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Introduction Sivelestat, a neutrophil elastase inhibitor, has been approved in Japan for the treatment of patients with acute lung injury (ALI) associated with systemic inflammatory response syndrome (SIRS). The Pharmaceuticals and Medical Devices Agency (PMDA) has ordered to conduct a postmarket clinical study in order to reevaluate the efficacy and safety of Sivelestat in actual clinical settings in Japan. Methods According to the PMDA’s order, we evaluated the efficacy and safety of Sivelestat in Japanese patients with ALI associated with SIRS using ventilator-free days (VFD) as the primary endpoint. The surrogate endpoints are ventilator-weaning rate, ICU discharge rate, and 180-day survival rate. Study design was an open-label, non-randomized, multi-center clinical trial. Sivelestat was intravenously administered at 0.2 mg/kg/h continuously for a maximum of 14 days. Sivelestat group and control group were compared by adjusting the outcome values using an inverse probability of treatment weighted method based on the propensity scores. Results Four hundred and four Sivelestat group patients and 177 control group patients were enrolled. The adjusted mean number of VFD was 15.7 and 12.1 in the Sivelestat group and control group, respectively ( P = 0.0022). Both the adjusted ventilator-weaning rate and ICU discharge rate were significantly higher in the Sivelestat group than in the control group ( P = 0.0028 and P = 0.019, respectively). The adjusted 180-day survival rate was significantly higher in the Sivelestat group than in the control group (71.8 percent vs. 56.3 percent). Conclusions Sivelestat contributed to early weaning from the mechanical ventilation, while showing no negative effect on the long-term outcomes of ALI associated with SIRS. The results of this study suggest the clinical usefulness of Sivelestat in this patient population.

Allergic bronchopulmonary mycosis, characterized by excessive mucus secretion, airflow limitation, bronchiectasis, and peripheral blood eosinophilia, is predominantly caused by a fungal pathogen, Aspergillus fumigatus. Using DNA microarray analysis of NCI-H292 cells, a human bronchial epithelial cell line, stimulated with fungal extracts from A. fumigatus, Alternaria alternata, or Penicillium notatum, we identified a mucin-related MUC5AC as one of the genes, the expression of which was selectively induced by A. fumigatus. Quantitative RT-PCR, ELISA, and histochemical analyses confirmed an induction of mucin and MUC5AC expression by A. fumigatus extracts or the culture supernatant of live microorganisms in NCI-H292 cells and primary cultures of airway epithelial cells. The expression of MUC5AC induced by A. fumigatus extracts diminished in the presence of neutralizing Abs or of inhibitors of the epidermal growth factor receptor or its ligand, TGF-α. We also found that A. fumigatus extracts activated the TNF-α–converting enzyme (TACE), critical for the cleavage of membrane-bound pro–TGF-α, and its inhibition with low-molecular weight inhibitors or small interfering RNA suppressed the expression of MUC5AC. The protease activity of A. fumigatus extracts was greater than that of other fungal extracts, and treatment with a serine protease inhibitor, but not with a cysteine protease inhibitor, eliminated its ability to activate TACE or induce the expression of MUC5AC mRNA in NCI-H292. In conclusion, the prominent serine protease activity of A. fumigatus, which caused the overproduction of mucus by the bronchial epithelium via the activation of the TACE/TGF-α/epidermal growth factor receptor pathway, may be a pathogenetic mechanism of allergic bronchopulmonary mycosis.

Purpose Bronchoscopic microsampling (BMS) is a novel and direct method with which to obtain epithelial lining fluid (ELF) from the lungs. Analysis of DNA hypermethylation of tumor suppressor genes (TSGs) is expected to be a sensitive tool for the early detection of lung cancer. It has been reported that the existence of EGFR mutations and EML4-ALK gene rearrangements are related to the sensitivity of corresponding kinase inhibitors. We aimed to evaluate the suitability of ELF as a sample for analyzing molecular changes specific for lung cancer. Patients and methods We collected ELF from 61 lung cancer patients by BMS from the airway close to the peripheral lung nodule and purified the nucleic acids. We performed methylation specific PCR in each ELF as well as matched serum and tumor tissue for TSGs for DNA methylation analysis. We also examined EGFR mutations and EML4-ALK rearrangement. Results The sensitivity for detecting DNA hypermethylation in ELF vs serum was 74.1% vs 18.5%. We found 60.1% of patients had at least one hypermethylation in ELF, while only 27.9% had it in serum. Of note, DNA hypermethylation was detected even in stage I patients (60.0%) and the detection rate was almost the same level in each stage. We also found the sensitivity for detecting EGFR mutation in ELF vs serum was 58.3% vs 8.3%. We detected an EML4-ALK fusion gene using ELF in one patient. Conclusions BMS is an alternative method to detect cancer specific genetic and epigenetic alterations and will be a useful complementary diagnostic tool for lung cancer. Summary Investigation of genetic and epigenetic changes associated with lung cancer has clinical importance for its diagnosis and management. The clinical usefulness of bronchoscopic microsampling (BMS) in lung cancer has not yet been evaluated. This study demonstrates that BMS could be useful for detecting lung cancer specific molecular changes and valuable for early diagnosis and determination of treatment options for lung cancer.

High-mobility group box 1 protein (HMGB1) is a late mediator of inflammatory responses that can cause acute lung injury. We examined the significance of serum HMGB1 elevation in the development of systemic inflammatory response syndrome (SIRS) and lung oxygenation impairment (LOI) after thoracic aortic aneurysm (TAA) repair. Serial measurements of the serum HMGB1 level and SIRS score for 7 days after surgery were determined in 20 patients with TAA who underwent surgical repair. Arterial oxygen tension was measured serially for at least 4 days after surgery, and LOI was defined as the lowest PaO(2)/FiO(2) ratio ≤ 200 mmHg. The serum HMGB1 level was markedly increased after surgery, peaking on day 2, and remained significantly elevated on day 7. Peak HMGB1 level positively correlated with SIRS duration and the cumulative SIRS score during postoperative days 1-7 (P = 0.0013 and P = 0.0004, respectively). Peak HMGB1 level and cumulative SIRS score were higher in patients with LOI than in those without (P = 0.01 and P = 0.044, respectively). Peak HMGB1 level was negatively correlated with the lowest PaO(2)/FiO(2) ratio (P = 0.0077) and positively correlated with postoperative length of hospitalization (P = 0.042). A greater serum HMGB1 elevation after TAA repair was associated with more severe SIRS and a higher incidence of LOI. HMGB1 might play a key role in the pathogenesis of SIRS and LOI after surgical TAA repair.

Little is known about the pathobiology of acute exacerbation of idiopathic pulmonary fibrosis (IPF), a condition that shares clinical and histopathological features with acute lung injury. Plasma biomarkers have been well studied in acute lung injury and have provided insight into the underlying disease mechanism. The objective of this study was to determine the plasma biomarker profile of acute exacerbation of IPF and compare this profile with that of stable IPF and acute lung injury. Plasma was collected from patients with stable IPF, acute exacerbation of IPF, and acute lung injury for measurement of biomarkers of cellular activity/injury (receptor for advanced glycation endproducts, surfactant protein D, KL-6, von Willebrand factor), systemic inflammation (IL-6), and coagulation/fibrinolysis (protein C, thrombomodulin, plasminogen activator inhibitor-1). Plasma from patients with acute exacerbation of IPF showed significant elevations in markers of type II alveolar epithelial cell injury and/or proliferation, endothelial cell injury, and coagulation. This profile differed from the biomarker profile in patients with acute lung injury. These findings support the hypothesis that type II alveolar epithelial cells are centrally involved in the pathobiology of acute exacerbation of IPF. Furthermore, they suggest that acute exacerbation of IPF has a distinct plasma biomarker profile from that of acute lung injury.

