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In Europe and America, the newly-developed, simultaneous measurement of diffusing capacity for CO (DLCO) and NO (DLNO) has replaced the classic DLCO measurement for detecting the pathophysiological abnormalities in the acinar regions. However, simultaneous measurement of DLCO and DLNO is currently not used by Japanese physicians. To encourage the use of DLNO in Japan, the authors reviewed aspects of simultaneously-estimated DLCO and DLNO from previously published manuscripts. The simultaneous DLCO-DLNO technique identifies the alveolocapillary membrane-related diffusing capacity (membrane component, DM) and the blood volume in pulmonary microcirculation (VC); VC is the principal factor constituting the blood component of diffusing capacity (DB,DB=θ·VC where θ is the specific gas conductance for CO or NO in the blood). As the association velocity of NO with hemoglobin (Hb) is fast and the affinity of NO with Hb is high in comparison with those of CO, θNO can be taken as an invariable simply determined by diffusion limitation inside the erythrocyte. This means that θNO is independent of the partial pressure of oxygen (PO2). However, θCO involves the limitations by diffusion and chemical reaction elicited by the erythrocyte, resulting in θCO to be a PO2-dependent variable. Furthermore, DLCO is determined primarily by DB (∼77%), while DLNO is determined equally by DM (∼55%) and DB (∼45%). This suggests that DLCO is more sensitive for detecting microvascular diseases, while DLNO can equally identify alveolocapillary membrane and microcirculatory abnormalities.

Chronic obstructive pulmonary disease (COPD) is often associated with co-morbidities. Metabolic disorders like hyperlipidemia and diabetes occur also in underweight COPD patients, although the mechanism is uncertain. Subcutaneous adipose tissue (SAT) plays an important role in energy homeostasis, since restricted capacity to increase fat cell number with increase in fat cell size occurring instead, is associated with lipotoxicity and metabolic disorders. The aim of this study is to show the protective role of SAT for the metabolic disorders in pulmonary emphysema of a murine model. We found ectopic fat accumulation and impaired glucose homeostasis with wasting of SAT in a murine model of elastase-induced pulmonary emphysema (EIE mice) reared on a high-fat diet. ONO-AE1-259, a selective E-prostanoid (EP) 2 receptor agonist, improved angiogenesis and subsequently adipogenesis, and finally improved ectopic fat accumulation and glucose homeostasis with restoration of the capacity for storage of surplus energy in SAT. These results suggest that metabolic disorders like hyperlipidemia and diabetes occured in underweight COPD is partially due to the less capacity for storage of surplus energy in SAT, though the precise mechanism is uncertained. Our data pave the way for the development of therapeutic interventions for metabolic disorders in emphysema patients, e.g., use of pro-angiogenic agents targeting the capacity for storage of surplus energy in the subcutaneous adipose tissue.

Recent evidence suggests that epithelial-mesenchymal transition (EMT) is involved in the pathogenesis of airway obstructive diseases, such as chronic obstructive pulmonary disease, asthma and bronchiolitis obliterans syndrome after lung transplantation. However, whether EMT occurs in an experimental model of airway fibrosis is not well known. We explored evidence of EMT in a murine model of airway fibrosis induced by repeated exposure to naphthalene. Mice were administered intraperitoneal injections of naphthalene or corn oil vehicle once weekly for 14 consecutive weeks. The animals were sacrificed 5days after the final injection of naphthalene or corn oil vehicle. EMT was evaluated in lung tissue sections using immunohistochemistry and immunofluorescence. Repeated naphthalene exposure induced loss of club cells, hyperplasia of epithelial cells and peribronchial fibrosis. However, we did not find any loss of E-cadherin expression or any acquisition of vimentin, S100A4 or αSMA in epithelial cells in control or naphthalene-exposed mice. These results suggest that EMT does not contribute significantly to naphthalene-induced airway fibrosis in mice.

