. Though the causes remain elusive and poorly understood in most patients, VACTERL association is thought to be due to defects in early embryogenesis and is likely genetically heterogeneous. We present data on 36 patients diagnosed with VACTERL association in addition to describing the phenotypic diversity of each component feature. Unique cases in our cohort include a patient with a 498.59 kb microdeletion in the 16p11.2 region and another with a 215 kb duplication in the 3p25.2 region. Our findings expand upon the current understanding of VACTERL association and guide future research aimed at determining its etiology.

ObjectiveThis study aimed to detect novel mesenchymal stem cell peptides/biomarkers of bronchopulmonary dysplasia (BPD) in the tracheal aspirate fluid (TAF) of preterm infants.Study designParticipants included infants less than 32 weeks' gestational age or birth weight under 1500 grams who required endotracheal intubation and mechanical ventilation within first 24 hours of life. TAF sample collection was performed at the time of the first clinically indicated routine suctioning. Standardization curves for human levels of osteopontin (Opn), macrophage colony stimulating factor 1 (Csf1), transforming growth factor beta 1 (TGF-β1), and secretory immunoglobulin A (sIgA) were generated for 15 enrolled participants.ResultsWe demonstrated that stem cell biomarkers are secreted into the TAF of preterm infants and their concentrations can be easily measured during the first week of life.ConclusionsFurther studies are warranted to determine a causal relationship between these biomarkers and BPD development and severity.

BackgroundTownes-Brocks syndrome (TBS) is a rare autosomal dominant condition characterized by renal, anal, limb, and auditory abnormalities. TBS diagnosis can be challenging in settings where genetic analysis is not readily available. TBS traits overlap with those of Goldenhar and VACTERL syndromes.Case presentationHere, we present the case of a 5-year-old Brazilian boy born with an anorectal abnormality, limb and external ears malformations, genitourinary anomalies, and a congenital heart defect. Genetic analysis revealed a SALL1 nonsense mutation. The case is discussed in the context of the current literature.ConclusionsBecause of the variability in TBS clinical presentation, genetic analysis is key to the differential diagnosis of TBS relative to phenotypically similar syndromes.

Background: Airtraq®-Airview (AA) and King Vision® (KV) video laryngoscopes are two new video laryngoscopes (VLS). Many studies have been carried out to compare the both regarding efficacy, ease of use etc., but the matter remains undecided. We compared AA to KV(channeled) primarily for total intubation time and secondarily for success rate, number of attempts, optimization maneuvers, percentage of glottis opening (POGO) score, Cormack-Lehane (CL) score, degree of difficulty of intubation (Likert scale) and adverse effects. Methodology: After obtaining institutional ethical committee approval and written informed consent from study subjects, this randomized control study was conducted in the Department of Anesthesiology & Critical Care, Mathura Das Mathur Hospital, Jodhpur from January 2019 to September 2019. Seventy ASA- I and II patients of age 20-60 y, weighing 40-70 kg, irrespective of their Mallampati class, posted for elective surgery under general anesthesia, were included in the study. Patients were randomly divided into two groups, Group AA (n = 35) intubated using AA and Group KC (n = 35) intubated using KV (channeled). Pregnant patients, patients with mouth opening less than 18 mm and any oral pathology, neck deformities and refusal to consent were excluded. Statistical analyses were performed using SPSS 22.0 software package (SPSS Inc., Chicago, IL, USA). Chi square test, Fisher’s exact test and Fisher-Freeman- Halton test were used. All data were summarized as mean ± SD for continuous variables, numbers and percentages for categorical variables. P < 0.05 was accepted as significant. Results: Total intubation time was significantly longer in group AA. Demographic and hemodynamic data of patients and intubation attempts, CL score, POGO score, degree of difficult intubation on Likert scale and adverse effects such as airway trauma in both the groups were comparable. Conclusions: Although both video laryngoscopes are similar in intubating conditions, intubation with King’s Vision® video laryngoscope was faster; hence might be preferable for intubation. [ABSTRACT FROM AUTHOR]

