Hirotoshi Matsui

Coordinated clearance of periciliary liquid and mucus from airway surfaces.

Airway surface liquid is comprised of mucus and an underlying, watery periciliary liquid (PCL). In contrast to the well-described axial transport of mucus along airway surfaces via ciliary action, theoretical analyses predict that the PCL is nearly stationary. Conventional and confocal microscopy of fluorescent microspheres and photoactivated fluorescent dyes were used with well-differentiated human tracheobronchial epithelial cell cultures exhibiting spontaneous, radial mucociliary transport to study the movements of mucus and PCL. These studies showed that the entire PCL is transported at approximately the same rate as mucus, 39.2+/-4.7 and 39.8+/-4.2 micrometer/sec, respectively. Removing the mucus layer reduced PCL transport by > 80%, to 4.8+/-0.6 micrometer/sec, a value close to that predicted from theoretical analyses of the ciliary beat cycle. Hence, the rapid movement of PCL is dependent upon the transport of mucus. Mucus-dependent PCL transport was spatially uniform and exceeded the rate expected for pure frictional coupling with the overlying mucus layer; hence, ciliary mixing most likely accelerates the diffusion of momentum from mucus into the PCL. The cephalad movement of PCL along airway epithelial surfaces makes this mucus-driven transport an important component of salt and water physiology in the lung in health and disease.

Loss of binding and entry of liposome-DNA complexes decreases transfection efficiency in differentiated airway epithelial cells.

The target cells for gene therapy of cystic fibrosis lung disease are the well differentiated cells that line airway lumens. Employing cultures of airway epithelial cells that grow like “islands” and exhibit a continuum of cellular differentiation, we studied the mechanisms that render well differentiated cells more difficult to transfect with cationic liposomes than poorly differentiated cells. The poorly differentiated cells at the edge of the islands were transfectable with liposome-DNA complexes (pCMVβ:LipofectACE = 1:5 (w/w)), whereas the more differentiated cells in the center of the islands were not. Evaluation of the steps leading to lipid-mediated transfection revealed that edge cells bound more liposome-DNA complexes, in part due to a more negative surface charge (as measured by cationized ferritin binding), and that edge cells internalized more liposome-DNA complexes than central cells. Edge cells exhibited receptor-mediated endocytosis of LDL, pinocytosis of 10-nm microspheres, and phagocytosis of 2-μm microspheres, whereas central cells were only capable of receptor-mediated endocytosis. Cytochalasin B, which inhibited pinocytosis by 65% and phagocytosis by 93%, decreased edge cell liposome-DNA complex entry by 50%. Potassium depletion, which decreased phagocytosis by >90% but had no effect on pinocytosis, inhibited edge cell liposome-DNA complex entry by 71%. These results indicate that liposome-DNA complexes enter edge cells via phagocytosis and that this pathway is not detectable in central cells. In conclusion, both reduced negative surface charge and absence of phagocytosis internalization pathways in relatively differentiated cells may explain differentiation-dependent decrements in cationic liposome-mediated gene transfer in airway epithelia.

Osmotic water permeabilities of cultured, well-differentiated normal and cystic fibrosis airway epithelia

Current hypotheses describing the function of normal airway surface liquid (ASL) in lung defense are divergent. One theory predicts that normal airways regulate ASL volume by modulating the flow of isosmotic fluid across the epithelium, whereas an alternative theory predicts that ASL is normally hyposmotic. These hypotheses predict different values for the osmotic water permeability (P(f)) of airway epithelia. We measured P(f) of cultures of normal and cystic fibrosis (CF) airway epithelia that, like the native tissue, contain columnar cells facing the lumen and basal cells that face a basement membrane. Xz laser scanning confocal microscopy recorded changes in epithelial height and transepithelial volume flow in response to anisosmotic challenges. With luminal hyperosmotic challenges, transepithelial and apical membrane P(f) are relatively high for both normal and CF airway epithelia, consistent with an isosmotic ASL. Simultaneous measurements of epithelial cell volume and transepithelial water flow revealed that airway columnar epithelial cells behave as osmometers whose volume is controlled by luminal osmolality. Basal cell volume did not change in these experiments. When the serosal side of the epithelium was challenged with hyperosmotic solutions, the basal cells shrank, whereas the lumen-facing columnar cells did not. We conclude that (a) normal and CF airway epithelia have relatively high water permeabilities, consistent with the isosmotic ASL theory, and the capacity to restore water on airway surfaces lost by evaporation, and (b) the columnar cell basolateral membrane and tight junctions limit transepithelial water flow in this tissue.