Objective Tumor cells experience hypoxia, acidosis, and hypoglycemia. Metabolic adaptation to glucose shortage is essential to maintain tumor cells’ survival because of their high glucose requirement. This study evaluated the hypothesis that acidosis might promote tumor survival during glucose shortage and if so, explored a novel drug targeting metabolic vulnerability to glucose shortage. Methods Cell survival and bioenergetics metabolism were assessed in lung cancer cell lines. Our in-house small-molecule compounds were screened to identify those that kill cancer cells under low-glucose conditions. Cytotoxicity against non-cancerous cells was also assessed. Tumor growth was evaluated in vivo using a mouse engraft model. Results Acidosis limited the cellular consumption of glucose and ATP, causing tumor cells to enter a metabolically dormant but energetically economic state, which promoted tumor cell survival during glucose deficiency. We identified ESI-09, a previously known exchange protein directly activated by cAMP (EAPC) inhibitor, as an anti-cancer compound that inhibited cancer cells under low-glucose conditions even when associated with acidosis. Bioenergetic studies showed that independent of EPAC inhibition, ESI-09 was a safer mitochondrial uncoupler than a classical uncoupler and created a futile cycle of mitochondrial respiration, leading to decreased ATP production, increased ATP dissipation, and fuel scavenging. Accordingly, ESI-09 exhibited more cytotoxic effects under low-glucose conditions than under normal glucose conditions. ESI-09 was also more effective than actively proliferating cells on quiescent glucose-restricted cells. Cisplatin showed opposite effects. ESI-09 inhibited tumor growth in lung cancer engraft mice. Conclusions This study highlights the acidosis-induced promotion of tumor survival during glucose shortage and demonstrates that ESI-09 is a novel potent anti-cancer mitochondrial uncoupler that targets a metabolic vulnerability to glucose shortage even when associated with acidosis. The higher cytotoxicity under lower-than-normal glucose conditions suggests that ESI-09 is safer than conventional chemotherapy, can target the metabolic vulnerability of tumor cells to low-glucose stress, and is applicable to many cancer cell types., Highlights • Tumor cells experience hypoxia, acidosis, and glucose shortage. • Tumor acidosis promotes metabolic adaptation to glucose shortage. • ESI-09, a known EAPC inhibitor, is a novel and safe mitochondrial uncoupler. • ESI-09 kills tumor cells during glucose shortage, even under acidosis conditions. • ES-09 targets metabolically dormant and chemotherapy-resistant tumor cells.

Objectives (1) To establish the general equation that describes relationship of DMCO/Vc versus DLNO/DLCO under conditions with no functional heterogeneities. (2) To examine the effects of functional heterogeneities, including parallel and series (stratified) heterogeneities, on DLNO/DLCO. Results and discussions: (1) Given that “true” θNO in pulmonary capillaries is represented by surface absorption-related θNO, relationship between DMCO/Vc and DLNO/DLCO does not differ significantly from that obtained on premise of infinite θNO. DLNO/DLCO decided physiologically may mirror morphometric DMCO/Vc actually working for gas exchange but not “total” morphometric ratio of DMCO/Vc. (2) There are three parallel heterogeneities that affect diffusing capacity (D)-related parameters. Of them, only the heterogeneity of D/VA, where VA is alveolar volume, underestimates DLCO and DLNO. DLNO/DLCO does not alleviate negative impact of D/VA heterogeneity, indicating that DMCO/Vc estimated from DLNO/DLCO does not mirror “true” morphometric DMCO/Vc in diseased lungs with D/VA maldistribution. (3) Stratified heterogeneity underrates morphometric DMCO, DMNO, and DMNO/DMCO maximally by 1.4 %, 2.8 %, and 1.4 %, respectively, under conditions similar to single-breath D measurements, suggesting that effect of stratified heterogeneity on D measures is no longer needed to be considered in normal subjects but may be in patients having lung diseases with destructive lesions of acinar structures.