ObjectivesEstablishing a definitive airway is often the first step in emergency department treatment of critically ill patients. Currently, there is no agreed upon consensus as to the most efficacious method of airway confirmation. Our objective was to determine the diagnostic accuracy of real-time sonography performed by resident physicians to confirm placement of the endotracheal tube during emergent intubation.MethodsWe performed a prospective cohort study of adult patients in the emergency department undergoing emergent endotracheal intubation. Thirty emergency medicine residents, who were blinded to end-tidal carbon dioxide detection results, performed real-time transverse tracheal sonography during intubation to evaluate correct endotracheal tube placement.ResultsSeventy-two patients were enrolled in the study. Sixty-eight instances (94.4%) were interpreted as correct placement in the trachea; 4 (5.6%) were interpreted as esophageal, of which 1 was a false-negative finding, therefore conferring sensitivity of 98.5% (95% confidence interval, 92.1%-99.9%) and specificity of 75.0% (95% confidence interval, 19.4%-99.4%) for correct placement. There was no significant difference in accuracy among resident sonographers with different levels of residency training.ConclusionsA simple transverse tracheal sonographic examination performed by emergency medicine resident physicians can be used as an adjunct to help confirm correct endotracheal tube placement during intubation. In our cohort, the level of training did not appear to affect the ability of residents to correctly identify the endotracheal tube position.

Objectives/hypothesisPhysiologically relevant, well-characterized in vitro vocal fold coculture models are needed to test the effects of various challenges and therapeutics on vocal fold physiology. We characterize a healthy state coculture model, created by using bronchial/tracheal epithelial cells and immortalized vocal fold fibroblasts. We also demonstrate that this model can be induced into a fibroplastic state to overexpress stress fibers using TGFβ1.Study designIn vitro.MethodsCell metabolic activity of immortalized human vocal fold fibroblasts incubated in different medium combinations was confirmed with an MTT (3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide) assay. Fibroblasts were grown to confluence, and primary bronchial/tracheal epithelial cells suspended in coculture medium were seeded directly over the base layer of the fibroblasts. Cells were treated with transforming growth factor β1 (TGFβ1) to induce myofibroblast formation. Cell shape and position were confirmed by live cell tracking, fibrosis was confirmed by probing for α smooth muscle actin (αSMA), and phenotype was confirmed by immunostaining for vimentin and E-cadherin.ResultsFibroblasts retain metabolic activity in coculture epithelial medium. Live cell imaging revealed a layer of epithelial cells atop fibroblasts. αSMA expression was enhanced in TGFβ1-treated cells, confirming that both cell types maintained a healthy phenotype in coculture, and can be induced into overexpressing stress fibers. Vimentin and E-cadherin immunostaining show that cells retain phenotype in coculture.ConclusionsThese data lay effective groundwork for a functional coculture model that retains the reproducibility necessary to serve as a viable diagnostic and therapeutic screening platform.Level of evidenceNA Laryngoscope, 127:E185-E192, 2017.

ObjectivesTo determine the feasibility of long-range optical coherence tomography (LR-OCT) as a tool to intraoperatively image and measure the subglottis and trachea during suspension microlaryngoscopy before and after endoscopic treatment of subglottic stenosis (SGS).MethodsLong-range optical coherence tomography of the adult subglottis and trachea was performed during suspension microlaryngoscopy before and after endoscopic treatment for SGS. The anteroposterior and transverse diameters, cross-sectional area (CSA), distance from the vocal cords, and length of the SGS were measured using a MATLAB software. Pre-intervention and postintervention airway dimensions were compared. Three-dimensional volumetric airway reconstructions were generated using medical image processing software (MIMICS).ResultsIntraoperative LR-OCT imaging was performed in 3 patients undergoing endoscopic management of SGS. Statistically significant differences in mean anteroposterior diameter (P < .01), transverse diameter (P < .001), and CSA (P < .001) were noted between pre-intervention and postintervention data. Three-dimensional airway models were viewed in cross-sectional format and via virtual "fly through" bronchoscopy.ConclusionsThis is the first report of intraoperative LR-OCT of the subglottic and tracheal airway before and after surgical management of SGS in humans. Long-range optical coherence tomography offers a practical means to measure the dimensions of SGS and acquire objective data on the response to endoscopic treatment of SGS.