BACKGROUND AND OBJECTIVE Whether beta(2)-adrenoceptor gene (ADRB2) polymorphisms are associated with airway responsiveness to beta(2)-agonist medications remains controversial, partly due to factors that may confound pharmacogenetic associations, including age, cigarette smoking and airway remodelling. To overcome these problems, we performed an analysis using parameters that reflected the specific bronchodilator response to beta(2)-agonists. METHODS The increases in FEV(1) after inhalation of procaterol hydrochloride (Delta FEV(1) procaterol) or oxitropium bromide (Delta FEV(1) oxitropium), and after sequential inhalation of procaterol and oxitropium (total airway reversibility), were measured in 81 Japanese patients with moderate to severe asthma. Approximately 3 kb of the DNA sequence of the coding and 5'-flanking regions of ADRB2 were genotyped by direct sequencing and PCR-restriction fragment length polymorphism assay. RESULTS The mean age of the participants was 54 years, and 38 (47%) were smokers. Although Delta FEV(1) procaterol and Delta FEV(1) oxitropium adjusted for predicted FEV(1) were not associated with ADRB2 polymorphisms, the ratio of Delta FEV(1) procaterol to total airway reversibility was significantly associated with the ADRB2 A46G genotype (P < 0.05). Patients who were homozygous for the A46 allele (arginine at amino acid 16) were more responsive than carriers of the G46 (glycine 16) allele (P = 0.008). Multivariate linear regression analysis showed that Delta FEV(1) procaterol was correlated with the number of A46 alleles (P = 0.014), and also with total airway reversibility (P < 0.001) and smoking index in current smokers (P = 0.009). CONCLUSIONS The ADRB2 A46G polymorphism was associated with a relatively greater bronchodilator responsiveness to beta(2)-agonists even in elderly asthmatic patients and smokers.

Sivelestat sodium hydrate is a selective inhibitor of neutrophil elastase, which is effective in acute lung injury associated with systemic inflammatory response syndrome. However, the effectiveness of sivelestat in sepsis has not been fully examined. In the present study, the effect of sivelestat on severe sepsis in a rat cecal ligation and puncture (CLP) model was investigated. Adult male Sprague-Dawley rats underwent CLP and were randomly divided into two groups: sivelestat-treated group and saline-treated controls. The serum concentrations of several inflammatory mediators were measured. Hematoxylin-eosin staining, and immunohistochemical staining for high-mobility group box chromosomal protein 1 (HMGB1), IL-8, and CD68 were performed on the lungs to assess pathological changes found 12 h after the CLP procedure. Treatment with sivelestat significantly improved the survival rate of the post-CLP septic animals (P = 0.030). Sivelestat also induced a significant reduction in serum IL-1beta (P = 0.038) and IL-10 (P = 0.008) levels in these CLP rats. Serum HMGB1 levels had no significant difference between the sivelestat-treated and the control group. The lungs from sivelestat-treated rats exhibited less severe pathological changes and decreased the numbers of HMGB1, IL-8, and CD68-positive cells (P < 0.001). Sivelestat significantly improved survival rate of rats with clinically relevant sepsis, possibly by attenuating sepsis-induced systemic inflammatory response and lung injury. This may explain the implicated health benefits of sivelestat in reducing morbidity and mortality from sepsis.

Katayama, M., Ishizaka, A., Sakamoto, M. et al. Intensive Care Med (2010) 36: 479. https://doi.org/10.1007/s00134-009-1719-6, Objective: Laminin-5, a cell adhesive molecule expressed solely by epithelium, is known to enhance epithelial cell migration and repair of injured epithelium, after its essential component γ2-chain is processed proteolytically. Our previous study revealed circulating levels of amino-terminal fragment of laminin γ2-chain (G2F) reflect epithelial tumor invasiveness in carcinoma patients, but its physiological role in alveolar epithelial injury remains unknown., Design: Sampling of epithelial lining fluids or pulmonary edema fluids from patients with acute lung injury (ALI) or related diseases was performed. Plasma samples were obtained from them at the time of disease onset or later. G2F concentrations were determined by immunoassay constructed by ourselves., Results: We found a significantly higher amount of G2F in pulmonary edema and epithelial lining fluids of patients with ALI, as compared with those with the other respiratory diseases. Their plasma levels were also elevated significantly early at the onset of ALI (mean ± SD; 147 ± 82 ng/ml in non-surviving and 90 ± 56 in surviving patients) as compared with those in the patients with cardiogenic pulmonary edema (59 ± 36) or idiopathic pulmonary fibrosis (37 ± 17), indicating alveolar epithelium rapidly secrete laminin-5 in ALI. At 5 days after onset, non-surviving patients maintained higher plasma concentrations (152 ± 84), but in contrast, the levels in surviving patients declined (71 ± 35), suggesting secretion of laminin-5 was suppressed, associated with recovery from ALI., Conclusion: Circulating G2F may be a biomarker for alveolar laminin-5 secreted early at disease onset in ALI, potentially regulating alveolar re-epithelialization.

Purpose To validate the feasibility of developing a radiotherapy unit with kilovoltage X-rays through actual irradiation of live rabbit lungs, and to explore the practical issues anticipated in future clinical application to humans through Monte Carlo dose simulation. Methods and Materials A converging stereotactic irradiation unit was developed, consisting of a modified diagnostic computed tomography (CT) scanner. A tiny cylindrical volume in 13 normal rabbit lungs was individually irradiated with single fractional absorbed doses of 15, 30, 45, and 60 Gy. Observational CT scanning of the whole lung was performed every 2 weeks for 30 weeks after irradiation. After 30 weeks, histopathologic specimens of the lungs were examined. Dose distribution was simulated using the Monte Carlo method, and dose–volume histograms were calculated according to the data. A trial estimation of the effect of respiratory movement on dose distribution was made. Results A localized hypodense change and subsequent reticular opacity around the planning target volume (PTV) were observed in CT images of rabbit lungs. Dose–volume histograms of the PTVs and organs at risk showed a focused dose distribution to the target and sufficient dose lowering in the organs at risk. Our estimate of the dose distribution, taking respiratory movement into account, revealed dose reduction in the PTV. Conclusions A converging stereotactic irradiation unit using kilovoltage X-rays was able to generate a focused radiobiologic reaction in rabbit lungs. Dose–volume histogram analysis and estimated sagittal dose distribution, considering respiratory movement, clarified the characteristics of the irradiation received from this type of unit.

