マウス内耳発生におけるTUNEL陽性死細胞の形態学的観察(第二報) : 微細構造による細胞死分類について

胎生期マウスの内耳に出現する死細胞にはTdT-mediated dUTP nick end-labeling (以下TUNEL)光顕レベルでアポトーシス細胞死(ACD)と非アポトーシス細胞死(NACD)を示すものが存在し,内耳全体の総死細胞数に対して前者は約90%,後者は約10%発現する.cis-diammine-dichloroplatinum(以下CDDP)負荷によって,総死細胞数に著変なくACDが約70%,NACDが約30%となり,NACDを示す死細胞の発現が増加することを見出し,前報に報告した(川崎医学会誌28(4) : 287-296,2002).本研究の目的は光顕的にTUNEL陽性死細胞が確認された切片を再包埋して電顕観察し,胎生期マウス内耳におけるACD, NACDについてその超微形態的特徴を明らかにすることである.続いて内耳全体を可及的に電顕観察することによってACD, NACDを示す死細胞に対する貧食処理様式,核正常であるためTUNEL法で検出不可能な死細胞存在の有無についても検討した.これらの結果から,ACDを示す死細胞はClarkeの分類によるtype1死細胞すなわちアポトーシスによる死細胞であり,NACDを示す死細胞はtype2死細胞すなわち自己貧食による死細胞で,さらにtype1死細胞は隣接細胞に貧食処理されていた.また,これらはCDDP負荷によっても変化しないことが明らかとなった.TdT-mediated dUTP nick end-labeling (TUNEL) positive dying cells in the inner ears of embryonic mice could be classified as involved in apoptotic cell death (ACD) and non-apoptotic cell death (NACD) by light microscopy. Approximately 90% of the inner ear dying cells were in ACD and remaining 10% were in NACD. Cis-diammine-dichloroplatinum (CDDP) decreased the ACD / NACD ratio as 70 / 30 without clear change in the number of total dying cells (Kawasaki Igakkai Shi 28(4) : 287-296, 2002). After observations under light microscope, using reembedding maneuver, the same TUNEL positive dying cells were observed by transmission electron microscopy (TEM). According to the classification of dying cells by Clarke (1990), the TUNEL positive dying cell\u27s images were analyzed. The phagocytosis of dying cells and TUNEL negative dying cells and TUNEL negative dying cells in the inner ears were observed by ordinary TEM. The results clearly showed that ACD involved in type 1 (apoptotic) dying cells and NACD involved in type2 (autophagic) dying cells in the inner ear of the mouse embryos, and that the type 1 dying cells were engulfed by neighboring cell. These images were not changed by the administration of CDDP

マウス内耳発生におけるTUNEL陽性死細胞の形態学的観察 : CDDP投与による変化について

昨今の細胞死研究において,核のDNA断片化を検出するTdT-mediated dUTP-biotin nick end labeling(以下TUNEL)法で陽性死細胞の中には形態学的"アポトーシス様"と判定される死細胞だけでなく"非アポトーシス様"と判定される死細胞も存在していることが明らかとなってきた.本研究は内耳形態変化が最も多く"自然細胞死"が高頻度に発現すると考えられる胎生12日目マウスの内耳を材料として,個体発生における"プログラム細胞死"で"アポトーシス様"と"非アポトーシス様"と判定される死細胞の占める比率を明らかにし,内耳毒性を有するcis-diammine-dichloroplatinum(以下のCDDP)投与の影響を観察した.対照群5匹5耳とCDDP負荷率(以下CDDP群)5匹5耳で観察されたTUNEL陽性死細胞を"アポトーシス"の定義に基づいて,光顕的にアポトーシス細胞死(以下ACD)と非アポトーシス細胞死(以下NACD)を示す死細胞に分類し,各検体における総死細胞数に対する比率(以下ACD率,NACD率)を算出した.その結果,対照群におけるACD率は約90%であり,NACD率は約10%であった.またCDDP群では対照群と比較して総死細胞数に著変は無かったが,前者が約70%,後者が約30%と発現率に変化が認められた.それによってACDは分裂・増殖と同様に発生期に必須であり,NACDの増加は内耳毒性に対する防御反応と推察された.In the recent studies of cell death, dying cells judged by the TdT-mediated dUTP nick end-la-beling (TUNEL) method have been classified into "apoptotic" and "non-apoptotic" cells. In this study, 12-day-old mouse embryos were used. The percentage of "apoptotic" and "non-apoptotic" cell among total dying cells in inner ear were calculated, and the effects on inner ear\u27s cell death of cis-diammine-dichloroplatinum (CDDP) were examined. Five inner ears from normal embryonic mice (Control group) and five inner ears from CDDP treated embryonic mice (CDDP group) were used. TUNEL positive dying cells occurring in apoptotic cell death (ACD) and non-apoptotic cell death (NACD) were classified by light microscopy, and the numbers in ACD and NACD in the whole inner ear were counted. About 90% of the inner ear dying cells of the Control group were ACD and about 10% were NACD. About 70% of the inner ear dying cells in the CDDP group were ACD and bout 30% were NACD. It was suggested that ACD essential to development of inner ear, and increased NACD might be defensive phenomenon against CDDP toxicity

