近藤 征史

Background: Several pre-clinical and clinical studies suggest a potential predictive role of epidermal growth factor receptor (EGFR) mutation in responsiveness to cytotoxic chemotherapy. The aim of this phase II study was to evaluate the efficacy and safety of pemetrexed-carboplatin combination as first-line chemotherapy in advanced non-squamous non-small cell lung cancer (NSCLC) limited to EGFR-wild-type cases. Patients and Methods: In this single-arm, multicenter clinical trial, patients received pemetrexed (500 mg/m(2)) and carboplatin (area under the curve=6) intravenously on day 1 every 3 weeks for three to six cycles. The objective response rate (ORR) was the primary end-point; secondary end-points included the disease control rate (DCR), progression-free survival (PFS), overall survival (OS) and safety. Results: A total of 54 patients were enrolled and 53 patients received therapy. No complete response was observed and partial response was observed in 19 (35.8%) cases, resulting in an ORR of 35.8% [95% confidence interval (CI)=23.1-50.2%]. Stable disease was observed in 20 (37.7%) patients and therefore the DCR was 73.6% (95% CI=59.7-84.7%). The median PFS was 5.4 months (95% CI=4.1-6.8 months) and the median OS was 12.7 months (95% CI=9.3-16.1 months). Treatment-related grade 3 or 4 hematological toxicities were neutropenia, leukopenia, anemia and thrombocytopenia in 35.8%, 11.3%, 30.2%, and 32.1% of patients, respectively. No grade 3 febrile neutropenia was observed, and grade 3 or 4 non-hematological toxicities were mild. There was no treatment-related death. Conclusion: The pemetrexed-carboplatin combination was effective and well-tolerated in patients with EGFR-wild-type non-squamous NSCLC (UMIN-CTR number: UMIN000003393).

Rheumatoid arthritis (RA)-related pulmonary disorders specifically airway abnormalities and interstitial pneumonia (IP) are important extra-articular manifestations. The forced oscillation technique (FOT) is a useful method to assess respiratory impedance, respiratory resistance (Rrs) and reactance (Xrs), at different oscillatory frequencies during tidal breathing. The aim of this study was to characterize the respiratory mechanics of patients with RA and to relate them to parameters of the pulmonary function test and findings of chest CT images. Respiratory impedance of RA patients (n = 69) was measured as a function of frequency from 4 to 36 Hz using the FOT device and compared with that of healthy subjects (n = 10). Data were retrospectively reviewed. Patients were female-dominant (60.9 %) and 95.7 % had abnormal CT findings including airway and parenchymal abnormalities. Thirty-seven of 69 patients (53.6 %) were smokers. Rrs was significantly frequency-dependent in RA patients but not in the healthy subjects. Xrs were significantly frequency-dependent in both RA and healthy groups. Rrs was significantly higher during an expiratory phase in both RA and healthy groups. Xrs was significantly lower (more negative) during an expiratory phase than that during an inspiratory phase in RA patients but not in healthy subjects. Xrs of the RA group was significantly more negative than that of the normal control. There was no difference in impedance parameters between the airway lesion dominant (n = 27) and IP dominant groups (n = 23) in the RA group. The impedance parameters of the RA group significantly correlated with most parameters of the pulmonary function test. In pulmonary function test results, % of the predicted value for forced expiratory flow from 25 to 75 % of forced vital capacity was significantly lower and % of the predicted value for diffusing capacity of the lung for carbon monoxide was higher in the airway lesion dominant group than those in the IP dominant group. Krebs von den Lungen-6, a serum indicator of IP, was significantly higher in the IP group than that in the airway lesion dominant group. Taken together, the impedance results reflect abnormalities in pulmonary functions and structures in patients with RA.