High-mobility group box 1 (HMGB1) is a nuclear protein that has been found to be a critical mediator of lethality in endotoxemia and sepsis. During the systemic inflammatory response, circulating levels of HMGB1 are increased, but in a delayed fashion compared with early inflammatory mediators. To counteract the inflammatory response of endotoxemia, a secondary anti-inflammatory response ensues in an attempt to prevent inflammation-induced tissue injury. One such cytoprotective gene that is induced during endotoxemia is heme oxygenase (HO)-1. HO-1, and its products of heme metabolism, possess anti-inflammatory and antioxidant properties to counter the damaging effects of endotoxemia. In the present study, we wanted to determine whether tissue and circulating levels of HMGB1 are increased further in the absence of HO-1 during endotoxemia, and whether this increase may contribute to the pathobiology of endotoxemia. Lung inflammation, HMGB1 protein levels, and expression of HMGB1 in inflammatory cells were increased in HO-1(-/-) mice compared with HO-1+/+ mice. After the administration of LPS, tissue levels of HMGB1 were not increased further in HO-1(-/-) mice; however, circulating levels of HMGB1 were higher when compared with HO-1+/+ mice. HO-1(-/-) mice treated with a carbon monoxide-releasing molecule or biliverdin showed a reduction in plasma HMGB1, which was associated with a marked improvement in survival. HO-1(-/-) mice given HMGB1-neutralizing antibody showed improvement in survival compared with control antibody. These data suggest that exaggerated circulating levels of HMGB1 contribute to endotoxin-induced mortality in the absence of HO-1.

Background: Patients with Mycobacterium avium-intracellulare complex (MAC) pulmonary disease often suffer from weight loss. Adipokines are factors secreted by adipocytes, including leptin and adiponectin, as well as some inflammatory cytokines, tumor necrosis factor-α (TNF-α) and interleukin 6 (IL-6). Body mass index (BMI) is known to be inversely correlated with adiponectin and positively with leptin, TNF-α, and IL-6. Objective: We aimed to evaluate the levels of serum adipokines, including adiponectin, leptin, TNF-α, and IL-6 in patients with MAC pulmonary disease. Methods: Forty consecutive patients with MAC pulmonary disease (8 males; median age 62 years; median BMI 18.1) were examined. Serum levels of adiponectin, leptin, TNF-α, and IL-6 were measured with ELISA. Age-, sex- and BMI-matched healthy subjects served as controls. Results: Serum adiponectin was significantly elevated in patients with MAC pulmonary disease compared with the controls (p < 0.01). In both the patients and controls, serum adiponectin levels were inversely correlated with BMI (p < 0.05). No significant correlation was observed between serum adiponectin levels and C-reactive protein or lung function. Serum leptin levels, which were positively correlated with BMI, did not differ between patients and controls. Serum levels of TNF-α and IL-6 were significantly greater in patients with MAC pulmonary disease than in controls. The levels of TNF-α and IL-6 were not correlated with BMI and other adipokines examined. Conclusion: The results of the present study indicate that, in patients with MAC pulmonary disease, adiponectin is inappropriately secreted and may play a role in the pathophysiology of the disease.

Clarithromycin (CAM) is widely accepted for the treatment of Mycobacterium avium complex (MAC) pulmonary diseases. This study measured (a) the concentrations of CAM and its active metabolite (14OH-CAM) in bronchial epithelial lining fluid (ELF) obtained by bronchoscopic microsampling (BMS), and (b) the minimal inhibitory concentrations (MIC) of CAM for each MAC isolate.We studied eight patients with MAC pulmonary disease treated with oral CAM, 400 (n=4) or 800 (n=4) mg/day. BMS was performed 3h after the last CAM dose, and the concentrations of CAM and 14OH-CAM were measured in ELF collected from the diseased and normal contralateral pulmonary segments, and in serum.The mean+/-SEM ELF concentrations of CAM (23.85+/-7.64 microg/ml) and CAM+14OH-CAM (28.71+/-8.37 microg/ml) in the 800 mg/day treatment group were significantly higher than in the 400mg/day group (7.48+/-2.58 microg/ml and 9.63+/-2.99 microg/ml, respectively; both p0.05), while the serum concentrations were similar in both groups. In both treatment groups, the mean ELF concentrations sampled from diseased and normal segments were higher than the MIC against MAC isolates. The mean ELF concentration of CAM in the 400mg/day treatment group was8 mircog/ml (the breakpoint concentration of CAM against M. avium recommended by the Clinical Laboratories Standards Institute), while the mean concentration in the 800 mg/day treatment group was8 microg/ml.These observations suggest that CAM, 800 mg/day, is an appropriate dose to treat MAC pulmonary disease, and prevent its spread from diseased to non-diseased lung segments.

Background Lung resection after induction chemotherapy and/or radiotherapy for down-staging of locally advanced lung cancer can be complicated with lethal interstitial pneumonia. We studied the effects of pneumonectomy on bleomycin-induced lung injury and fibrosis in mice. Methods The mice underwent left pneumonectomy or a sham thoracotomy after intratracheal administration of saline or bleomycin. Lung permeability index, wet-to-dry weight ratio, histological changes, collagen contents, and concentrations of inflammatory mediators and cell counts in broncho-alveolar lavage (BAL) fluid were assessed in the residual right lung 7 d after surgery. Results The index of capillary permeability, lung water content, and inflammatory cell counts in BAL fluid were significantly increased by pneumonectomy. These measurements were highest in the mice with both pneumonectomy and intratracheal administration of bleomycin. Similarly, fibrotic change in lung pathology, as well as an increase in lung collagen content, was most prominent in the mice exposed to both interventions. The BAL fluid concentrations of interleukin-1β, interleukin-6, RANTES, and high mobility group box 1 were significantly increased by pneumonectomy and enhanced by the additional administration of bleomycin. Conclusions The results of this study indicate that pneumonectomy alone causes noncritical lung injury, which amplifies the inflammatory response to bleomycin and promotes lung fibrosis. Several inflammatory mediators appear to be involved in the exacerbation of bleomycin-induced lung injury and fibrosis.

Background: CC chemokines play important roles in the pathogenesis of interstitial lung diseases. Elevated CC chemokine levels have been observed in bronchoalveolar lavage (BAL) fluid of patients with idiopathic pulmonary fibrosis (IPF). Objectives:We aimed to examine whether the levels of four CC chemokines, i.e. monocyte chemoattractant protein-1 (MCP-1/CCL2), macrophage inflammatory protein-1α (MIP-1α/CCL3), thymus- and activation-regulated chemokine (TARC/CCL17), and macrophage-derived chemokine (MDC/CCL22), in BAL fluid are predictive of the prognosis of IPF patients. Methods: We compared the chemokine levels of patients alive 5 years after diagnosis and those who had died. Lung function data, CT scores, and serum markers were also compared. Results: Among 39 patients (29 males, median age, 60 years), 19 patients (48%) died within 5 years after the diagnosis. Whereas percent vital capacity was not different, percent lung diffusion capacity for carbon monoxide was significantly higher in the surviving patients than in the nonsurviving patients (p < 0.01). Median CCL2 levels of surviving and nonsurviving patients were 154.3 (interquartile range, IQR: 67.3–381.8) and 427.2 (IQR: 329.2–1184.1) pg/ml, respectively (p < 0.02). CCL3 levels in BAL fluid did not differ between the surviving and nonsurviving patients. CCL17 was detected in BAL fluid of 7 patients, 6 of whom died within 5 years. CCL22 was detectable in BAL fluid of 10 patients, only 1 of whom survived. Serum levels of KL-6 and lactate dehydrogenase did not differ between the surviving and nonsurviving patients. Conclusion: Elevated levels of CCL2, CCL17 and CCL22 in BAL fluid might be predictive of a poor outcome in patients with IPF.