Associations of CT evaluations of antigravity muscles, emphysema and airway disease with longitudinal outcomes in patients with COPD

Multiple CT indices are associated with disease progression and mortality in patients with COPD, but which indices have the strongest association remain unestablished. This longitudinal 10-year observational study (n=247) showed that the emphysema severity on CT is more closely associated with the progression of airflow limitation and that a reduction in the cross-sectional area of erector spinae muscles (ESMCSA) on CT is more closely associated with mortality than the other CT indices, independent of patient demographics and pulmonary function. ESMCSA is a useful CT index that is more closely associated with long-term mortality than emphysema and airway disease in patients with COPD

Physiological Impairments on Respiratory Oscillometry and Future Exacerbations in Chronic Obstructive Pulmonary Disease Patients without a History of Frequent Exacerbations

Respiratory oscillometry allows measuring respiratory resistance and reactance during tidal breathing and may predict exacerbations in patients with chronic obstructive pulmonary disease (COPD). While the Global Initiative for Chronic Obstructive Lung Disease (GOLD) advocates the ABCD classification tool to determine therapeutic approach based on symptom and exacerbation history, we hypothesized that in addition to spirometry, respiratory oscillometry complemented the ABCD tool to identify patients with a high risk of exacerbations. This study enrolled male outpatients with stable COPD who were prospectively followed-up over 5 years after completing mMRC scale and COPD assessment test (CAT) questionnaires, post-bronchodilator spirometry and respiratory oscillometry to measure resistance, reactance, and resonant frequency (Fres), and emphysema quantitation on computed tomography. Total 134 patients were classified into the GOLD A, B, C, and D groups (n = 48, 71, 5, and 9) based on symptoms on mMRC and CAT and a history of exacerbations in the previous year. In univariable analysis, higher Fres was associated with an increased risk of exacerbation more strongly than other respiratory oscillometry indices. Fres was closely associated with forced expiratory volume in 1 sec (FEV1). In multivariable Cox-proportional hazard models of the GOLD A and B groups, either lower FEV1 group or higher Fres group was associated with a shorter time to the first exacerbation independent of the GOLD group (A vs B) and emphysema severity. Adding respiratory oscillometry to the ABCD tool may be useful for risk estimation of future exacerbations in COPD patients without frequent exacerbation history

Low serum free light chain is associated with risk of COPD exacerbation

Background: Most exacerbations of chronic obstructive pulmonary disease (COPD) are triggered by respiratory tract infections. Adaptive immunity via antibody production is important in preventing infections. Impaired antibody production is reported to be associated with an increased risk of exacerbations of COPD. In the present study, we elucidated whether reduced free light chains (FLCs), which are excessive amounts of light chains produced during antibody synthesis and can be used to estimate systemic antibody production, may be a promising biomarker to predict the risk of exacerbations of COPD. Methods: We enrolled stable male patients with COPD and prospectively observed them for 2 years. At baseline, serum combined FLC (cFLC; sum of kappa and lambda values) and pulmonary function were evaluated. Exacerbation was defined as a worsening of symptoms requiring treatments with antibiotics, corticosteroids or both. Results: 63 patients with stable COPD were enrolled (72.8±8.1 years, GOLD A/B/C/D=24/28/6/5), and 51 patients completed the 2-year follow-up. Serum cFLC was 31.1 mg·L−1 on average and ranged widely (1.4 to 89.9 mg·L−1). The patients with low cFLC (below the mean−sd, n=6) experienced a significantly shorter time to the first exacerbation of COPD (p<0.0001 by the log-rank test). A multivariate Cox proportional hazard model, including the COPD assessment test score, % predicted forced expiratory volume in 1 s (FEV1 % pred), and number of previous exacerbations demonstrated that low cFLC and low FEV1 % pred were independently and significantly correlated with the risk for exacerbations of COPD. Conclusion: Low cFLC may be a B-cell-associated novel biomarker associated with risk of COPD exacerbation

p38 mitogen-activated protein kinase determines the susceptibility to cigarette smoke-induced emphysema in mice