Objective Endobronchial ultrasonography with a guide sheath (EBUS-GS) and virtual bronchoscopic navigation (VBN) improves the diagnostic yield in patients with peripheral pulmonary lesions (PPLs). Most previous reports on EBUS-GS-guided transbronchial biopsy (TBB) have included patients with benign and malignant diseases. We aimed to determine the factors that predicted a successful diagnosis by EBUS-GS-guided TBB diagnostic in patients with small peripheral lung cancer, with a focus on the high-resolution computed tomography (HRCT) findings before bronchoscopy. Methods We retrospectively reviewed the medical records of 173 consecutive patients with 175 small (<= 30 mm) PPLs who were diagnosed with primary lung cancer between June 2010 and October 2013 at Nagoya University Hospital. All patients underwent EBUS-GS-guided TBB with VBN using a ZioStation computer workstation (Ziosoft, Osaka, Japan). We analyzed the patient characteristics, HRCT findings, diagnostic yield, and the diagnostic factors in small peripheral lung carcinoma. Results The EBUS probe position was within the PPL in 83 of the 175 lesions (47%) and 112 (64.0%) cases were successfully diagnosed by EBUS-GS-guided TBB. A univariate analysis revealed that the following factors were associated with a significantly higher diagnostic yield: CT bronchus sign positivity, a lesion of >20 mm in diameter, a solid nodule, and a probe position that was within the lesion. The following factors were not significant: the lesion location, the number of biopsies, and the lung cancer histology. A multivariate analysis revealed that the following factors significantly affected the diagnostic yield: CT bronchus sign positivity [ odds ratio (OR) =2.479]; a probe position that was within the lesion (OR=2.542); and a solid nodule (OR=2.304). Conclusion The significant factors that were significantly associated with a successful diagnosis using EBUS-GS-guided TBB in small peripheral lung carcinoma were as follows: CT bronchus sign positivity, a solid nodule, and a probe position that was within the lesion.

Both pro- and anti-oncogenic roles of miR-221 and miR-222 microRNAs are reported in several types of human cancers. A previous study suggested their oncogenic role in invasiveness in lung cancer, albeit only one cell line (H460) was used. To further evaluate involvement of miR-221 and miR-222 in lung cancer, we investigated the effects of miR-221 and miR-222 overexpression on six lung cancer cell lines, including H460, as well as one immortalized normal human bronchial epithelial cell line, HBEC4. miR-221 and miR-222 induced epithelial-to-mesenchymal transition (EMT)-like changes in a minority of HBEC4 cells but, unexpectedly, both the microRNAs rather suppressed their invasiveness. Consistent with the prior report, miR-221 and miR-222 promoted growth in H460; however, miR-221 suppressed growth in four other cell lines with no effects in one, and miR-222 suppressed growth in three cell lines but promoted growth in two. These are the first results to show tumor-suppressive effects of miR-221 and miR-222 in lung cancer cells, and we focused on clarifying the mechanisms. Cell cycle and apoptosis analyses revealed that growth suppression by miR-221 and miR-222 occurred through intra-S-phase arrest and/or apoptosis. Finally, lung cancer cell lines transfected with miR-221 or miR-222 became more sensitive to the S-phase targeting drugs, possibly due to an increased S-phase population. In conclusion, our data are the first to show tumor-suppressive effects of miR-221 and miR-222 on lung cancer, warranting testing their potential as therapeutics for the disease.

© 15CBMS-0001. In the present study, we propose immuno-wall lab-on-a-chip companion diagnostic devices for epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKI) such as erlotinib and gefitinib. The lysates of cytological samples including pleural effusion in lung cancer patients were successfully analyzed within 20 minutes. This is the first experiment demonstrating the detection of mutated EGFRs in the pleural effusion by microdevices. Our devices have a great potential to become the next generation companion diagnostic devices which overcome the problems of currently available methods.