Objectives To propose new physical constants for NO and CO (Krogh diffusion constant ratio (KDNO/CO) and specific blood conductance for NO (θNO)) for calculating DMCO and Vc, according to Roughton-Forster’s equation (Roughton and Forster, J. Appl. Physiol. 11: 290–302, 1957) from simultaneous DLNO and DLCO measurements. Results and conclusions (1) The Graham’s law is unacceptable for determining KDNO/CO because CO does not fulfil all the conditions of an “ideal” gas. We have re-estimated KDNO/CO in a new way based on difference in molar volumes of two gases (molar volume theory). The KDNO/CO thus decided is 2.34. (2) θNO measured with rapid-reaction, constant-flow method by Carlsen and Comroe (J. Gen. Physiol. 42: 83–107, 1958) may be underestimated by about 40 % due to unstirred water layer surrounding the erythrocyte. (3) Erythrocyte θO2 can be harvested from O2 release kinetics in presence of high concentration of dithionite, which effectively removes the unstirred water layer-elicited effect. Multiplication of erythrocyte θO2 by erythrocyte KDNO/O2 equals erythrocyte θNO, the value of which is 6.2 mL/min/mmHg/(mL⋅blood). According to the concepts of Kang et al. (RESPNB. 241: 62–71, 2017) and Borland et al. (RESPNB. 241: 58–61, 2017), in vitro θNO decided from rapid-mixing experiments may mirror bulk absorption of NO by erythrocytes. (4) In pulmonary capillaries, NO uptake takes place predominantly in the surface rim of the erythrocyte. This surface absorption of NO increases the θNO 10-fold versus bulk absorption of NO to about 60 mL/min/mmHg/(mL⋅blood).

Incorporation of 5-bromo-2′-deoxyuridine (BrdU) into proliferating cells has been used to label dividing cells in many tissues. Although BrdU has been shown to be genotoxic, teratogenic and mutagenic, such adverse effects have largely been ignored by researchers. We determined whether long-term BrdU exposure causes any histopathological changes in the lungs of mice. Eight-week-old male C57/BL6J mice were administered BrdU by intraperitoneal injection on 3 consecutive days of each week for 14 weeks. While no obvious structural changes such as tissue damage, fibrosis, emphysema, airway remodeling, vascular thickening or tumorigenesis were noted, a moderate degree of macrophage infiltration was observed in the airways and lung parenchyma in the lungs of the mice exposed repeatedly to BrdU (BrdU-exposed mice). The proliferative activities of the airway and alveolar epithelial and mesenchymal cells were reduced in the BrdU-exposed mice, although the numbers of these cells in the lungs were maintained. Double immunofluorescence study of the lungs of the BrdU-exposed mice showed overexpression of IL-6 in the airway epithelial and alveolar wall cells, some of which were also double-positive for BrdU. These results indicate that long-term exposure to BrdU inhibits cell proliferation and induces low-grade inflammation in the lungs of mice. Our findings underscore the need for caution in the interpretation of studies that involve long-term exposure to BrdU.

Background and objectives The pathophysiologic aspects of parasympathetic nerve (PN) function during sleep in patients with obstructive sleep apnea (OSA) studied by classical power spectrum analysis on heart rate variability (HRV) are highly controversial. The controversy is attributed to methodologic concerns, such as poor time resolution involved in power spectrum analysis. We aimed to establish the appropriate method for the investigation of PN function in OSA patients with apneas and hypopneas using instantaneous time–frequency analysis with complex demodulation (CD) and sufficient time resolution. Methods A total of 30 patients with PSG-confirmed mild to severe OSA were recruited for the analysis of frequency spectra contained in R-R intervals (RRI) of overnight electrocardiograph (ECG) tracings. High-frequency (HF) domains ranging between 0.15 and 0.40 Hz were selected for analysis. Among these domains, the HF domain with the maximum instantaneous amplitude was defined as the main HF peak and was used as the surrogate marker of PN discharge. Based on density spectrum array (DSA) map for main HF peak constructed with a time scale of 1 s and a frequency resolution of 0.002 Hz (HF-DSA map), the shift in central frequency (CF) of main HF peak over time was continuously monitored. When the main HF peak with the same CF lasted for more than 20 s or 5 min on HF-DSA map, the PN function was considered to be stable or very stable. The measurements were then repeated after continuous positive airway pressure (CPAP) treatment. Results The extent of PN-evoked modulation of RRI was enhanced in nonrapid eye movement (NREM) sleep, though the stability was reduced in both NREM and rapid eye movement (REM) sleep. These peculiar behaviors of PN function were reversed by CPAP treatment. Conclusion We found that instantaneous time–frequency analysis allowed estimation of transitional changes in PN function during sleep in OSA patients.