Protein disulfide isomerase (PDI) family members regulate protein folding and calcium homeostasis in the endoplasmic reticulum (ER). The PDI family member anterior gradient (AGR) 3 is expressed in the airway, but the localization, regulation, and function of AGR3 are poorly understood. Here we report that AGR3, unlike its closest homolog AGR2, is restricted to ciliated cells in the airway epithelium and is not induced by ER stress. Mice lacking AGR3 are viable and develop ciliated cells with normal-appearing cilia. However, ciliary beat frequency was lower in airways from AGR3-deficient mice compared with control mice (20% lower in the absence of stimulation and 35% lower after ATP stimulation). AGR3 deficiency had no detectable effects on ciliary beat frequency (CBF) when airways were perfused with a calcium-free solution, suggesting that AGR3 is required for calcium-mediated regulation of ciliary function. Decreased CBF was associated with impaired mucociliary clearance in AGR3-deficient airways. We conclude that AGR3 is a specialized member of the PDI family that plays an unexpected role in the regulation of CBF and mucociliary clearance in the airway.

The airway epithelia initiate and modulate the inflammatory responses to various pathogens. The cystic fibrosis transmembrane conductance regulator-mediated Cl(-) secretion system plays a key role in mucociliary clearance of inhaled pathogens. We have explored the effects of Toxoplasma gondii, an opportunistic intracellular protozoan parasite, on Cl(-) secretion of the mouse tracheal epithelia. In this study, ATP-induced Cl(-) secretion indicated the presence of a biphasic short-circuit current (Isc) response, which was mediated by a Ca(2+)-activated Cl(-) channel (CaCC) and the cystic fibrosis transmembrane conductance regulator. However, the ATP-evoked Cl(-) secretion in T. gondii-infected mouse tracheal epithelia and the elevation of [Ca(2+)]i in T. gondii-infected human airway epithelial cells were suppressed. Quantitative reverse transcription-PCR revealed that the mRNA expression level of the P2Y2 receptor (P2Y2-R) increased significantly in T. gondii-infected mouse tracheal cells. This revealed the influence that pathological changes in P2Y2-R had on the downstream signal, suggesting that P2Y2-R was involved in the mechanism underlying T. gondii infection in airways. These results link T. gondii infection as well as other pathogen infections to Cl(-) secretion, via P2Y2-R, which may provide new insights for the treatment of pneumonia caused by pathogens including T. gondii.

ImportanceThis study reviews a single center's experience of performing staged laryngotracheoplasty (LTP) for the treatment of laryngotracheal stenosis with the ultimate goal of attaining long-term airway patency without restenosis.ObjectiveTo identify staged LTP as an efficacious surgical treatment option for laryngotracheal stenosis.Design, setting, and participantsFrom January 2000 to January 2012, patients at a tertiary care academic institution presenting with diagnoses of laryngeal or laryngotracheal stenosis were retrospectively identified. Medical records from adult patients were inspected, and patient demographics, clinical data, and clinical outcomes were recorded. All patients undergoing staged LTP were initially included. Patients with history of head and neck malignant neoplasm were excluded.InterventionsStaged LTP.Main outcomes and measuresThe primary outcome was long-term decannulation, defined as decannulation for duration of at least 6 months.ResultsSixty-one patients were included in this study. The mean (SD) patient age was 47.1 (16.7) at the time of first-stage LTP and had a mean (range) follow-up of 5.32 (0.5-17.3) years from the first-stage reconstruction. Etiology of stenosis included prolonged intubation in 27 patients (44%), autoimmune disease in 9 (15%), idiopathic causes in 11 (18%), blunt laryngeal trauma in 10 (16%), and other causes in 4 (7%). Forty-nine patients (80%) were successfully decannulated, while to date 12 (20%) remain tracheostomy or tympanostomy tube dependent. Univariate analyses showed no significant association between decannulation and age (P = .35), sex (P = .52), history of intubation (P = .22), surgeon (P = .20), etiology of stenosis (P = .91), or length of stenosis (P = .31). Multivariate logistic regression analysis showed a significant inverse relationship between grade of stenosis and probability of decannulation (P = .01).Conclusions and relevanceStaged LTP is an option for the reconstruction laryngotracheal stenosis. Our experience shows excellent decannulation rates in the selected patients with stenosis, many of whom have failed treatment with other surgical modalities.