Neutrophil elastase (NE) has been recognized as a double-edge sword as it can be both host-defensive and pro-inflammatory depending on its tissue level and microenvironment. The present study was designed to examine whether inhibition of NE activity by sivelestat, a specific NE inhibitor with low cellular permeability, is beneficial for acute lung injury induced by Streptococcus pneumoniae in hamsters. Intratracheal inoculation of live S. pneumoniae (1.5 x 10(7) CFU/Lung) into hamsters caused acute lung injury characterized by an increase in lung alveolar permeability and leukocytes accumulation in the lungs. These pathophysiological changes, which were associated with elevation of NE activity in the bronchoalveolar lavage fluid (BALF), were transient but remained high 4-22 h post-bacterial inoculation. Intravenous infusion of sivelestat at 3mg/kg/h 0-22 h after bacterial inoculation reduced the increase in NE activity and lung alveolar permeability, as indicated by leakage of pre-injected human serum albumin into BALF, without affecting the number of leukocytes in BALF and lung interstitial tissue. Interestingly, the number of bacteria in BALF and lung interstitial tissue was also reduced by sivelestat to less than 10% of the control values. Furthermore, S. pneumoniae-induced decrease in the level of surfactant protein D (SP-D), a protein that protects against bacterial infection, was preserved by sivelestat treatment with a significant negative correlation between NE activity and SP-D content in BALF. These results suggest that sivelestat is beneficial in acute lung injury associated with lung bacterial infection and that this inhibitor rather decreases infection by preserving host defense system.

Metallothionein (MT) is a free radical scavenger induced by inflammatory stimuli; however, its roles in inflammation have not been fully investigated. In the present study, we genetically determined the role of MT in ozone (O(3))-induced lung inflammation using MT-I/II null (-/-) mice. Subacute (65 h) exposure to O(3) (0.3 ppm) induced lung inflammation and enhanced vascular permeability, which was significantly greater in MT(-/-) than in corresponding wild-type mice. Electron microscopically, O(3) exposure induced vacuolar degeneration of pulmonary endothelial and epithelial cells, and interstitial edema with focal loss of the basement membrane, which was more prominent in MT(-/-) than in wild-type mice. O(3) -induced lung expression of interleukin-6 was significantly greater in MT(-/-) than in wild-type mice; however, lung expression of the chemokines examined was comparable in both genotypes of mice in the presence of O(3). Following O(3) exposure, the formation of oxidative stress-related molecules/adducts, such as heme oxidase-1, inducible nitric oxide synthase, 8-hydroxy-2'-deoxyguanosine, and nitrotyrosine, in the lung was significantly greater in MT(-/-) than in wild-type mice. Collectively, MT protects against O(3)-induced lung inflammation, at least partly, via the regulation of pulmonary endothelial and epithelial integrity and its antioxidative property.

There are reports indicating that thromboxane A(2) receptors (TP receptors) may stimulate the eosinophil accumulation in the lower airways of asthmatics, however, the mechanisms behind such an effect remain unknown. We quantified the synthesis of eosinophil chemotactic activity and eosinophilic CC chemokines, including CCL5, CCL7, CCL8, CCL11, CCL13, CCL24, and CCL26 in primary cultures of human bronchial smooth muscle cells (BSMC) stimulated with a prostanoid TP receptor agonist, IBOP (10(-9)-10(-7) M). The activation of prostanoid TP receptors in BSMC induced the release of potent eosinophil chemoattractant(s) in the presence of interleukin (IL)-4. CCL11/eotaxin-1 was the only synthesis significantly increased by IBOP co-stimulated with IL-4, and pretreatment with an anti-CCL11 antibody abrogated the eosinophil chemotactic activity released from IBOP/IL-4-stimulated BSMC. The effect of IBOP was also completely blocked by pretreatment with a prostanoid TP receptor-specific antagonist, AA-2414. IBOP had no effect on the expression of IL-4 receptor-alpha, or on the IL-4-induced phosphorylation of STAT6 in BSMC. In conclusion, activation of prostanoid TP receptors in a Th2-dominant microenvironment might exacerbate the eosinophilic inflammation of the airways by synthesis and release of CCL11 from BSMC.

Despite intensive research as to the pathogenesis of lipopolysaccharide (LPS)-related inflammation with coagulatory disturbance, their exacerbating factors have not been well explored. This study examined the effects of pulmonary exposure to two types of nano-sized materials (carbon nano-tubes: CNT [single-wall: SWCNT, and multi-wall: MWCNT]) on lung inflammation and consequent systemic inflammation with coagulatory disturbance induced by pulmonary exposure to LPS in mice and their cellular mechanisms in vitro. ICR male mice were divided into 6 experimental groups that intra-tracheally received the vehicle, two types of CNT (4 mg/kg), LPS (33 μ g/kg), or LPS plus either type of CNT. Twenty-four hours after treatment, both types of CNT alone induced lung inflammation with enhanced lung expression of proinflammatory cytokines, but did not synergistically exacerbate lung inflammation elicited by LPS. SWCNT significantly induced/ enhanced pulmonary permeability and hyperfibrinogenemia and reduced activated protein C in the absence or presence of LPS, whereas MWCNT did moderately. Both CNT moderately, but not significantly, elevated circulatory levels of proinflammatory cytokines and chemokines. In the presence of LPS, CNT tended to elevate the levels of the mediators with an overall trend, which was more prominent with SWCNT than with MWCNT. In vitro study showed that both CNT amplified LPS-induced cytokine production from peripheral blood monocytes. These results suggest that CNT can facilitate systemic inflammation with coagulatory disturbance, at least in part, via the activation of mononuclear cells, which is accompanied by moderate enhancement of acute lung inflammation related to LPS.

Introduction Since the invention of the pulse oximeter, physicians often or even routinely perform venous blood gas analysis (VBGA). However, it has not been generally agreed that the application of VBGA is practically meaningful in routine clinical situations such as in an ED. Methods We measured venous-arterial P co 2 difference ((v-a)P co 2) and arterial-venous pH difference ((a-v)pH), and analyzed the physiological factors that affect these differences in healthy volunteers and hyperventilation patients. Results In healthy volunteers, both (v-a)P co 2 and (a-v)pH increased during finger exercise or hyperventilation in an intensity-dependent manner. Doppler echography indicated that increases in (v-a)P co 2 and (a-v)pH during hyperventilation are induced by reduction of peripheral blood flow. Approximately 40% of patients with untreated respiratory alkalosis were found to be incorrectly diagnosed if based only on VBGA. Conclusions It must be noted that VBGA may lead to overestimation of acidosis and to underestimation of respiratory alkalosis when extremities muscles are active or patients are hyperventilating. Physicians should keep these limitations in mind when conducting VBGA.

Introduction Epidermal growth factor receptor (EGFR) mutations are strong determinants of tumor response to EGFR tyrosine kinase inhibitors in non-small cell lung cancers (NSCLCs). Currently available methods of EGFR mutation detection rely on direct sequencing. Here, we describe the use of an alternative way to screen EGFR mutations. Methods A total of 109 frozen tumor specimens from NSCLC patients were obtained. For mutational analysis of EGFR exons 18, 19, and 21, reverse transcription-polymerase chain reaction was performed on the cDNA using original primers designed for restriction fragment length polymorphism (RFLP). Results EGFR mutations were detected in 37 patients (34%) by both RFLP and direct sequencing except one case in which it was detected only by RFLP. EGFR mutations were more frequently observed to be significant by multivariate analysis in patients with adenocarcinoma (OR = 5.56), no-smoking history (OR = 4.34), and 65-year-old or younger (OR = 2.64), but not in women (OR = 1.14). Among 37 patients, 18 were treated with gefitinib and 9 responded to the treatment. One patient without any mutation responded. Conclusion RFLP is a useful method for screening EGFR mutations and can also be applied to predicting the sensitivity of NSCLC patients to EGFR-tyrosine kinase inhibitors.