Although lung age calculated backward from regression formulas constructed for FEV1 estimation is widely used, it possesses a couple of faults. We developed novel equations predicting lung age from varied spirometric parameters (spirometry-derived lung age (SDL-age)). Applying multiple regression analysis, equations predicting SDL-age were invented using data from 8015 never-smokers with normal spirometry (group I). Validation was made based on data from 6398 never-smokers with normal spirometry (group II). Equations were further applied for 446 subjects with airflow limitation. FEV1, FEV1/FVC, FEF50, and PEF were selected as explanatory variables for reference value of SDL-age. Normal limits of difference between SDL-age and chronological-age were ±13.4 years in the male and ±15.0 years in the female. Established equations predicted SDL-age of group II. SDL-age was older than chronological-age only in subjects with severe airflow limitation. Novel regression equations allowing prediction of reference value of SDL-age and normal limits of difference between SDL-age and chronological-age were elaborated in both genders.

We investigated whether systemically administered EP2 receptor agonists would stimulate angiogenesis in the emphysematous lungs of mice. Saline or porcine pancreatic elastase was intratracheally administered to C57BL/6J mice to induce the formation of emphysematous lesions, and 4 weeks later the mice were intraperitoneally injected with an EP2 receptor agonist, ONO-AE1-259 or PGE2, or saline on days 1-5 each week for 3 weeks. Intraperitoneal ONO-AE1-259 in the mice in the intratracheal saline group increased pulmonary capillary volume to 114.9% of the control value (P0.05), and when administered to the intratracheal elastase group, ONO-AE1-259 partially restored pulmonary capillary volume, from 70.9% of the control value to 88.3% of the control value (P0.05). Intraperitoneal PGE2 tended to increase pulmonary capillary volume in the mice in the intratracheal saline group (P=0.07) but not in the intratracheal elastase group. Intraperitoneal ONO-AE1-259 and PGE2 each increased the concentration of vascular endothelial growth factor (VEGF) and the number of endothelial progenitor cells (EPCs) in circulating blood. These results suggest that systemically administered ONO-AE1-259 stimulates pulmonary angiogenesis in an elastase-induced murine model of emphysema.

Recent evidence implicates increased oxidative stress as an important mechanism of the pulmonary inflammation that occurs in cigarette smokers. Since cigarette smoke (CS) contains and generates a large amount of reactive oxygen species (ROS) that elicit pulmonary inflammation, antioxidants may become effective therapeutic agents for CS-related inflammatory lung diseases, such as chronic obstructive pulmonary disease. Platinum nanoparticles stabilized with polyacrylate to form a stable colloid solution (PAA-Pt) are a new class of antioxidants that has been shown to efficiently quench ROS. In the present study we investigated the therapeutic effects of PAA-Pt on pulmonary inflammation in smoking mice. PAA-Pt or saline was administered intranasally to DBA/2 mice, which were then exposed to CS or control air daily for 3 days. Mice were sacrificed 4 h after their final exposure to CS or control air. CS exposure caused depletion of antioxidant capacity, NFκB activation, and neutrophilic inflammation in the lungs of mice, and intranasal administration of PAA-Pt prior to CS exposure was found to inhibit these changes. Intranasal administration of PAA-Pt alone did not elicit pulmonary inflammation or toxicity. In in vitro experiments, treatment of alveolar-type-II-like A549 cells with PAA-Pt inhibited cell death after exposure to a CS extract. These results suggest that platinum nanoparticles act as antioxidants that inhibit pulmonary inflammation induced by acute cigarette smoking.