Airway smooth muscle (ASM) cells within the airway walls are continually exposed to mechanical stimuli, and exhibit various functions in response to these mechanical stresses. ATP acts as an extracellular mediator in the airway. Moreover, extracellular ATP is considered to play an important role in the pathophysiology of asthma and chronic obstructive pulmonary disease. However, it is not known whether ASM cells are cellular sources of ATP secretion in the airway. We therefore investigated whether mechanical stretch induces ATP release from ASM cells. Mechanical stretch was applied to primary human ASM cells cultured on a silicone chamber coated with type I collagen using a stretching apparatus. Concentrations of ATP in cell culture supernatants measured by luciferin-luciferase bioluminescence were significantly elevated by cyclic stretch (12 and 20% strain). We further visualized the stretch-induced ATP release from the cells in real time using a luminescence imaging system, while acquiring differential interference contrast cell images with infrared optics. Immediately after a single uniaxial stretch for 1 second, strong ATP signals were produced by a certain population of cells and spread to surrounding spaces. The cyclic stretch-induced ATP release was significantly reduced by inhibitors of Ca2+-dependent vesicular exocytosis, 1,2-bis(o-aminophenoxy) ethane-N, N, N', N'-tetraacetic acid tetraacetoxymethyl ester, monensin, N-ethylmaleimide, and bafilomycin. In contrast, the stretch-induced ATP release was not inhibited by a hemichannel blocker, carbenoxolone, or blockade of transient receptor potential vanilloid 4 by short interfering RNA transfection or ruthenium red. These findings reveal a novel property of ASM cells: mechanically induced ATP release may be a cellular source of ATP in the airway.

One cause of progressive pulmonary fibrosis is dysregulated wound healing after lung inflammation or damage in patients with idiopathic pulmonary fibrosis and severe acute respiratory distress syndrome. The mechanical forces are considered to regulate pulmonary fibrosis via activation of lung fibroblasts. In this study, the effects of mechanical stretch on the intracellular Ca2+ concentration ([Ca2+](i)) and ATP release were investigated in primary human lung fibroblasts. Uniaxial stretch (10-30% in strain) was applied to fibroblasts cultured in a silicone chamber coated with type I collagen using a stretching apparatus. Following stretching and subsequent unloading, [Ca2+](i) transiently increased in a strain-dependent manner. Hypotonic stress, which causes plasma membrane stretching, also transiently increased the [Ca2+](i) The stretch-induced [Ca2+](i) elevation was attenuated in Ca2+-free solution. In contrast, the increase of [Ca2+](i) by a 20% stretch was not inhibited by the inhibitor of stretch-activated channels GsMTx-4, Gd3+, ruthenium red, or cytochalasin D. Cyclic stretching induced significant ATP releases from fibroblasts. However, the stretch-induced [Ca2+](i) elevation was not inhibited by ATP diphosphohydrolase apyrase or a purinergic receptor antagonist suramin. Taken together, mechanical stretch induces Ca2+ influx independently of conventional stretch-sensitive ion channels, the actin cytoskeleton, and released ATP. (C) 2014 Elsevier Inc. All rights reserved.

Idiopathic pulmonary fibrosis (IPF) is associated with an independent increased risk of lung carcinogenesis. The benefit of chemotherapy for extensive-stage small-cell lung cancer (ED-SCLC) in cases of IPF remains unknown. This study was conducted to elucidate the efficacy of chemotherapy for ED-SCLC in patients with IPF. This was a retrospective observational study of ED-SCLC patients with IPF (all with distant metastasis) who received systemic chemotherapy. The response rate, toxicity, overall survival, and progression-free survival (PFS) were investigated. Eleven patients treated with chemotherapy between January 2005 and December 2011 were the subjects of this study. The overall response rate with the 1st regimen was 63.6 %. The median overall survival was 7.0 months, and the median PFS was 4.7 months. Our results suggest that ED-SCLC patients with IPF may benefit from chemotherapy. A prospective study will be needed to confirm this in the future.

Background: Newer more advanced techniques in bronchoscopy may require longer procedure times, although a standard protocol for sedation during prolonged bronchoscopy has not yet been defined. Methods: We designed a prospective, non-randomized, single-arm study (UMIN trial number 000003971) using patient questionnaires and vital sign monitoring to assess the efficacy and safety of a standardized midazolam dosing protocol based on gender and age for use during bronchoscopy. The loading dose of midazolam was 0.075mg/kg for men ≤65 years old and women ≤70 and 0.05mg/kg for men ≥66 years and women ≥71 years, with subsequent doses of one-half the loading dose to be administered every 20min. The primary endpoint was tolerability and secondary endpoints included anxiety and recall of procedure, willingness to undergo repeat procedure, and complications. Safety was evaluated in terms of monitored changes in blood pressures, ECG, oxygen saturation, and CO2 content in expiration during the procedure. Results: A total of 204 patients were included in the study. Overall, 163 patients (79.9%) reported "no distress" during the procedure, 185 patients (90.7%) reported "no anxiety," and 175 (85.8%) replied that they would accept a repeat procedure, if necessary. The mean minimum oxygen saturation was 90.2% and the mean maximum expiratory CO2 level was 37.7mmHg. There were no serious complications related to the protocol. Conclusions: The midazolam dosing protocol examined in this study was safe and effective. It is simple, and it could easily be translated to routine clinical practice. © 2013 The Japanese Respiratory Society.