BACKGROUND: There is a need for agents that suppress inflammation and progression of chronic obstructive pulmonary disease. p38 mitogen-activated protein kinase (p38 MAPK) has been associated with this disorder, and several inhibitors of this cascade are in clinical trials for its treatment, but their efficacy and utility are unknown. This study evaluated the relationship between p38 MAPK activation and susceptibility to cigarette smoke (CS)-induced emphysema, and whether its inhibition ameliorated the lung inflammation and injury in murine models of cigarette smoke exposure. METHODS: In acute and chronic CS exposure, the activation and expression of p38 MAPK in the lungs, as well as lung inflammation and injury (proteinase production, apoptosis, and oxidative DNA damage), were compared between two mouse strains: C57BL/6 (emphysema-susceptible) and NZW (emphysema-resistant). The selective p38 MAPK inhibitor SB203580 (45 mg/kg) was administrated intra-peritoneally to C57BL/6 mice, to examine whether it ameliorated cigarette smoke-induced lung inflammation and injury. RESULTS: Acute CS-induced lung inflammation (neutrophil infiltration, mRNA expressions of TNF-α and MIP-2), proteinase expression (MMP-12 mRNA), apoptosis, and oxidative DNA damage were significantly lower in NZW than C57BL/6 mice. p38 MAPK was significantly activated and up-regulated by both acute and chronic CS exposure in C57BL/6 but not NZW mice. mRNA expression of p38 MAPK was also upregulated in C57BL/6 by chronic CS exposure and tended to be constitutively suppressed in NZW mice. SB203580 significantly attenuated lung inflammation (neutrophil infiltration, mRNA expressions of TNF-α and MIP-2, protein levels of KC, MIP-1α, IL-1β, and IL-6), proteinase expression (MMP-12 mRNA), oxidative DNA damage, and apoptosis caused by acute CS exposure. CONCLUSIONS: Cigarette smoke activated p38 MAPK only in mice that were susceptible to cigarette smoke-induced emphysema. Its selective inhibition ameliorated lung inflammation and injury in a murine model of cigarette smoke exposure. p38 MAPK pathways are a possible molecular target for the treatment of chronic obstructive pulmonary disease

Disproportionally Impaired Diffusion Capacity Relative to Airflow Limitation in COPD

Forced expiratory volume in 1 s (FEV₁) is a standard physiological index of chronic obstructive pulmonary disease (COPD), but reflects emphysema and vascular abnormalities less sensitively than diffusion capacity for carbon monoxide (D_LCO). This study tested whether a disproportionally impaired D_LCO relative to FEV₁ (FEV₁ z-score>-3 and D_LCO z-score≤-3) is a common functional COPD phenotype associated with distinct clinical and structural features and the prognosis of two cohorts. The cross-sectional analyses of the Korea COPD Subgroup Study (KOCOSS) cohort (multicenter study in Korea) included 743 males with COPD whose D_LCO was available. The cross-sectional and longitudinal analyses of the Kyoto University Cohort (single-center study in Japan) included 195 males with COPD who were prospectively followed for 10 years. A disproportionally impaired D_LCO relative to FEV₁ was observed in 29% and 31% of patients in the KOCOSS and Kyoto University cohorts, respectively. In the multivariable analysis, the disproportionally impaired D_LCO was associated with worse symptoms, shorter 6-minute walking distance, paraseptal and centrilobular emphysema on computed tomography, and reduced arterial oxygen and carbon dioxide pressures compared to the reference (FEV₁ z-score>-3 and D_LCO z-score>-3). In the multivariable Cox proportional hazard model, a higher long-term mortality was observed in the disproportionally impaired D_LCO group than in the reference group (hazard ratio [95% confidence interval] = 3.09 [1.52–6.29]) and similar to the D_LCO z-score≤-3 and FEV₁ z-score≤-3 group. The disproportionally impaired D_LCO relative to FEV₁ is common and associated with increased symptoms, emphysema, arterial blood gas abnormalities, and increased long-term mortality in patients with COPD

Quantitative measurement of airway dimensions using ultra-high resolution computed tomography

Background: Quantitative measurement of airway dimensions using computed tomography (CT) is performed in relatively larger airways due to the limited resolution of CT scans. Nevertheless, the small airway is an important pathological lesion in lung diseases such as chronic obstructive pulmonary disease (COPD) and asthma. Ultra-high resolution scanning may resolve the smaller airway, but its accuracy and limitations are unclear. Methods: Phantom tubes were imaged using conventional (512 × 512) and ultra-high resolution (1024 × 1024 and 2048 × 2048) scans. Reconstructions were performed using the forward-projected model-based iterative reconstruction solution (FIRST) algorithm in 512 × 512 and 1024 × 1024 matrix scans and the adaptive iterative dose reduction 3D (AIDR-3D) algorithm for all scans. In seven subjects with COPD, the airway dimensions were measured using the 1024 × 1024 and 512 × 512 matrix scans. Results: Compared to the conventional 512 × 512 scan, variations in the CT values for air were increased in the ultra-high resolution scans, except in the 1024×1024 scan reconstructed through FIRST. The measurement error of the lumen area of the tube with 2-mm diameter and 0.5-mm wall thickness (WT) was minimal in the ultra-high resolution scans, but not in the conventional 512 × 512 scan. In contrast to the conventional scans, the ultra-high resolution scans resolved the phantom tube with ≥ 0.6-mm WT at an error rate of < 11%. In seven subjects with COPD, the WT showed a lower value with the 1024 × 1024 scans versus the 512 × 512 scans. Conclusions: The ultra-high resolution scan may allow more accurate measurement of the bronchioles with smaller dimensions compared with the conventional scan