Background Proteomics is increasingly leading to biomarker discovery in human disease. Epithelial lining fluid (ELF), until now only recovered indirectly, diluted in BAL fluid, is an attractive sample for lung disease proteomics. The direct recovery of undiluted ELF is now possible using a bronchoscopic microsampling (BMS) probe. In this preliminary study of anesthetized ventilated rabbits, we applied this probe to recover ELF and to analyze the resulting samples with the aim of determining their potential in lung disease biomarker discovery. Methods In order to do so, a method was devised and evaluated in preliminary experiments both for nonbronchoscopic use of the probe and for recovering undiluted ELF from probe tips. To verify the proteomic potential of the sample, the recovered ELF was separated by one-dimensional polyacrylamide gel electrophoresis, and the resulting lane was cut into multiple fractions, each of which was digested and analyzed by liquid chromatography tandem mass spectrometry. The identified proteins were then searched against Medline for association with broad categories of lung disease. Results Nonbronchoscopic use of the probe allowed successful ELF sampling and the recovery of undiluted ELF from probe tips. Proteomic analysis showed that ELF contains many proteins that have already been reported as being associated with lung disease as well as proteins potentially correlated with lung disease. Conclusions This preliminary study of undiluted ELF, as recovered by the BMS probe, shows that it may be an ideal sample for lung proteomics. The potential application of this sampling technique in various lung diseases will need to be confirmed by future studies.

Background and objective: Cathepsin S is involved in the pathogenesis of COPD in murine models overexpressing interferon (IFN)-γ and IL-13. It is widely accepted that genetic factors partly influence susceptibility to COPD; however, the association of genetic polymorphisms in the cathepsin S gene with COPD has not been reported previously. In this study, functional polymorphisms in the 5′-flanking region of the human cathepsin S gene were identified and their association with COPD phenotypes was investigated. Methods: Genetic polymorphisms were detected by single strand conformation polymorphism analysis and direct sequencing. Genotypes and haplotypes were determined using amplification refractory mutation systems and single strand conformation polymorphism analysis. A promoter-reporter assay was used to compare transcriptional activities between the haplotypes on stimulation with IFN-γ. Haplotype analysis was performed on 204 smoking and 104 non-smoking Japanese subjects. Results: Four novel polymorphisms were detected in the 5′-flanking region and three major haplotypes were identified. The haplotypes were associated with distinct promoter activities on stimulation with IFN-γ and there was a trend indicating that higher transcriptional activity was linked to less pulmonary emphysema. Conclusion: Novel functional polymorphisms were identified in the cathepsin S gene, which has a possible association with pulmonary emphysema in the Japanese population.

In order to examine whether ATP-sensitive potassium channel Kir6.2 is involved in hypoxic ventilatory responses, especially in hypoxic ventilatory depression (HVD), and whether the involvement shows age-dependence, we measured the hypoxic ventilatory response in the Kir6.2-knockout mouse (Kir6.2−/−) in an unanesthetized unrestrained state by means of pressure plethysmography in the 2nd and 4th postnatal weeks, and compared the response with that of its wild type counterpart, the C57BL6/J mouse. In the 4th postnatal week, but not in the 2nd week, the Kir6.2−/− exhibited a larger and longer initial augmentation and a weaker subsequent depression of respiratory frequency and ventilation in response to hypoxia (FIO 2 = 0.12 in N 2 ). These findings suggest that Kir6.2 is involved in HVD of the mouse at a certain point during the postnatal development.

Interleukin-6 (IL-6) is known to be involved in the pathogenesis of various inflammatory diseases, but its role in bleomycin (BLM)-induced lung injury and subsequent fibrotic changes remains to be determined. We evaluated the role of IL-6 in the lung inflammatory changes induced by BLM using wild-type (WT) and IL-6-deficient (IL-6(-/-)) mice. The mice were treated intratracheally with 1 mg/kg BLM and killed 2, 7, or 21 days later. Lung Inflammation in the acute phase (Days 2 and 7) was assessed by differential cell counts in bronchoalveolar lavage (BAL) fluid and cytokine levels in the lung. Lung fibrotic changes were evaluated on Day 21 by histopathology and collagen assay. On Day 2, BLM administration induced significant increases in the numbers of total cells, macrophages, and neutrophils in BAL fluid, which were attenuated in IL-6(-/-) mice (P0.05). Lung pathology also showed inflammatory cell accumulation, which was attenuated in the IL-6(-/-) mice compared with WT mice. In WT mice, elevated levels of TGF-beta(1) and CCL3 were observed 2 and 7 days after BLM challenge, respectively. On Day 7, BLM-induced inflammatory cell accumulation did not differ between the genotypes. Lung pathology 21 days after BLM challenge revealed significant fibrotic changes with increased collagen content, which was attenuated in IL-6(-/-) mice. Although the TGF-beta(1) level in the lung did not differ between the genotypes on Day 21, CCL3 was significantly lower in IL-6(-/-) mice. These results indicate that IL-6 may play an important role in the pathogenesis of BLM-induced lung injury and subsequent fibrotic changes.

Besides lowering cholesterol, statins exert multiple effects, such as anti-inflammatory activity and improvement of endothelial cell function. We examined whether simvastatin (SS) protects against the development of elastase-induced pulmonary emphysema in mice by using mean linear intercepts of alveoli (Lm) as a morphometric parameter of emphysema. After injection of intratracheal elastase on day 0, C57BL/6 mice were treated daily with SS (SS+ group) or PBS (SS− group) for 2 wk. A 21% decrease in Lm on day 7 was observed in the SS+ group vs. the SS− group. Anti-inflammatory effects of SS were observed as a decrease in percentage of neutrophils up to day 3, and in hydroxyproline concentration on day 3, in bronchoalveolar lavage fluid (BALF). SS also increased the number of proliferating cell nuclear antigen (PCNA)-positive alveolar epithelial cells between days 3 and 14. To confirm the role of statins in promoting proliferation of alveolar cells, mice were treated with SS (SS+) vs. PBS (SS−) for 12 days, starting 3 wk after elastase administration. After SS treatment, Lm decreased by 52% and PCNA-positive alveolar epithelial cells increased compared with the SS− group. Concentrations of vascular endothelial growth factor in BALF and endothelial nitric oxide synthase protein expression in pulmonary vessels tended to be higher in the SS+ group vs. the SS− group in this protocol. In conclusion, SS inhibited the development of elastase-induced pulmonary emphysema in mice. This therapeutic effect was due not only to anti-inflammation but also to the promotion of alveolar epithelial cell regeneration, partly mediated by restoring endothelial cell functions.