AimThe usefulness and safety of endobronchial ultrasound transbronchial needle aspiration (EBUS-TBNA) have been established recently, but no study has evaluated whether or not aging increases the risk of the procedure. In the present study, we aimed to assess the usefulness and safety of EBUS-TBNA in older patients. MethodsThe medical records and database of 109 patients who received EBUS-TBNA between 2008 and 2011 at Nagoya University Hospital, Nagoya, Japan were reviewed retrospectively. All patients underwent bronchoscopy under light sedation with midazolam. A total of 34 patients were aged 70 years or older (the older group) and 75 were aged 69 years or younger (the younger group). We analyzed patients' characteristics, changes of clinical parameters, usage doses of midazolam and lidocaine, procedure duration, geographic data of biopsied lymph nodes, diagnostic yield, and complications in both groups. ResultsThere were more comorbidities in the older group. Four patients (11.8%) in the older group had poor performance status (2-3). Systolic blood pressure at baseline was significantly higher in the older group. There were no statistical differences between the two groups in some clinical parameters (minimum oxygen saturation [SpO(2)], reduction in SpO(2), maximum oxygen supplementation, elevation of systolic blood pressure, increase of heart rate) during the procedure. Diagnostic performance in older patients was similar to that found in younger patients. There was no difference in the frequency of complications between both groups. ConclusionSafety and usefulness of EBUS-TBNA in older people were comparable with those in younger people. Geriatr Gerontol Int 2013; 13: 986-992.

Migration of airway smooth muscle (ASM) cells plays an important role in the pathophysiology of airway hyperresponsiveness and remodeling in asthma. It has been reported that prostaglandin (PG)E-2 inhibits migration of ASM cells. Although PGE(2) regulates cellular functions via binding to distinct prostanoid EP receptors, the role of EP receptor subtypes in mechanisms underlying cell migration has not been fully elucidated. We investigated the role of EP receptors in the inhibitory effects of PGE(2) on the migration of human ASM cells. Migration induced by platelet-derived growth factor (PDGF)-BB (10 ng/ml, 6 h) was assessed by a chemotaxis chamber assay. PDGF-BB-induced cell migration was inhibited by PGE(2), the specific EP2 agonist ONO-AE1-259-01, the specific EP4 agonist ONO-AE1-329, and cAMP-mobilizing agents. The inhibition of cell migration by PGE(2) was significantly reversed by a blockade of EP2 and EP4 receptors using antagonists or transfection with siRNAs. Moreover, PGE(2), the EP2 agonist, and the EP4 agonist significantly increased phosphorylation of small heat shock protein 20, one of the protein substrates for protein kinase A(PKA), with depolymerization of actin. In contrast, the EP3 agonist ONO-AE-248 significantly promoted baseline cell migration without affecting PDGF-BB-induced cell migration. In summary, activation of EP2 and EP4 receptors and subsequent activation of the cAMP/PKA pathway are the main mechanisms of inhibition of ASM cell migration by PGE(2). HSP20 phosphorylation by PKAis possibly involved in this mechanism. Conversely, EP3 is potent in promoting cell migration. EP receptor subtypes may be novel therapeutic target molecules in airway remodeling and asthma.