Respiratory viral infections such as human rhinovirus (HRV) can lead to substantial morbidity and mortality, especially in people with underlying lung diseases such as asthma and COPD. One proposed strategy to detect viral infections non-invasively is by volatile organic compound (VOC) assessment via analysis of exhaled breath. The epithelial cells are one of the most important cell lines affected during respiratory infections as they are the first line of pathogen defense. Efforts to discover infection-specific biomarkers can be significantly aided by understanding the VOC emanations of respiratory epithelial cells. Here we test the hypothesis that VOCs obtained from the headspace of respiratory cell culture will differentiate healthy cells from those infected with HRV. Primary human tracheobronchial cells were cultured and placed in a system designed to trap headspace VOCs. HRV-infected cells were compared to uninfected control cells. In addition, cells treated with heat-killed HRV and poly(I:C), a TLR3 agonist, were compared to controls. The headspace was sampled with solid-phase microextraction fibers and VOCs were analyzed by gas chromatography/mass spectrometry. We determined differential expression of compounds such as aliphatic alcohols, branched hydrocarbons, and dimethyl sulfide by the infected cells, VOCs previously associated with oxidative stress and bacterial infection. We saw no major differences between the killed-HRV, poly(I:C), and control cell VOCs. We postulate that these compounds may serve as biomarkers of HRV infection, and that the production of VOCs is not due to TLR3 stimulation but does require active viral replication. Our novel approach may be used for the in vitro study of other important respiratory viruses, and ultimately it may aid in identifying VOC biomarkers of viral infection for point-of-care diagnostics.

PurposeTracheoesophageal puncture (TEP) is an effective rehabilitation method for postlaryngectomy speech and has already been described as a procedure that is safely performed in the office. We review our long-term experience with office-based TEP over the past 7 years in the largest cohort published to date.Materials and methodsA retrospective chart review was performed of all patients who underwent TEP by a single surgeon from 2005 through 2012, including office-based and operating room procedures. Indications for the chosen technique (office versus operating room) and surgical outcomes were evaluated.ResultsFifty-nine patients underwent 72 TEP procedures, with 55 performed in the outpatient setting and 17 performed in the operating room, all without complication. The indications for performing TEPs in the operating room included 2 primary TEPs, 14 due to concomitant procedures requiring general anesthesia, and 1 due to failed attempt at office-based TEP. Nineteen patients with prior rotational or free flap reconstruction successfully underwent office-based TEP.ConclusionsTEP in an office-based setting with immediate voice prosthesis placement continues to be a safe method of voice rehabilitation for postlaryngectomy patients, including those who have previously undergone free flap or rotational flap reconstruction. Office-based TEP is now our primary approach for postlaryngectomy voice rehabilitation.