Background C-reactive protein (CRP), a prototypical acute phase protein, is increased as much as 1000-fold during acute inflammation, suggesting its biological role in that process. CRP can be modified at sites of inflammation where proteases have been released by neutrophils migrating to tissues. In this study, we investigated whether native CRP and neutrophil elastase-digested products of CRP modulate the rate of neutrophil apoptosis. Methods Neutrophils were isolated from venous blood of healthy volunteers and incubated with either native CRP (1, 10, or 30 μg/mL) or CRP digested with neutrophil elastase (1 U/mL). After 6 and 24 h of incubation, neutrophils were harvested and examined for apoptosis under light microscopy. Results We found that CRP degradation products generated by neutrophil elastase, but not native CRP, promoted neutrophil apoptosis and cell death. The rate of apoptosis was not affected by the addition of elastase that was not incubated with CRP. Conclusions These results suggest that CRP degradation products generated by neutrophil elastase promote neutrophil apoptosis. Cleavage of CRP by neutrophil elastase may offer protection from inflammatory injury.

The method predicting lung age was originally proposed by Morris and Temple (1985). In their method, lung age was estimated by counting back the regression formula predicting normal value of FEV1. Since the normal value of FEV1 at a given age is not unique and exists within a certain range defined as 95% confidence interval, the backward value of lung age calculated with the original method includes statistical and physiological problems. Analyzing the problems related to the original method, we have developed a novel method with revising age-elicited variation in FEV1. When the original method is applied, the lung age of a person with measured FEV1 beyond upper-limit-of-normal (ULN) results in being remarkably young (sometimes, below zero), while that of a person with FEV1 below lower-limit-of-normal (LLN) is estimated as being very elderly (sometimes, over 100). On the other hand, the novel method, in which age-related variation in FEV1 is reliably corrected, predicts a wide range of lung age even when measured FEV1 is significantly above ULN or below LLN.

Summary Background Th2 cytokines play an important part in the pathogenesis of asthma. Our aim was to study the effect of suplatast tosilate, a selective Th2 cytokine inhibitor, on asthma control and asthma exacerbations during reduction of inhaled corticosteroid dose in patients with steroid-dependent asthma. Methods 85 patients with moderate to severe asthma taking high doses (⩾1500 μg per day) of inhaled beclometasone dipropionate, were assigned suplatast tosilate (100 mg three times daily) or placebo for 8 weeks in a double-blind, randomised, parallel-group, multicentre trial. During the first 4 weeks, other medications remained unchanged (add-on phase); during the next 4 weeks, the doses of beclometasone were halved (steroid-reduction phase). Main outcome measures were pulmonary function, asthma symptoms, and use of β 2 -agonists. Findings Data were available from 77 patients. During the add-on phase, suplatast tosilate treatment, compared with placebo, was associated with higher forced expiratory volume in 1 s (mean difference between groups for changes from baseline at week 4, 0·20 L [95% CI 0·16–0·24], p=0·043), morning peak expiratory flow (18·6 L/min [14·1–23·1], p=0·037), and less diurnal variation in peak expiratory flow rate, asthma symptom scores (7·1 [6·6–7·6], p=0·029), and serum concentrations of eosinophil cationic protein and IgE. In the steroid-reduction phase, pulmonary function, asthma symptoms, and use of β 2 -agonist deteriorated significantly more in the placebo group than in the suplatast group. Interpretation Treatment with a Th2 cytokine inhibitor in steroid-dependent asthma improves pulmonary function and symptom control, and allows a decrease in dose of inhaled corticosteroid without significant side-effects. Some improvements in pharmacokinetics are, however, needed.