Background and objective: Vascular endothelial growth factor (VEGF) is known to contribute to the development of pulmonary oedema, and has been suggested to have a protective role against lung injury. To determine the role of VEGF in acute lung injury (ALI) and ARDS, VEGF levels were measured in lung epithelial lining fluid (ELF) collected from patients with ALI/ARDS. Methods: Forty patients with ALI/ARDS underwent bronchoscopic microsampling to collect ELF on days 0 (onset of ALI/ARDS), 1, 3, 5, 7 and 10, unless the patient was extubated or had died. Twelve patients, who underwent bronchoscopy for examination of small, peripheral pulmonary nodules, served as controls. Results: The initial (day 0) levels of VEGF in ELF of the ALI/ARDS patients who survived and those who did not were 5.5 ng/mL (IQR: 2.3–19.7) and 1.7 ng/mL (IQR: 0.0–6.4), respectively. On days 0, 5, 7 and 10, the VEGF levels in ELF were significantly greater in survivors than in non-survivors (P

Background and objective: BAL is an established technique for measuring antibiotic concentrations in the epithelial lining fluid (ELF) of the bronchiolar-alveolar regions. However, the results may not reflect concentrations in bronchial regions. Bronchoscopic microsampling (BMS) is a technique for repeated sampling of bronchial ELF. The objective of the present study was to determine the time versus concentration profile of clarithromycin and its active metabolite, 14-hydroxy-clarithromycin, in bronchial ELF, as determined by BMS. Methods: BMS was performed at 1, 2, 3, 5 and 10 h after a single oral administration of 200 mg clarithromycin in five healthy volunteers. BAL was performed 3 h after administration to determine clarithromycin concentrations in alveolar ELF and alveolar macrophages (AM). Results: The maximum concentration (Cmax) of clarithromycin was 0.36 ± 0.07 mg/L in serum and 1.44 ± 0.49 mg/L in bronchial ELF (P

Background: A new bronchoscopic microsampling (BMS) probe has recently been developed, which allows the minimally invasivecollection of epithelial lining fluid (ELF) simultaneously from multiple sites. While the number of immunocompetent patients with pulmonary Mycobacterium avium complex(MAC) disease is growing, the precise pathogenesis of the disease remains to be clarified. Objectives: This study aimed to measure biochemical markers in the ELF of patients with pulmonary MAC disease using BMS. Methods: We studied 8 healthy volunteers (controls) and 8 patients with pulmonary MAC disease. In the patients, ELF was collected from visibly abnormal (involved) and normal (uninvolved) sites using high-resolution computed tomography (HRCT). Results: The concentration of IFN-γ (means ± SEM) was significantly higher in patient ELF sampled from involved (26.62 ± 9.66 pg/ml) than from uninvolved sites (5.33 ± 1.44 pg/ml; p < 0.05) or from normal controls (Conclusions: The specific inflammatory reaction caused by MAC infection might not be limited to sites that were visibly abnormal on HRCT. Although further improvement has to be considered, BMS may help to increase the understanding of the pathogenesis of various respiratory disorders.

In this report, we describe two cases of small-cell lung cancer (SCLC) presenting with fulminant hepatic failure as the initial manifestation. Neither of the patients had a previous history of liver disease, evidence of viral infection, or exposure to hepatotoxins. Both presented with hepatomegaly and an increase in the serum LDH level. Although they had a history of smoking and respiratory symptoms such as dyspnea on exertion, their most striking initial symptoms were those of hepatic failure, which made it difficult to suspect the underlying primary lung cancer. In one case, contrast-enhanced CT of the abdomen revealed hypovascular nodules that were not visible on plain CT. After a rapidly deteriorating course, both patients died, 8 and 13 days after admission, respectively. Autopsy revealed diffuse infiltration of the hepatic sinusoids and extensive replacement of the liver parenchyma by metastatic small-cell carcinoma. We conclude that metastatic SCLC should be considered in the differential diagnosis in patients of advanced age with a smoking history presenting with rapidly progressive liver failure associated with hepatomegaly and elevation of the serum LDH.

Respiratory RNA viruses responsible for the common cold often worsen airway inflammation and bronchial responsiveness, two characteristic features of human asthma. We studied the effects of dsRNA, a nucleotide synthesized during viral replication, on airway inflammation and bronchial hyperresponsiveness in murine models of asthma. Intratracheal instillation of poly I:C, a synthetic dsRNA, increased the airway eosinophilia and enhanced bronchial hyperresponsiveness to methacholine in OVA-sensitized, exposed rats. These changes were associated with induction of cyclooxygenase-2 (COX-2) expression and COX-2-dependent PGD2 synthesis in the lungs, particularly in alveolar macrophages. The direct intratracheal instillation of PGD2 enhanced the eosinophilic inflammation in OVA-exposed animals, whereas pretreatment with a dual antagonist against the PGD2 receptor-(CRTH2) and the thromboxane A2 receptor, but not with a thromboxane A2 receptor-specific antagonist, nearly completely eliminated the dsRNA-induced worsening of airway inflammation and bronchial hyperresponsiveness. CRTH2-deficient mice had the same degree of allergen-induced airway eosinophilia as wild-type mice, but they did not exhibit a dsRNA-induced increase in eosinophil accumulation. Our data demonstrate that COX-2-dependent production of PGD2 followed by eosinophil recruitment into the airways via a CRTH2 receptor are the major pathogenetic factors responsible for the dsRNA-induced enhancement of airway inflammation and responsiveness.

The aim of this study was to establish an animal experimental model of pulmonary stereotactic irradiation and clarify the morphological patterns of pulmonary radiation injury with computed tomography and the histopathological features. Tiny spherical regions in the lungs of seven anesthetized rabbits were irradiated stereotactically with a single fractional dose of 21–60 Gy. Subsequently, the irradiated lungs were observed biweekly with computed tomography (CT) for 24 weeks. Radiation injury of the lung was examined histopathologically in one specimen. Localized hypodense changes were observed 7–15 weeks after irradiation in three rabbits irradiated with 60 Gy, and the findings persisted beyond that time. The electron density ratios in the lung fields obtained from the CT images were shown to be decreasing, corresponding to the hypodensity changes. No clear increased density opacity was observed in any rabbit in the 60-Gy irradiated group. Severe localized fibrotic change was observed in the histopathological specimens. Specific localized hypodensity changes were found in only three rabbits irradiated with 60 Gy, the highest dose we employed.

Prolonged exposure to hyperoxia causes lung inflammation, but the role of Toll-like receptor 4 (TLR4) in hyperoxia-induced signal transduction remains unclear.We evaluated neutrophil accumulation, signal transduction and cytokine production during hyperoxia, comparing TLR4 mutant (C3H/HeJ) and wild type (C3H/HeN) mice.The mice were exposed to 80% oxygen in a hyperoxic chamber for 0 (control), 48, or 96 h. After the exposure, bronchoalveolar lavage (BAL) was performed for differential cell counting and cytokine measurement. In lung homogenate, activation of NF-kappaB and STAT1 was also examined.In C3H/HeJ mice, hyperoxia-induced neutrophil accumulation in BAL fluid was significantly decreased compared with C3H/HeN. Hyperoxia for 96 h caused NF-kappaB translocation in C3H/HeN mice, which was significantly attenuated in C3H/HeJ mice (p0.05). In contrast, STAT1 activation occurred as early as after 48 h of oxygen exposure, which did not differ between the two strains. The levels of TNF-alpha, IL-6, and KC in BAL fluid were increased after oxygen exposure, which was suppressed by the lack of TLR4 signaling.These results suggest that TLR4-dependent NF-kB activation may be an important process of the upregulation of proinflammatory mediators and subsequent neutrophil accumulation into the lung during hyperoxia.