Background and objective: Bronchiolitis obliterans (BO) has been reported to develop following ingestion of Sauropus androgynus (SA), a leafy shrub distributed in Southeast Asia. Little is known about direct effects of SA on airway resident cells or haematopoietic cells in vitro. Identification of the SA component responsible for the development of BO would be an important key to elucidate its mechanism. We sought to elucidate the direct effects of SA on airway resident cells or haematopoietic cells and identify the SA element responsible for the pathogenesis of BO. Methods: SA dry powder was partitioned into fractions by solvent extraction. Human and murine monocytic cells, epithelial cells and endothelial cells were cultured with SA solution or fractions eluted from SA. We also investigated the effect of SA in vivo using a murine BO syndrome (BOS) model. Results: The aqueous fraction of SA induced significant increases of inflammatory cytokine and chemokine production from monocytic lineage cells. This fraction also induced significant apoptosis of endothelial cells and enhanced intraluminal obstructive fibrosis in allogeneic trachea allograft in the murine BOS model. We found individual differences in tumour necrosis factor α (TNF-α) production from monocytes of healthy controls stimulated by this aqueous fraction of SA, whereas it induced high-level TNF-α production from monocytes of patients with SA-induced BO. Conclusions: These results suggest that an aqueous fraction of SA may be responsible for the pathogenesis of BO. Ingestion of Sauropus androgynus (SA) as dry powder or fresh juice has been reported to induce constrictive bronchiolitis obliterans (BO), but the pathogenesis of this condition is unknown. This study suggests that an aqueous fraction of SA could be responsible for development of SA-induced BO. © 2012 Asian Pacific Society of Respirology.

TIMELESS (TIM) is a mammalian homolog of a Drosophila circadian rhythm gene, but its circadian properties in mammals have yet to be determined. TIM appears to be essential for replication protection and genomic stability. Recently, the involvement of TIM in human malignancies has been reported; therefore, we investigated the role of TIM in lung cancer. Microarray expression analysis of lung cancer cell lines showed that TIM expression was elevated 3.7-fold (P<0.001) in non-small cell lung cancer cell lines (n=116) compared to normal lung controls (n=59). In addition, small cell lung cancer cell lines (n=29) expressed TIM at levels 2.2-fold (P<0.001) higher than non-small cell lung cancer. Western blot analysis of 22 lung cancer cell lines revealed that all of them expressed TIM protein and that 20 cell lines (91%) expressed TIM protein at higher levels than a normal control line. Remarkably, immunohistochemistry of 30 surgically resected lung cancer specimens showed that all lung cancer specimens but no matched normal lung tissues were positive for TIM expression. Moreover, immunohistochemistry of surgically resected specimens from 88 consecutive patients showed that high TIM protein levels correlated with poor overall survival (P=0.013). Mutation analysis for TIM in 23 lung cancer cell lines revealed no mutation. TIM knockdown suppressed proliferation and clonogenic growth, and induced apoptosis in H157 and H460 cells. Taken together, our findings suggest that TIM could be useful as a diagnostic and prognostic marker for lung cancer and targeting it would be of high therapeutic value for this disease.

Background: Idiopathic pulmonary fibrosis (IPF) is the most common type of idiopathic interstitial pneumonia and is associated with an independent increased risk of lung carcinogenesis. The benefit of chemotherapy for lung cancer in cases of IPF remains unknown. Objectives: This study was conducted to elucidate the efficacy of chemotherapy for advanced non-small cell lung cancer (NSCLC) in patients with IPF. Methods: Advanced (i.e. stage IIIB and IV) NSCLC patients with IPF who received systemic chemotherapy were studied. Response rate, toxicity, overall survival and progression-free survival were investigated. Results: Between January 2000 and December 2009, 21 patients were enrolled in this study and treated with chemotherapy. The overall response rate with the 1st regimen was 42.9%. The median overall survival was 11.4 months, the 1-year survival rate was 28.6% and the median PFS was 5.4 months. Conclusions: This study showed that advanced NSCLC patients with IPF may benefit from chemotherapy; well-controlled studies are still needed to clarify the efficacy. Copyright (C) 2012 S. Karger AG, Basel