Background: Cyclooxygenase products of arachidonic acid may play a part in bronchoconstriction and airway inflammation in asthma. Objective: We sought to determine the effect of inhaled indomethacin on asthma control and asthma exacerbations during reduction of inhaled corticosteroids in patients with moderate-to-severe steroid-dependent asthma. Methods: We conducted a double-blind, randomized, parallel-group, multicenter study in 38 patients with asthma taking high doses (≥1500 μg/d) of beclomethasone dipropionate (BDP). After a run-in period, patients were assigned inhaled indomethacin (50 mg/d) or placebo for 6 weeks, during which the daily doses of BDP were reduced to half at week 2 and then to one third of the baseline dose at week 4. Results: Data were available from 34 patients. After the reduction of BDP doses, FEV 1 , peak expiratory flow, asthma symptoms, and exhaled nitric oxide concentrations deteriorated in both treatment groups, but these effects were less pronounced in the indomethacin group compared with the placebo group. During the 6-week treatment period, 89% of the patients receiving placebo had relapse of asthma, whereas only 38% of those receiving inhaled indomethacin did so ( P = .003). Conclusion: Inhalation of indomethacin can reduce asthma exacerbations induced by reduction of high-dose inhaled corticosteroid in steroid-dependent asthma. (J Allergy Clin Immunol 2000;105:1134-9.)

Neutrophil accumulation in the lung is implicated in the pathogenesis of pulmonary emphysema and chronic bronchitis associated with cigarette smoking. To determine whether nicotine contributes to this accumulation through the prolongation of neutrophil survival, we examined the survival rates of isolated neutrophils cultured with or without nicotine. We found that nicotine prolonged neutrophil survival in a dose-dependent fashion, with a maximum effect at 10(-6) mol/L. The survival rate at 72 hours was 35.6% +/- 1.2% in medium with 10(-6) mol/L nicotine, compared with 15.5% +/- 0.5% in control medium (mean +/- SEM; p < 0.01), as determined by trypan blue dye exclusion. This prolongation was brought about by suppression of apoptosis, as evidenced by both transmission electron and fluorescence microscopy, and was associated with the preservation of neutrophil functions such as chemotaxis and O2- generation. The prolongation of survival caused by nicotine was abrogated by the addition of Pro-Lys-Arg-NH2, a competitive inhibitor of the specific binding of nicotine to noncholinergic receptors on neutrophils. However, the prolongation of survival caused by nicotine was not suppressed in the presence of K-252b, an inhibitor of protein kinase C. These findings suggest that nicotine prolongs neutrophil survival through noncholinergic nicotine receptors and new protein synthesis, without activation of protein kinase C.

Abbreviations: AM 5 alveolar macrophages; CS 5 cigarette smoke A poptosis is a critical mechanism controlling cellularity in various tissues. It is so far unknown whether apoptosis plays a role in cigarette smoking-related pulmonary diseases. It is, however, reported that inhaled toxic materials such as silica and particle matters induce apoptosis of alveolar macrophages (AMs). This study was aimed to test hypothesis that cigarette smoke (CS) may induce apoptosis of AMs. In lung tissue specimens obtained from current smokers with pulmonary emphysema, approximately 0.3% of the AM population were found to be positive for terminal deoxynucleotidyl transferase-mediated nucleotide nick end-labeling and immunostaining with monoclonal anti-single-stranded DNA (ApoStain; Alexis Corp; San Diego, CA). In in vitro studies, mouse, rat, and human AMs, and human blood monocyte-derived macrophages cultured with aqueous cigarette smoke extracts underwent apoptosis as evidenced by light and electron microscopy, and terminal deoxynucleotidyl transferase-mediated nucleotide nick end-labeling. This apoptosis was associated with increased oxidative stress, Bax protein accumulation, mitochondrial dysfunction, and mitochondrial cytochrome c release, but was independent of p53, Fas, and caspase activation. The cigarette smoke extract-induced apoptosis was inhibited by glutathione, ascorbic acid, and a-tocopherol, which are antioxidants known to be present in respiratory tract lining fluids. In in vivo studies where rats were exposed to the smoke from 10 cigarettes over 5 h in an exposure chamber, approximately 3% of AMs obtained by BAL showed apoptosis after 24 h. To evaluate the role of lung antioxidants in CS-induced AM apoptosis in vivo, glutathione-depleted rats produced by administration of buthionine sulfoximine were exposed to CS. There was a significant increase in CS-induced AM apoptosis in glutathione-depleted rats compared with control rats. These results may provide information to explain macrophage dysfunction and lung diseases in cigarette smokers.