Airways are exposed to myriad environmental and damaging agents such as reactive oxygen species (ROS), which also have physiological roles as signaling molecules that regulate stem cell function. However, the functional significance of both steady and dynamically changing ROS levels in different stem cell populations, as well as downstream mechanisms that integrate ROS sensing into decisions regarding stem cell homeostasis, are unclear. Here, we show in mouse and human airway basal stem cells (ABSCs) that intracellular flux from low to moderate ROS levels is required for stem cell self-renewal and proliferation. Changing ROS levels activate Nrf2, which activates the Notch pathway to stimulate ABSC self-renewal and an antioxidant program that scavenges intracellular ROS, returning overall ROS levels to a low state to maintain homeostatic balance. This redox-mediated regulation of lung stem cell function has significant implications for stem cell biology, repair of lung injuries, and diseases such as cancer.

Respiratory disease is the third leading cause of death in the industrialized world. Consequently, the trachea, lungs, and cardiopulmonary vasculature have been the focus of extensive investigations. Recent studies have provided new information about the mechanisms driving lung development and differentiation. However, there is still much to learn about the ability of the adult respiratory system to undergo repair and to replace cells lost in response to injury and disease. This Review highlights the multiple stem/progenitor populations in different regions of the adult lung, the plasticity of their behavior in injury models, and molecular pathways that support homeostasis and repair.

A continuously mixed series of microbial communities inhabits various points of the respiratory tract, with community composition determined by distance from colonization sources, colonization rates, and extinction rates. Ecology and evolution theory developed in the context of biogeography is relevant to clinical microbiology and could reframe the interpretation of recent studies comparing communities from lung explant samples, sputum samples, and oropharyngeal swabs. We propose an island biogeography model of the microbial communities inhabiting different niches in human airways. Island biogeography as applied to communities separated by time and space is a useful parallel for exploring microbial colonization of healthy and diseased lungs, with the potential to inform our understanding of microbial community dynamics and the relevance of microbes detected in different sample types. In this perspective, we focus on the intermixed microbial communities inhabiting different regions of the airways of patients with cystic fibrosis.

RationaleLymphatic vessels in the respiratory tract normally mature into a functional network during the neonatal period, but under some pathological conditions they can grow as enlarged, dilated sacs that result in the potentially lethal condition of pulmonary lymphangiectasia.ObjectiveWe sought to determine whether overexpression of the lymphangiogenic growth factor (vascular endothelial growth factor-C [VEGF-C]) can promote lymphatic growth and maturation in the respiratory tract. Unexpectedly, perinatal overexpression of VEGF-C in the respiratory epithelium led to a condition resembling human pulmonary lymphangiectasia, a life-threatening disorder of the newborn characterized by respiratory distress and the presence of widely dilated lymphatics.Methods and resultsAdministration of doxycycline to Clara cell secretory protein-reverse tetracycline-controlled transactivator/tetracycline operator-VEGF-C double-transgenic mice during a critical period from embryonic day 15.5 to postnatal day 14 was accompanied by respiratory distress, chylothorax, pulmonary lymphangiectasia, and high mortality. Enlarged sac-like lymphatics were abundant near major airways, pulmonary vessels, and visceral pleura. Side-by-side comparison revealed morphological features similar to pulmonary lymphangiectasia in humans. The condition was milder in mice given doxycycline after age postnatal day 14 and did not develop after postnatal day 35. Mechanistic studies revealed that VEGF recptor (VEGFR)-3 alone drove lymphatic growth in adult mice, but both VEGFR-2 and VEGFR-3 were required for the development of lymphangiectasia in neonates. VEGFR-2/VEGFR-3 heterodimers were more abundant in the dilated lymphatics, consistent with the involvement of both receptors. Despite the dependence of lymphangiectasia on VEGFR-2 and VEGFR-3, the condition was not reversed by blocking both receptors together or by withdrawing VEGF-C.ConclusionsThe findings indicate that VEGF-C overexpression can induce pulmonary lymphangiectasia during a critical period in perinatal development.

