Toshiya Irokawa

Hyposecretion, not hyperabsorption, is the basic defect of cystic fibrosis airway glands.

Human airways and glands express the anion channel cystic fibrosis transmembrane conductance regulator, CFTR, and the epithelial Na+ channel, ENaC. Cystic fibrosis (CF) airway glands fail to secrete mucus in response to vasoactive intestinal peptide or forskolin; the failure was attributed to loss of CFTR-mediated anion and fluid secretion. Alternatively, CF glands might secrete acinar fluid via CFTR-independent pathways, but the exit of mucus from the glands could be blocked by hyperabsorption of fluid in the gland ducts. This could occur because CFTR loss can disinhibit ENaC, and ENaC activity can drive absorption. To test these two hypotheses, we measured single gland mucus secretion optically and applied ENaC inhibitors to determine whether they augmented secretion. Human CF glands were pretreated with benzamil and then stimulated with forskolin in the continued presence of benzamil. Benzamil did not rescue the lack of secretion to forskolin (50 glands, 6 CF subjects) nor did it increase the rate of cholinergically mediated mucus secretion from CF glands. Finally, neither benzamil nor amiloride increased forskolin-stimulated mucus secretion from porcine submucosal glands (75 glands, 7 pigs). One possible explanation for these results is that ENaC within the gland ducts was not active in our experiments. Consistent with that possibility, we discovered that human airway glands express Kunitz-type and non-Kunitz serine protease inhibitors, which might prevent proteolytic activation of ENaC. Our results suggest that CF glands do not display excessive, ENaC-mediated fluid absorption, leaving defective, anion-mediated fluid secretion as the most likely mechanism for defective mucus secretion from CF glands.

Nitric oxide regulation of glycoconjugate secretion from feline and human airways in vitro

To determine whether nitric oxide (NO) regulates mucus secretion from airway submucosal glands which are the main source of human airway secretion, we examined the effects of NO synthase inhibitors (L-NAME and L-NMMA) on mucus glycoprotein (MGP) secretion from feline and human airway explants (with epithelium) and isolated submucosal glands. MGP secretion was estimated by measuring trichloroacetic-acid (TCA) precipitable [H3]-glycoconjugates using secretory indices. NO synthase inhibitors alone did not alter significantly MGP secretion from explants or isolated glands. Pretreatment with NO synthase inhibitors significantly inhibited both methacholine (MCh) and bradykinin (BK)-induced secretion from isolated glands, but not significantly inhibit the secretion from explants. The inhibition by L-NAME was reversed by the addition of L-arginine in both MCh- and BK-induced secretions from isolated glands. Further, a NO generator isosorbide dinitrate induced a significant increase in the secretion. These findings suggest that endogenous NO has a stimulatory action in airway submucosal gland secretion and directly regulates the secretion from submucosal glands independently of superficial epithelial cells.

Application of impulse oscillometry for within-breath analysis in patients with chronic obstructive pulmonary disease: pilot study

Background The impulse oscillometry is increasingly used for assessing the oscillatory mechanics of the respiratory system. The within-breath behaviour of the oscillatory mechanics in chronic obstructive pulmonary disease (COPD) is a well-known physiological feature. The purpose of this study was to develop a new approach for assessing this feature using impulse oscillometry. Methods The oscillatory mechanics were assessed by a commercially available impulse oscillometry device. The respiratory system resistance (Rrs) and reactance (Xrs) were measured during tidal breathing in patients with COPD (n=39) and healthy subjects (n=5). Selected data, the Rrs at 5 Hz (R5), Rrs at 20 Hz (R20), Xrs at 5 Hz (X5), and resonant frequency of Xrs (Fres) every 0.2 s, were extracted from the device. These data were divided into eight time fractions during the respiratory cycle to form averaged respiratory phases. Results The time courses of the R5 and X5 were notably dependent on the respiratory cycles in patients with COPD, while there was little such dependency in healthy subjects. Irrespective of respiratory phase, R5 and Fres increased, and X5 fell to a more negative level in patients with COPD in a severity-dependent fashion. The increase in the R5 and negative level in the X5 were more prominent in the middle of the expiratory phase. The severity dependence in the R20 was relatively small compared with that in the R5. Conclusions The results of this study suggest that impulse oscillometry can assess the within-breath behaviour of the oscillatory mechanics with high temporal resolution, which may be helpful for evaluating the severity of COPD. Further studies are needed to reveal which biomarkers obtained with this approach would be suitable for evaluating the airway obstruction.