Objective Endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) is a minimally invasive technique with a high diagnostic yield used in the investigation of mediastinal diseases including sarcoidosis. Although previous reports have discussed the echoic features of metastatic mediastinal lymph nodes in lung cancer, few have addressed those features of mediastinal lymph nodes with sarcoidosis. We therefore investigated whether the echoic features of lymph nodes with sarcoidosis are distinct when compared to those of metastatic lymph nodes in lung cancer. Methods This retrospective analysis was held in one university hospital between April 2007 and June 2011. EBUS-guided biopsies were performed on 219 patients, and thus resulting in sarcoidosis diagnoses in 53 patients. We quantitatively analyzed the echoic morphologic features of 42 lymph nodes from 34 sarcoidosis patients and 59 lymph nodes from 44 patients with lung cancer using digital image analyzing software. Results In patients with sarcoidosis, 64.3% of the lymph nodes had a round shape, 71.4% had a distinct margin, and 88.1% exhibited homogeneous echogenicity. A germinal center structure was observed in 71.4% of the cases. In the context of shape and margin, no significant difference could be observed between sarcoidosis and lung cancer metastasis. However, homogeneous low echogenicity and the presence of a germinal center structure were observed in sarcoidosis more frequently than in lung cancer. Conclusion Homogeneous low echogenicity and the presence of a germinal central structure may be distinctive echoic features of lymph nodes with sarcoidosis. Analyzing the echogenicity of the mediastinal lymph nodes may help to distinguish sarcoidosis from lung cancer.

Malignant pleural mesothelioma (MPM) is a highly aggressive neoplasm arising from the mesothelial cells lining the parietal pleura and it exhibits poor prognosis. Although there has been significant progress in MPM treatment, development of more efficient therapeutic approaches is needed. BMAL1 is a core component of the circadian clock machinery and its constitutive overexpression in MPM has been reported. Here, we demonstrate that BMAL1 may serve as a molecular target for MPM. The majority of MPM cell lines and a subset of MPM clinical specimens expressed higher levels of BMAL1 compared to a nontumorigenic mesothelial cell line (MeT-5A) and normal parietal pleural specimens, respectively. A serum shock induced a rhythmical BMAL1 expression change in MeT-5A but not in ACC-MESO-1, suggesting that the circadian rhythm pathway is deregulated in MPM cells. BMAL1 knockdown suppressed proliferation and anchorage-dependent and independent clonal growth in two MPM cell lines (ACC-MESO-1 and H290) but not in MeT-5A. Notably, BMAL1 depletion resulted in cell cycle disruption with a substantial increase in apoptotic and polyploidy cell population in association with downregulation of Wee1, cyclin B and p21(WAF1/CIP1) and upregulation of cyclin E expression. BMAL1 knockdown induced mitotic catastrophe as denoted by disruption of cell cycle regulators and induction of drastic morphological changes including micronucleation and multiple nuclei in ACC-MESO-1 cells that expressed the highest level of BMAL1. Taken together, these findings indicate that BMAL1 has a critical role in MPM and could serve as an attractive therapeutic target for MPM.

This study investigated the physiological effects of inhaled corticosteroids, which are used widely to treat asthma. The application of fluticasone propionate (FP, 100 mu M) induced sustained increases in the short-circuit current (ISC) in human airway Calu-3 epithelial cells. The FP-induced ISC was prevented by the presence of H89 (10 mu M, a protein kinase A inhibitor) and SQ22536 (100 mu M, an adenylate cyclase inhibitor). The FP-induced responses involved bumetanide (a Na+-K+-2Cl(-) cotransporter inhibitor)-sensitive and 4,4'-dinitrostilbene-2,2'-disulfonic acid (an inhibitor of HCO32- dependent anion transporters)-sensitive components, both of which reflect basolateral anion transport. Further, FP augmented apical membrane Cl- current (I-Cl), reflecting cystic fibrosis transmembrane conductance regulator (CFTR)-mediated conductance, in the nystatin-permeabilized monolayer. In ISC and ICl responses, FP failed to enhance the responses to forskolin (10 mu M, an adenylate cyclase activator). Nevertheless, we found that FP synergistically increased cytosolic cAMP concentrations in combination with forskolin. All these effects of FP were reproduced with the use of budesonide. Collectively, inhaled corticosteroids such as FP and budesonide stimulate CFTR-mediated anion transport through adenylate cyclase-mediated mechanisms in a nongenomic fashion, thus sharing elements of a common pathway with forskolin. However, the corticosteroids cooperate with forskolin for synergistic cAMP production, suggesting that the corticosteroids and forskolin do not compete with each other to exert their effects on adenylate cyclase. Considering that such synergism was also observed in the FP/salmeterol combination, these nongenomic aspects may play therapeutic roles in mucus congestive airway diseases, in addition to genomic aspects that are generally recognized.