The diagnosis of pneumocystis pneumonia (PCP) is difficult because it requires microscopic examination to identify pneumocystis from induced sputum or BAL fluid.To evaluate the usefulness of four serum markers-lactate dehydrogenase (LDH), (1--3) beta-D-glucan (beta-D-glucan), KL-6, and C-reactive protein (CRP)-in the diagnosis of PCP.Case-control retrospective study.We reviewed the medical records of 295 consecutive patients who underwent BAL for the diagnosis of PCP. Differential cell counts in BAL fluid and serum levels of LDH, beta-D-glucan, KL-6, and CRP were examined. Oxygenation index was determined using arterial oxygen tension and inspiratory oxygen concentration.Based on the microscopic examination of BAL fluid, 57 patients were PCP positive and 238 patients were PCP negative. There were no significant differences in cell count or differentials in BAL fluid between the positive and negative cases. Serum levels of LDH, beta-D-glucan, and KL-6 were significantly higher in PCP-positive patients (p0.01). Receiver operating characteristic curves suggest that beta-D-glucan was the most reliable indicator. The cut-off level of beta-D-glucan was estimated to be 31.1 pg/mL, with which the positive and negative predictive values were 0.610 and 0.980, respectively. In PCP-positive patients, the oxygenation index was decreased and correlated with LDH. Both LDH and beta-D-glucan levels were correlated with the proportion of neutrophils in BAL fluid.Serum beta-D-glucan is a reliable marker for the diagnosis of PCP. Since BAL procedure is invasive, measuring beta-D-glucan should be considered as a primary modality for a diagnosis of PCP, especially for patients with severe respiratory failure.

Bronchoalveolar lavage (BAL) is an established technique for measuring antibiotic concentrations in the epithelial lining fluid (ELF) of the bronchiolar and alveolar regions; however, the results may not reflect concentrations in bronchial regions. Bronchoscopic microsampling (BMS) is a technique that makes it possible to obtain multiple samples from bronchial ELF.BMS and BAL were used to analyze the pharmacokinetics of gatifloxacin in bronchial ELF from healthy young adult subjects and adult patients with chronic bronchitis.Bronchial ELF samples were obtained by BMS at 1, 2, 3, 4, 6, 10, and 24 hours after administration of a single oral dose of gatifloxacin 200 mg in healthy young adult (aged 20-25 years) subjects, and at 1, 2, 4, and 10 hours after a single dose in patients with chronic bronchitis (agedor =20 years). At least 1 month after the initial BMS, alveolar (BAL) and bronchial (BMS) ELF samples were obtained from another group of healthy subjects 2 hours after administration of a single oral dose of gatifloxacin 200 mg for comparison of gatifloxacin concentrations in samples obtained by the 2 techniques.Bronchial ELF samples were obtained from 8 healthy subjects and 5 patients with chronic bronchitis; alveolar ELF samples were obtained from a separate group of 5 healthy subjects. For the healthy subjects, the mean (SD) AUC(0-24) in serum and bronchial ELF, corrected for mg/kg doses, was 4.6 (1.1) and 7.6 (3.5) mg x h/L, respectively. In the patients with chronic bronchitis, the AUC(0-10) in serum and bronchial ELF, corrected for mg/kg doses, was 3.9 (0.8) and 4.1 (1.5) mg x h/L. The C(max) in serum and bronchial ELF, corrected for mg/kg doses, was 0.6 (0.2) and 1.4 (0.8) mg/L in healthy subjects and 0.7 (0.2) and 0.7 (0.2) mg/L in patients with chronic bronchitis. In healthy subjects, the C(max) and AUC(0-24) were significantly higher in bronchial ELF than in serum (both, P0.05). Gatifloxacin concentrations were significantly lower in bronchial ELF obtained by BMS than in alveolar ELF obtained by BAL (P0.05).Based on the findings of this study in small numbers of healthy young adult volunteers and patients with chronic bronchitis, BMS appears to be a promising method for measuring drug concentrations and determining the pharmacokinetic profile of gatifloxacin in bronchial ELF. Additional studies are needed to correlate measured concentrations obtained by BMS with clinical and/or microbiologic outcomes in larger populations.

Extramedullary hematopoiesis predominantly occurs in the liver, spleen, and lymph nodes with hemolytic anemia. Occurrence with osteopetrosis tarda in the paravertebral region is very rare. We discuss the examination of the third known case of paravertebral extramedullary hematopoiesis arising with osteopetrosis tarda.

It has been demonstrated that cytokines play a central role in mediating host responses to endotoxemia. In this study, we examined and compared the effects of E. coli sepsis and the cytokines

Although the endothelial expression of various adhesion molecules substantially differs between pulmonary microvessels, their importance for neutrophil and lymphocyte sequestration in ventilator-induced lung injury (VILI) has not been systematically analyzed. We investigated the kinetics of polymorphonuclear cells (PMN) and mononuclear cells (MN) in the acinar microcirculation of the isolated rat lung with VILI by real-time confocal laser fluorescence microscopy, with or without inhibition of ICAM-1, VCAM-1, or P-selectin by monoclonal antibodies (MAb). Adhesion molecules in each microvessel were estimated by intravital fluorescence microscopy or immunohistochemical staining. In high tidal volume-ventilated lungs, 1) ICAM-1, VCAM-1, and P-selectin were differently upregulated in venules, arterioles, and capillaries; 2) venular PMN rolling was improved by inhibition of ICAM-1, VCAM-1, or P-selectin, whereas arteriolar PMN rolling was improved by ICAM-1 or VCAM-1 inhibition; 3) capillary PMN entrapment was ameliorated only by anti-ICAM-1 MAb; and 4) MN rolling in venules and arterioles and MN entrapment in capillaries were improved by ICAM-1 and VCAM-1 inhibition. In conclusion, the contribution of endothelial adhesion molecules to abnormal leukocyte behavior in VILI-injured microcirculation is microvessel and leukocyte specific. ICAM-1- and VCAM-1-dependent, but P-selectin-independent, arteriolar PMN rolling, which is expected to reflect the initial stage of tissue injury, should be taken as a phenomenon unique to ventilator-associated lung injury.

The whole gene deletion CYP2A6*4 , the defect of the main nicotine oxidase, contributes to limiting lifelong and daily cigarette consumption. However, the effects on smoking habits of CYP2A6*7 and *9 , two major functional polymorphisms common in Asian populations, have not been reported. The present study examined the relationship between polymorphisms *4 , *7 and *9 with the smoking habits of 200 Japanese smokers who visited the Keio University Hospital (Tokyo, Japan). The allele frequencies of *1 (wild type), *4 , *7 and *9 were 52, 17, 11 and 20%, respectively. When the three polymorphisms were considered simultaneously, the percentages of homozygous wild type, heterozygote, and homozygous mutants and compound heterozygotes were 26.0, 52.5 and 21.5%, respectively. Homozygous mutants and compound heterozygotes (n = 43) smoked fewer cigarettes daily than heterozygotes (n = 105) and homozygous wild-type individuals (n = 52). Smokers with *7 / *7 , *9/*9 or *7 / *9 had lower daily cigarette consumption than smokers with *1/*1 . In conclusion, polymorphisms *4 , *7 and *9 of CYP2A6 were detected in approximately three out of four Japanese smokers, and their daily cigarette consumption was genetically modulated by these functional polymorphisms.

Diesel exhaust particles (DEP) are associated with respiratory disease and exposure to diesel exhaust induces an inflammatory response associated with marked leukocytic infiltration in the lung. This study examined whether neutrophils are activated by the active component of DEP (methanol extract of DEP [me-DEP]). The authors demonstrated that neutrophils exposed to me-DEP had increased levels of the f-actin content, the surface expression of adhesion molecules, and the release of interleukin (IL-8) and leukotriene B4 (LTB4), superoxide, and matrix metalloproteinase (MMP-9). Thus, the author conclude that DEP exposure activates neutrophils and that these activated neutrophils could contribute to the adverse respiratory health effects associated with DEP and to the pathogenesis of chronic inflammatory lung diseases.