Mesenchymal Stem Cells Correct Inappropriate Epithelial–mesenchyme Relation in Pulmonary Fibrosis Using Stanniocalcin-1

Current hypotheses suggest that aberrant wound healing has a critical role in the pathogenesis of idiopathic pulmonary fibrosis (IPF). In these hypotheses, continuous TGF-β1 secretion by alveolar epithelial cells (AECs) in abnormal wound healing has a critical role in promoting fibroblast differentiation into myofibroblasts. Mesenchymal stem cells (MSCs) home to the injury site and reduce fibrosis by secreting multifunctional antifibrotic humoral factors in IPF. In this study, we show that MSCs can correct the inadequate-communication between epithelial and mesenchymal cells through STC1 (Stanniocalcin-1) secretion in a bleomycin-induced IPF model. Inhalation of recombinant STC1 shows the same effects as the injection of MSCs. Using STC1 plasmid, it was possible to enhance the ability of MSCs to ameliorate the fibrosis. MSCs secrete large amounts of STC1 in response to TGF-β1 in comparison to AECs and fibroblasts. The antifibrotic effects of STC1 include reducing oxidative stress, endoplasmic reticulum (ER) stress, and TGF-β1 production in AECs. The STC1 effects can be controlled by blocking uncoupling protein 2 (UCP2) and the secretion is affected by the PI3/AKT/mTORC1 inhibitors. Our findings suggest that STC1 tends to correct the inappropriate epithelial-mesenchymal relationships and that STC1 plasmid transfected to MSCs or STC1 inhalation could become promising treatments for IPF.

Signature current of SO2-induced bronchitis in rabbit.

To investigate abnormalities of airway epithelial ion transport underlying chronic inflammatory airway diseases, we performed electrophysiological, histological, and molecular biological experiments using rabbits exposed to SO2 as a model of bronchitis. By comparison with control, the SO2-exposed trachea exhibited decreased short circuit current (Isc) and conductance associated with increased potential difference. In normal trachea, apical ATP induced a transient Isc activation followed by a suppression, whereas the bronchitis model exhibited a prolonged activation without suppression. This pathological ATP response was abolished by diphenylamine 2-carboxylate or Cl--free bath solution. A significant increase in net Cl- flux toward the lumen was observed after ATP in our bronchitis model. Isoproterenol or adenosine evoked a sustained Isc increase in SO2-exposed, but not in normal, tracheas. The Northern blot analysis showed a strong expression of cystic fibrosis transmembrane conductance regulator (CFTR) mRNA in SO2-exposed epithelium. The immunohistochemical study revealed a positive label of CFTR on cells located luminally only in SO2-exposed rabbits. We concluded that the prolonged ATP response in our bronchitis model was of a superimposed normal and adenosine-activated current. The latter current was also activated by isoproterenol and appeared as a signature current for the bronchitis model airway. This was likely mediated by CFTR expressed in the course of chronic inflammation.

Measurement of Fluid Secretion from Intact Airway Submucosal Glands

Human airways are kept sterile by a mucosal innate defense system that includes mucus secretion. Mucus is secreted in healthy upper airways primarily by submucosal glands and consists of defense molecules mixed with mucins, electrolytes, and water and is also a major component of sputum. Mucus traps pathogens and mechanically removes them via mucociliary clearance while inhibiting their growth via molecular (e.g., lysozyme) and cellular (e.g., neutrophils, macrophages) defenses. Fluid secretion rates of single glands in response to various mediators can be measured by trapping the primary gland mucus secretions in an oil layer, where they form spherical bubbles that can be optically measured at any desired interval to provide detailed temporal analysis of secretion rates. The composition and properties of the mucus (e.g., solids, viscosity, pH) can also be determined. These methods have now been applied to mice, ferrets, cats, pigs, sheep, and humans, with a main goal of comparing gland secretion in control and CFTR-deficient humans and animals.