It is suggested that migration of airway smooth muscle (ASM) cells plays an important role in the pathogenesis of airway remodeling in asthma. Increases in intracellular Ca2+ concentrations ([Ca2+](i)) regulate most ASM cell functions related to asthma, such as contraction and proliferation. Recently, STIM1 was identified as a sarcoplasmic reticulum (SR) Ca2+ sensor that activates Orai1, the Ca2+ channel responsible for store-operated Ca2+ entry (SOCE). We investigated the role of STIM1 in [Ca2+](i) and cell migration induced by platelet-derived growth factor (PDGF)-BB in human ASM cells. Cell migration was assessed by a chemotaxis chamber assay. Human ASM cells express STIM1, STIM2, and Orai1 mRNAs. SOCE activated by thapsigargin, an inhibitor of SR Ca2+-ATPase, was significantly blocked by STIM1 siRNA and Orai1 siRNA but not by STIM2 siRNA. PDGF-BB induced a transient increase in [Ca2+](i) followed by sustained [Ca2+](i) elevation. Sustained increases in [Ca2+](i) due to PDGF-BB were significantly inhibited by a Ca2+ chelating agent EGTA or by siRNA for STIM1 or Orai1. The numbers of migrating cells were significantly increased by PDGF-BB treatment for 6 h. Knockdown of STIM1 and Orai1 by siRNA transfection inhibited PDGF-induced cell migration. Similarly, EGTA significantly inhibited PDGF-induced cell migration. In contrast, transfection with siRNA for STIM2 did not inhibit the sustained elevation of [Ca2+](i) or cell migration induced by PDGF-BB. These results demonstrate that STIM1 and Orai1 are essential for PDGF-induced cell migration and Ca2+ influx in human ASM cells. STIM1 could be an important molecule responsible for airway remodeling.

Background. The role of ZEB1, a master epithelial-to-mesenchymal transition gene, in malignant pleural mesothelioma (MPM) is unclear. Methods. The expression of ZEB1, E-cadherin, vimentin, and epithelial cell adhesion molecule (EpCAM) in 18 MPM cell lines and a normal pleural mesothelial cell line MeT-5A was determined by quantitative real-time polymerase chain reaction and Western blot testing. RNA interference-mediated transient and/or stable knockdown of ZEB1 and EpCAM was performed. Microarray expression analysis was performed with a TORAY-3D gene chip. Growth was evaluated by colorimetric proliferation and colony formation assays. Luciferase reporter assay was performed to access the effects of ZEB1 knockdown on EpCAM promoter activity. Results. Most MPM cell lines exhibited mesenchymal phenotype and expressed ZEB1. Transient ZEB1 knockdown suppressed growth in all four cell lines studied (ACC-MESO-1, H2052, Y-MESO-8A, Y-MESO-29) while stable ZEB1 knockdown suppressed growth only in Y-MESO-29. Genome-wide gene expression analysis revealed that EpCAM was the most prominently up-regulated gene by both transient and stable ZEB1 knockdown in ACC-MESO-1, with more marked up-regulation in stable knockdown. We hypothesized that EpCAM up-regulation counteracts the stable ZEB1 knockdown-induced growth inhibition in ACC-MESO-1. Transient EpCAM knockdown suppressed growth dramatically in ACC-MESO-1 cells expressing shZEB1 but only modestly in those expressing shGFP, supporting our hypothesis. Luciferase reporter assay showed that ZEB1 knockdown resulted in increased EpCAM promoter activity. EpCAM was also up-regulated in Y-MESO-29 expressing shZEB1, but this EpCAM up-regulation did not counteract ZEB1knockdown-induced growth suppression, suggesting that the counteracting effects of EpCAM may be cellular context dependent. Conclusions. RNA interference-mediated ZEB1 knockdown may be a promising therapeutic strategy for MPM, but one has to consider the possibility of diminished growth inhibitory effects of long-term ZEB1 knockdown, possibly as a result of EpCAM up-regulation and/or other gene expression changes resulting from ZEB1 knockdown.