A transgenic ferret model of cystic fibrosis has recently been generated. It is probable that malfunction of airway mucous glands contributes significantly to the airway pathology of this disease. The usefulness of the ferret model may therefore depend in part on how closely the airway glands of ferrets resemble those of humans. Here, we show that in the ferret trachea glands are commonest in its most ventral aspect and disappear about half way up the lateral walls; they are virtually absent from the dorsal membranous portion. Further, the aggregate volume of glands per unit mucosal surface declines progressively by about 60% between the larynx and the carina. The average frequency of glands openings for the ferret trachea as a whole is only about one-fifth that in humans (where gland openings are found at approximately the same frequency throughout the trachea). Glands in the ferret trachea are on average about one-third the size of those in the human. Therefore, the aggregate volume of tracheal glands (per unit mucosal surface area) in the ferret is only about 6% that in humans. As in other mammalian species, airway glands in the ferret disappear at an airway internal diameter of ∼1 mm, corresponding approximately in this species to airway generation 6.

These studies used bi-transgenic Clara cell secretory protein (CCSP)/IL-1β mice that conditionally overexpress IL-1β in Clara cells to determine whether IL-1β can promote angiogenesis and lymphangiogenesis in airways. Doxycycline treatment induced rapid, abundant, and reversible IL-1β production, influx of neutrophils and macrophages, and conspicuous and persistent lymphangiogenesis, but surprisingly no angiogenesis. Gene profiling showed many up-regulated genes, including chemokines (Cxcl1, Ccl7), cytokines (tumor necrosis factor α, IL-1β, and lymphotoxin-β), and leukocyte genes (S100A9, Aif1/Iba1). Newly formed lymphatics persisted after IL-1β overexpression was stopped. Further studies examined how IL1R1 receptor activation by IL-1β induced lymphangiogenesis. Inactivation of vascular endothelial growth factor (VEGF)-C and VEGF-D by adeno-associated viral vector-mediated soluble VEGFR-3 (VEGF-C/D Trap) completely blocked lymphangiogenesis, showing its dependence on VEGFR-3 ligands. Consistent with this mechanism, VEGF-C immunoreactivity was present in some Aif1/Iba1-immunoreactive macrophages. Because neutrophils contribute to IL-1β-induced lung remodeling in newborn mice, we examined their potential role in lymphangiogenesis. Triple-transgenic CCSP/IL-1β/CXCR2(-/-) mice had the usual IL-1β-mediated lymphangiogenesis but no neutrophil recruitment, suggesting that neutrophils are not essential. IL1R1 immunoreactivity was found on some epithelial basal cells and neuroendocrine cells, suggesting that these cells are targets of IL-1β, but was not detected on lymphatics, blood vessels, or leukocytes. We conclude that lymphangiogenesis triggered by IL-1β overexpression in mouse airways is driven by VEGF-C/D from macrophages, but not neutrophils, recruited by chemokines from epithelial cells that express IL1R1.

Optical coherence tomography (OCT) is a nondestructive imaging technique offering high temporal and spatial resolution, which makes it a natural choice for assessing tissue mechanical properties. We have developed methods to mechanically analyze the compliance of the rabbit trachea in vivo using tissue deformations induced by tidal breathing, offering a unique tool to assess the behavior of the airways during their normal function. Four-hundred images were acquired during tidal breathing with a custom-built endoscopic OCT system. The surface of the tissue was extracted from a set of these images via image processing algorithms, filtered with a bandpass filter set at respiration frequency to remove cardiac and probe motion, and compared to ventilatory pressure to calculate wall compliance. These algorithms were tested on elastic phantoms to establish reliability and reproducibility. The mean tracheal wall compliance (in five animals) was 1.3±0.3×10(-5) (mm Pa)(-1). Unlike previous work evaluating airway mechanics, this new method is applicable in vivo, noncontact, and loads the trachea in a physiological manner. The technique may have applications in assessing airway mechanics in diseases such as asthma that are characterized by significant airway remodeling.