Cholinomimetic action of macrolide antibiotics on airway gland electrolyte secretion

We investigated the acute effects of erythromycin (EM) and its derivatives on ionic currents in airway glands from feline tracheae. Therapeutic concentrations of EM or clarithromycin (CAM) attenuated the whole cell currents evoked by ACh in a competitive manner. The maximally stimulated inward Cl- currents were reduced to 54 and 83% and the outward K+ currents to 55 and 84% of control values by EM and CAM, respectively, whereas the responses induced by phenylephrine, norepinephrine, caffeine, or ionomycin were unaffected by EM, CAM, or EM523, a synthetic derivative of EM. K+ channels in excised outside-out patches were not influenced by macrolides. Although therapeutic concentrations of macrolides showed no effect on the baseline currents, high concentrations of macrolides alone evoked currents mimicking the ACh response, which were abolished completely by atropine. We concluded that macrolides act as a partial agonist on cholinergic receptors, resulting in a reduction of Cl- secretion at pharmacological doses of the agents, which may exhibit a pronounced effectiveness on hypertrophied and/or cholinergically sensitized submucosal glands in pathological airways.We investigated the acute effects of erythromycin (EM) and its derivatives on ionic currents in airway glands from feline tracheae. Therapeutic concentrations of EM or clarithromycin (CAM) attenuated the whole cell currents evoked by ACh in a competitive manner. The maximally stimulated inward Cl-currents were reduced to 54 and 83% and the outward K+ currents to 55 and 84% of control values by EM and CAM, respectively, whereas the responses induced by phenylephrine, norepinephrine, caffeine, or ionomycin were unaffected by EM, CAM, or EM523, a synthetic derivative of EM. K+ channels in excised outside-out patches were not influenced by macrolides. Although therapeutic concentrations of macrolides showed no effect on the baseline currents, high concentrations of macrolides alone evoked currents mimicking the ACh response, which were abolished completely by atropine. We concluded that macrolides act as a partial agonist on cholinergic receptors, resulting in a reduction of Cl- secretion at pharmacological doses of the agents, which may exhibit a pronounced effectiveness on hypertrophied and/or cholinergically sensitized submucosal glands in pathological airways.

Efavirenz-induced neurological symptoms in rare homozygote CYP2B6 *2/*2 (C64T).

Some of the HIV-1-infected patients who were given highly active anti-retroviral therapy (HAART) including efavirenz (EFV) presented adverse central nervous system (CNS) symptoms such as fatigue and insomnia. The incidence of adverse CNS symptoms is associated with hepatic cytochrome P450 isozymes (CYP2B6) polymorphisms. For example, CYP2B6 *6 (G516T and A785G) and *7 (G516T, A785G and C1459T) prolonged the EFV half-life despite discontinuation of EFV. CYP2B6 *2/*2 (C64T) is extremely rare and there have been no data describing the EFV plasma concentrations in C64T homozygous patients, who developed adverse CNS symptoms. C64T homozygous possibly has some catalytic defects.

Bacterial Hazards of Sludge Brought Ashore by the Tsunami after the Great East Japan Earthquake of 2011

Bacterial Hazards of Sludge Brought Ashore by the Tsunami after the Great East Japan Earthquake of 2011: Koji WADA, et al. Department of Public Health, Kitasato University School of Medicine—

Sequential Granulocyte-Macrophage Colony-Stimulating Factor Inhalation after Whole-Lung Lavage for Pulmonary Alveolar Proteinosis. A Report of Five Intractable Cases

&NA; Autoimmune pulmonary alveolar proteinosis (aPAP) is a rare disease characterized by the excessive accumulation of surfactant proteins within the alveolar spaces and by higher titers of autoantibodies to granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) in the serum and bronchoalveolar lavage fluid. The antibodies inhibit the maturation and phagocytosis of alveolar macrophages. Although the standard therapy for aPAP has been whole‐lung lavage (WLL), this procedure is invasive and needs to be repeated for several years. GM‐CSF inhalation therapy is a new procedure for treating aPAP and can induce remission with less invasiveness, although it is generally less effective in severe cases. We evaluated five cases with remarkable improvement by using sequential GM‐CSF inhalation therapy after WLL; however, the treatment failed when this therapy preceded WLL. Therefore, sequential GM‐CSF inhalation after WLL may reinforce the efficiency of WLL in patients with severe aPAP.