A physical linkage between cystic fibrosis airway surface dehydration and Pseudomonas aeruginosa biofilms

A vexing problem in cystic fibrosis (CF) pathogenesis has been to explain the high prevalence of Pseudomonas aeruginosa biofilms in CF airways. We speculated that airway surface liquid (ASL) hyperabsorption generates a concentrated airway mucus that interacts with P. aeruginosa to promote biofilms. To model CF vs. normal airway infections, normal (2.5% solids) and CF-like concentrated (8% solids) mucus were prepared, placed in flat chambers, and infected with an ≈5 × 103 strain PAO1 P. aeruginosa. Although bacteria grew to 1010 cfu/ml in both mucus concentrations, macrocolony formation was detected only in the CF-like (8% solids) mucus. Biophysical and functional measurements revealed that concentrated mucus exhibited properties that restrict bacterial motility and small molecule diffusion, resulting in high local bacterial densities with high autoinducer concentrations. These properties also rendered secondary forms of antimicrobial defense, e.g., lactoferrin, ineffective in preventing biofilm formation in a CF-like mucus environment. These data link airway surface liquid hyperabsorption to the high incidence of P. aeruginosa biofilms in CF via changes in the hydration-dependent physical–chemical properties of mucus and suggest that the thickened mucus gel model will be useful to develop therapies of P. aeruginosa biofilms in CF airways.

Reduced Three-Dimensional Motility in Dehydrated Airway Mucus Prevents Neutrophil Capture and Killing Bacteria on Airway Epithelial Surfaces

Cystic fibrosis (CF) lung disease is characterized by persistent lung infection. Thickened (concentrated) mucus in the CF lung impairs airway mucus clearance, which initiates bacterial infection. However, airways have other mechanisms to prevent bacterial infection, including neutrophil-mediated killing. Therefore, we examined whether neutrophil motility and bacterial capture and killing functions are impaired in thickened mucus. Mucus of three concentrations, representative of the range of normal (1.5 and 2.5% dry weight) and CF-like thickened (6.5%) mucus, was obtained from well-differentiated human bronchial epithelial cultures and prepared for three-dimensional studies of neutrophil migration. Neutrophil chemotaxis in the direction of gravity was optimal in 1.5% mucus, whereas 2.5% mucus best supported neutrophil chemotaxis against gravity. Lateral chemokinetic movement was fastest on airway epithelial surfaces covered with 1.5% mucus. In contrast, neutrophils exhibited little motility in any direction in thickened (6.5%) mucus. In in vivo models of airway mucus plugs, neutrophil migration was inhibited by thickened mucus (CF model) but not by normal concentrations of mucus (“normal” model). Paralleling the decreased neutrophil motility in thickened mucus, bacterial capture and killing capacity were decreased in CF-like thickened mucus. Similar results with each mucus concentration were obtained with mucus from CF cultures, indicating that inhibition of neutrophil functions was mucus concentration dependent not CF source dependent. We conclude that concentrated (“thick”) mucus inhibits neutrophil migration and killing and is a key component in the failure of defense against chronic airways infection in CF.

A replication-deficient adenovirus enhances liposome-mediated nucleic acid transfer into a stable cell line expressing T7 RNA polymerase

Liposome-mediated transfer of nucleic acids into a cell line expressing bacteriophage T7 RNA polymerase was enhanced by addition of a replication-deficient adenovirus (Ad5-259A) to transfection mixtures. Increasing quantities of Ad5-259A resulted in a dose-related (up to 30-fold) enhancement of reporter gene activity expressed in BT7-H cells transfected with plasmid DNA containing the reporter sequence fused to the internal ribosome entry site of encephalomyocarditis virus. Similarly, Ad5-259A enhanced reporter gene expression 7-fold following transfection of DNA containing the reporter sequence under transcriptional control of the Rous sarcoma virus long terminal repeat. Addition of Ad5-295A to transfection mixtures increased the proportion of cells staining positively for reporter gene activity, from 2 to 25% when the reporter was expressed via the T7 polymerase and from 20 to 50% when the reporter was under the control of a eucaryotic promoter. Thus, Ad5-259A enhanced reporter protein activities expressed by cytoplasmic T7-directed transcription and cap-independent initiation of translation, or nuclear transcription and cap-dependent translation. Transfection enhancement was blocked by neutralizing antibody to Ad5, and is most likely related to the endosome-disrupting activities of the virus. Adenovirus enhancement of liposome-mediated transfection provides a useful method for efficient nucleic acid transfer into eucaryotic cells.