To determine the role of innate immune responses in controlling influenza A virus replication, rhesus macaques (RM) were administered pegylated IFN-alpha prior to virus challenge. Systemic and mucosal pegylated IFN-alpha administration induced expression of the interferon-stimulated genes (ISG) MxA and OAS in the airways. RM treated with IFN-alpha 24 hours prior to influenza virus challenge had significantly lower peak vRNA levels in the trachea compared to untreated animals. In addition to blunting viral replication, IFN-alpha treatment minimized the weight loss and spike in body temperature after influenza infection of RM. These results confirm the importance of IFN-alpha induced innate immune responses in the rapid control of influenza A virus replication in primates.

Dosimetry provides information linking environmental exposures to sites of deposition, removal from these sites, and translocation of deposited materials. Dosimetry also aids in extrapolating laboratory animal and in vitro data to humans. Recent progress has shed light on: properties of particles in relation to their fates in the body; influence of age, gender, body size, and lung diseases on inhaled particle doses; particle movement to the brain via the olfactory nerves; and particle deposition hot spots in the respiratory tract. Ultrafine size has emerged as an important dosimetric characteristic. Particle count, composition, and surface properties are recognized as potentially important toxicology-related considerations. Differences in body size influence airway sizes, inhaled particle deposition, specific ventilation, and specific doses (e.g. per unit body mass). Related to body size, age, gender, species, and strain are also dosimetric considerations. Diseases, such as chronic obstructive pulmonary disease (COPD) and bronchitis, produce uneven doses within the respiratory tract. Traditional concepts of the translocation and clearance of deposited particles have been challenged. Ultrafine particles can translocate to the brain via olfactory nerves, and from the lung to other organs. The clearance rates of particles from tracheobronchial airways are slowed by respiratory tract infections, but newer evidence implies that slow particle clearance from this region also exists in healthy lungs. Finally, hot spots of particle deposition are seen in hollow models, lung tissue, and dosimetric simulations. Local doses to groups of epithelial cells can be much greater than those to surrounding cells. The new insights challenge dosimetry scientists.

Objective/hypothesisLaser-induced damage of tracheal wall microstructures might contribute to recurrence after bronchoscopic treatment of tracheal strictures. The purpose of this study was to demonstrate how multimodal imaging using white light bronchoscopy (WLB), endobronchial ultrasound (EBUS), and optical coherence tomography (OCT) might identify in vivo airway wall changes before and resulting from Nd:YAG laser ablation and dilation of tracheal stenosis.Study designCase study.MethodsCommercially available WLB, high frequency EBUS using a 20-MHz radial probe and time-domain, frontal imaging OCT systems were used to characterize the extent, morphology, and airway wall microstructures at the area of hypertrophic fibrotic tissue formation before, during and after treatment of postintubation tracheal stenosis.ResultsWLB revealed the location of a complex, extensive, severe stricture. EBUS showed a homogeneous layer overlying a hyperechogenic layer corresponding to disrupted cartilage. OCT showed a homogeneous light backscattering layer and absence of layered microstructures, confirming absence in close proximity of normal airway wall. After laser ablation, OCT of charred tissue showed high backscattering and shadowing artifacts. OCT of noncharred tissue showed a thinner, homogeneous, light backscattering layer. EBUS showed thinner but persistent hypertrophic tissue suggesting incomplete treatment. WLB revealed improved airway patency postprocedure and recurrence 3 weeks later.ConclusionsEBUS identified cartilage disruption and residual hypertrophic tissue, the evidence of which might contribute to recurrence. OCT revealed homogeneous light backscaterring representing persistent noncharred hypertrophic tissues but it did not visualize cartilage disruption. Future studies are warranted to confirm whether these technologies can help guide bronchoscopic treatments.