A small GTPase, Rho, plays key roles in cell adhesion, motility, and contraction after stimulation. Among Rho effectors isolated, the family of Rho-associated coiled-coil-forming protein kinases (ROCK) is implicated in Rho-mediated cell adhesion and smooth muscle contraction. The effect of a specific inhibitor of ROCK, Y-27632, was evaluated in a murine model of acute lung injury induced by intravenous injection of Escherichia coli endotoxin (lipopolysaccharide [LPS]). Lung edema was evaluated by measuring extravascular leakage of radio-labeled serum albumin, and neutrophil emigration into the lung parenchyma by morphometric observation and measuring myeloperoxidase activity. Pretreatment with Y-27632 attenuated both lung edema and neutrophil emigration after LPS. We also measured albumin transfer through cultured endothelial cell monolayers on a porous filter. Tumor necrosis factor-alpha significantly increased albumin transfer, which was attenuated by pretreatment with Y-27632. Fluorescence microscopy revealed that morphologic changes in endothelial cells induced by tumor necrosis factor-alpha were inhibited by Y-27632. In contrast, the increased fraction of neutrophils with polymerized actin after formyl-methionyl-leucyl-phenylalanine was not altered by Y-27632. These data suggest that ROCK may play an important role in the pathogenesis of LPS-induced lung injury and that ROCK inhibition could attenuate cytoskeletal rearrangement of endothelial cells, leading to decreased neutrophil emigration into the lung parenchyma.

The effects of tacrolimus on murine acute lung injury were tested, especially in comparison to dexamethasone. Acute lung injury was induced by intratracheal instillation of bleomycin. Oral tacrolimus significantly improved survival rates of bleomycin-exposed mice, while cyclosporin A or dexamethasone did not. After instillation of bleomycin (day 0), a migration of neutrophils into alveolar spaces peaked on day 3, with concomitant increases of chemokines. On day 6, marked morphological changes in the lungs were observed. All these changes were significantly inhibited by tacrolimus. Furthermore, DNA ladder and immunohistochemical analyses of lungs showed that apoptosis of lung cells appeared on day 6 and was abolished only by the treatment of tacrolimus. These results suggest that both anti-inflammatory and anti-apoptotic action of tacrolimus contribute to improvement of bleomycin-induced acute lung injury.

This study was performed to examine the putative role of high mobility group box (HMGB) protein in the pathogenesis of acute lung injury (ALI). Observations were made (1) in 21 patients who were septic with ALI and 15 patients with normal lung function and (2) in a mouse model 24 hours after intratracheal instillation of lipopolysaccharide (LPS). The concentrations of HMGB1 were increased in plasma and lung epithelial lining fluid of patients with ALI and mice instilled with LPS. LPS-induced ALI was mitigated by anti-HMGB1 antibody. Although this protein was not detected in the plasma of control humans or mice, the concentrations of HMGB1 in lung epithelial lining fluid or in bronchoalveolar lavage fluid were unexpectedly high. The nuclear expression of HMGB1 was apparent in epithelial cells surrounding terminal bronchioles in normal mice, whereas its nuclear and cytoplasmic expression was observed in alveolar macrophages in LPS-instilled mice. Lung instillation of HMGB2 did not cause as much inflammation as HMGB1. Extracellular HMGB1 may play a key role in the pathogenesis of clinical and experimental ALI. However, its expression in normal airways is noteworthy and suggests that it also plays a physiologic role in the lung.

Mast cell-derived prostaglandin D(2) (PGD(2)) is one of the essential modulators of eosinophilic airway inflammation in asthma and allergic rhinitis. Two G protein-coupled receptors for PGD(2), prostaglandin D(2) receptor (DP) and chemoattractant receptor-homologous molecule expressed on Th(2) cells (CRTH2), are both expressed on the surface of eosinophils, and CRTH2 has been demonstrated to mediate PGD(2)-induced eosinophil mobilization in vitro. However, it has not yet been determined whether PGD(2) and its receptors mediate in vivo eosinophil trafficking into the airways or other organs. We demonstrated that intratracheal administration of PGD(2) in rats pretreated with systemic interleukin-5 (IL-5) injection induced marked airway eosinophilia, determined by the differential counts of cells in bronchoalveolar lavage (BAL) fluid and lung histology, within 2 h. Systemic IL-5 alone significantly increased the number of eosinophils in the peripheral blood but showed no effect on airway eosinophilia. Three CRTH2-specific agonists (13,14-dihydro-15-keto-PGD(2), 11-deoxy-11-methylene-15-keto-PGD(2), and indomethacin) demonstrated equivalent induction of BAL eosinophilia to that of PGD(2), but a DP agonist (BW 245C [5-(6-carboxyhexyl)-1-(3-cyclohexyl-3-hydroxypropyl)-hydantoin]) or a thromboxane A(2) receptor (TP) agonist ([1S-1alpha,2beta(5Z), 3alpha(1E,3R*),4alpha)]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxabicyclo-[2.2.1]heptan-2-yl]-5-heptenoic acid) showed no effect. PGD(2) or CRTH2 agonist-induced BAL eosinophilia was almost completely inhibited by pretreatment with a CRTH2/TP antagonist, ramatroban [BAY-u3405; (+)-(3R)-3-(4-fluorobenzenesulfonamido)-1,2,3,4-tetra-hydrocarbazole-9-propionic acid], whereas a TP-specific antagonist, SQ29,548 (5-heptenoic, 7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]-hept-2-yl]-[1S-[1alpha,2alpha(Z),3alpha,4alpha]]), or a DP-specific antagonist, BW A868C [3-benzyl-5-(6-carboxyhexyl)-1-(2-cyclohexy-2-hydroxyethylamino)-hydantoin], did not inhibit the effects of PGD(2). These results suggest that CRTH2 plays a significant role in the eosinophil trafficking from the bloodstream into the airways in PGD(2)-related airway inflammation.

KL-6 is a pulmonary epithelial mucin more prominently expressed on the surface membrane of alveolar type II cells when these cells are proliferating, stimulated, and/or injured. We hypothesized that high levels of KL-6 in epithelial lining fluid and plasma would reflect the severity of lung injury in patients with acute lung injury (ALI). Epithelial lining fluid was obtained at onset ( day 0) and day 1 of acute respiratory distress syndrome (ARDS)/ALI by bronchoscopic microsampling procedure in 35 patients. On day 0, KL-6 and albumin concentrations in epithelial lining fluid were significantly higher than in normal controls ( P < 0.001), and the concentrations of KL-6 in epithelial lining fluid ( P < 0.002) and in plasma ( P < 0.0001) were higher in nonsurvivors than in survivors of ALI/ARDS. These observations were corroborated by the immunohistochemical localization of KL-6 protein expression in the lungs of nonsurvivors with ALI and KL-6 secretion from cultured human alveolar type II cells stimulated by proinflammatory cytokines. Because injury to distal lung epithelial cells, including alveolar type II cells, is important in the pathogenesis of ALI, the elevation of KL-6 concentrations in plasma and epithelial lining fluid could be valuable indicators for poor prognosis in clinical ALI.