A successful pembrolizumab treatment case of lung adenocarcinoma after becoming resistant to ALK-TKI treatment due to G1202R mutation

BACKGROUND In current guidelines, the role of immune checkpoint inhibitors is not yet determined in the treatment strategy for NSCLC harboring ALK translocations. CASE A 51-year-old woman with lung adenocarcinoma harboring ALK translocation was treated with alectinib until PD. After the second (CDDP/PEM) and third (crizotinib) line treatment, a second biopsy was performed, revealing PD-L1 tumor proportion score of 70-80% and G1202R mutation of ALK. Pembrolizumab was selected for the fourth line, leading to PR for more than 6 months. CONCLUSIONS While alectinib might induce resistance to ALK-TKI, it could increase PD-L1 positive cells to become sensitive to PD-1/PD-L1 inhibitors.

Clinical Features and Prognosis of Nontuberculous Mycobacterial Pleuritis

Background: While nontuberculous mycobacterial (NTM) pleuritis rarely complicates pulmonary NTM infection, high mortality has been reported in case reports and small studies. Objectives: The purpose of this study was to clarify the clinical features and treatment outcomes of pulmonary NTM infection cases accompanied by NTM pleuritis. Methods: Medical records of 1,044 patients with pulmonary NTM disease were retrospectively reviewed to select patients complicated by NTM-proven pleuritis. We investigated clinical characteristics, pathogens, pleural effusion examinations, radiographic findings, treatments, and clinical course of the NTM pleuritis patients. Results: Among 1,044 cases with pulmonary NTM, NTM pleuritis occurred in 15 cases (1.4%). The mean age was 69 years with a performance status of mostly 2 or better (80.0%), and 6 cases (40.0%) were complicated by pneumothorax. Subpleural cavities were radiologically detected in 11 cases (73.3%), and extrapulmonary air-fluid level was detected in 14 cases (93.3%). Eleven patients were treated with combinations of 2–4 antimycobacterial drugs, including clarithromycin, and 2 patients were treated with isoniazid, rifampicin, and ethambutol. Chest tube drainage was performed in 11 cases, and surgical approach was added in 6 cases. The pleural effusion of 2 patients treated with only antimycobacterial medications gradually deteriorated. Two patients died from NTM pleuritis, and 1 patient died from pneumonitis during a mean of 1.8 years of follow-up. Conclusions: Comorbid NTM pleuritis was difficult to treat by medical therapy alone and resulted in a poor prognosis. In addition to antimycobacterial agents, chest tube drainage and surgical procedures in the early stages should be considered to treat NTM pleuritis.

Pathology of Airway Disease in Rheumatoid Arthritis

This chapter describes the lesions in the airways of the lungs, particularly those observed in bronchiolitis obliterans, in patients with rheumatoid arthritis (RA). RA-associated bronchiolitis obliterans has two different morphological presentations: (1) endobronchiolitis obliterans (EBO), in which the lesions are primarily present in the lumen of the airway, with minimal destruction of the airway wall, and (2) cellular destructive bronchiolitis (CDB), in which transmural inflammation causes destruction of the airway wall. The clinical presentations of EBO are markedly different from those of CDB, and they are generally characterized by distinct pathologies. Notably, a few cases of RA-associated bronchiectasis have been found to represent secondary lesions of CDB.

Pulmonary Adenocarcinoma, Harboring Both an EGFR Mutation and ALK Rearrangement, Presenting a Stable Disease to Erlotinib and a Partial Response to Alectinib

A 63-year-old woman with pulmonary adenocarcinoma (stage IIIB) that was positive for an epidermal growth factor receptor (EGFR) mutation and an anaplastic lymphoma kinase (ALK) rearrangement was treated with erlotinib as the first-line treatment, resulting in a stable disease. Due to skin rashes, fatigue and anorexia, erlotinib was suspended on erlotinib day 44. Alectinib was administered as the second-line treatment, exhibiting a partial response. On alectinib day 56, drug-induced lung injury forced suspension of alectinib, which was cured with corticosteroid therapy. ALK-tyrosine kinase inhibitors may be more effective for patients positive for both EGFR mutation and ALK rearrangement than other agents.