The thin layer of liquid at the surface of airway epithelium, the airway surface liquid (ASL), is important in normal airway physiology and in the pathophysiology of cystic fibrosis. At present, the best method to measure ASL depth involves scanning confocal microscopy after staining with an aqueous-phase fluorescent dye. We describe here a simple, noninvasive imaging method to measure ASL depth by reflectance imaging of an epithelial mucosa in which the surface is illuminated at a 45-degree angle by an elongated 13-microm wide rectangular beam produced by a 670-nm micro-focus laser. The principle of the method is that air-liquid, liquid-liquid, and liquid-cell interfaces produce distinct specular or diffuse reflections that can be imaged to give a micron-resolution replica of the mucosal surface. The method was validated using fluid layers of specified thicknesses and applied to measure ASL depth in cell cultures and ex vivo fragments of pig trachea. In addition, the method was adapted to measure transepithelial fluid transport from the dynamics of fluid layer depth. Compared with confocal imaging, ASL depth measurement by surface laser reflectance microscopy does not require dye staining or costly instrumentation, and can potentially be adapted for in vivo measurements using fiberoptics.

There are two main epithelial cell types in the secretory tubules of mammalian glands: serous and mucous. The former is believed to secrete predominantly water and antimicrobials, the latter mucins. Primary cultures of human airway gland epithelium have been available for almost 20 yr, but they are poorly differentiated and lack clear features of either serous or mucous cells. In this study, by varying growth supports and media, we have produced cultures from human airway glands that in terms of their ultrastructure and secretory products resemble either mucous or serous cells. Of four types of porous-bottomed insert tested, polycarbonate filters (Transwells) most strongly promoted the mucous phenotype. Coupled with the addition of epidermal growth factor (EGF), this growth support produced "mucous" cells that contained the large electron-lucent granules characteristic of native mucous cells, but lacked the small electron-dense granules characteristic of serous cells. Furthermore, they showed high levels of mucin secretion and low levels of release of lactoferrin and lysozyme (markers of native serous cells). By contrast, growth on polyethylene terephthalate filters (Cyclopore) in medium lacking EGF produced "serous" cells in which small electron-dense granules replaced the electron-lucent ones, and the cells had high levels of lactoferrin and lysozyme but low levels of mucins. Measurements of transepithelial resistance and short-circuit current showed that both "serous" and "mucous" cell cultures possessed tight junctions, had become polarized, and were actively secreting Cl.

ObjectiveInhaled nitric oxide (iNO) is a potential new therapy for prevention of bronchopulmonary dysplasia and brain injury in premature infants. This study examined dose-related effects of iNO on NO metabolites as evidence of NO delivery.Study designA subset of 102 premature infants in the NO CLD trial, receiving 24 days of iNO (20 p.p.m. decreasing to 2 p.p.m.) or placebo, were analyzed. Tracheal aspirate (TA) and plasma samples collected at enrollment and at intervals during study gas were analyzed for NO metabolites.ResultiNO treatment increased NO metabolites in TA at 20 and 10 p.p.m. (1.7- to 2.3-fold vs control) and in plasma at 20, 10, and 5 p.p.m. (1.6- to 2.3-fold). In post hoc analysis, treated infants with lower metabolite levels at entry had an improved clinical outcome.ConclusioniNO causes dose-related increases in NO metabolites in the circulation as well as lung fluid, as evidenced by TA analysis, showing NO delivery to these compartments.

A fast-scan-rate swept laser for optical coherence tomography (OCT) is suitable to record and analyze a 3-D image volume. However, the whole OCT system speed is limited by data streaming, processing, and storage. In this case, postprocessing is a common technique. Endoscopic clinical applications prefer onsite diagnosis, which requires a real-time technique. Parallel digital signal processors were applied to stream and process data directly from a data digitizer. A real-time system with 20-kHz axial line speed, which was limited only by our swept laser scan rate, was implemented. To couple with the system speed, an endoscope based on an improved 3-D microelectromechanical motor (diameter 1.5 mm, length 9.4 mm) was developed. In vivo 3-D imaging of the human airway was demonstrated.