Background: Sphingosine-1-phosphate (S1P), a lysophospholipid released from inflammatory cells, causes cell migration by increasing cytokines and chemokines. This study was designed to determine whether S1P causes adherence of eosinophils to pulmonary endothelial cells via enhancement of adhesion molecule expression. Methods: Expression of VCAM-1 and ICAM-1 was assessed by RT-PCR and Western blot analysis in human pulmonary microvasucular endothelial cells (HPMVECs). The number of adherent eosinophils to HPMVECs was calculated according to adhesion assay. Results: Pre-treatment of HPMVECs with S1P increased mRNA and protein expression of VCAM-1, in contrast, did not dramatically increase those expression of ICAM-1. The maximal expression of these adhesion molecules in mRNA and protein was observed 4 and 8 h after exposure to Si P, respectively. Pre-treatment with S1P also activated RhoA, a monomeric G protein; the ability of S1P to enhance the expression of VCAM-1 was attenuated by RhoA related inhibitors such as Y-27632, 03 exoenzyme, and GGTI-286. The effects of S1P on VCAM-1 were attenuated by pre-incubation with pertussis toxin, which catalyzes the ADP-ribosylation of G(i), a heterotrimeric G protein. After HPMVECs were treated with S1P, adhesion of human eosinophilic leukemic cell line (EoL-1) cells to HPMVECs was enhanced in a concentration-dependent manner. Augmented adherence of EoL-1 cells by S1P was also attenuated by Y-27632 and pertussis toxin. S1P causes adherence of eosinophils to pulmonary endothelium via RhoA activation. Conclusions: S1P may act as a lipid mediator in asthma. The RhoA/Rho-kinase pathway may be a therapeutic target for preventing eosinophil infiltration to the airway.

Background: Autoimmune pancreatitis is characterized by diffuse swelling of the pancreas and a high serum immunoglobulin (Ig) G4 concentration. Histopathologically, dense infiltration of lymphocytes and IgG4-positive plasma cells with fibrosis are seen in the pancreas. Although allergic diseases complicating autoimmune pancreatitis have been reported, the clinical features of bronchial asthma complicated by autoimmune pancreatitis remain unclear. Case Summary: We report three cases of bronchial asthma preceding the onset of type 1 autoimmune pancreatitis by 3 months to 30 years. All three cases were males with high serum IgG, IgG4, and IgE concentrations. The radioallergosorbent tests were positive for common allergens such as mites and house dust. One case had a pulmonary manifestation that proved to be an inflammatory pseudotumor of the lung with an accumulation of IgG4-positive plasma cells. The asthma symptom was ameliorated by oral prednisolone therapy for autoimmune pancreatitis, and when the corticosteroid doses were reduced, asthma became worse in all three cases. Discussion: It is possible that atopy and increased Th2 cell activity are related to a higher coincidence of IgG4-related diseases such as type 1 autoimmune pancreatitis. Because the present cases are few in number, further studies are necessary.

非ステロイド性抗炎症薬(NSAID)やステロイド薬はがん疼痛の緩和治療薬として頻用されているが、胃腸障害を惹起しやすい薬物として知られている。そのため実地臨床ではNSAIDやステロイド薬による胃腸障害に対して予防的に胃酸分泌抑制薬が経験的に使用されているが、その使用方法は医師による個人差が大きい。本研究では、肺がんの疼痛緩和治療を目的としてNSAIDおよびオピオイドを定期的に処方されていた入院患者83名を対象として、緩和治療における胃酸分泌抑制薬の使用状況について解析し、呼吸器内科医師25名に胃酸分泌抑制薬の処方に関する意識調査を行った。レトロスペクティブ調査の結果、NSAID+ステロイド薬併用群において胃酸分泌抑制薬が予防的に処方されていた割合は、NSAID単独群のそれに比べて有意に高かった。医師の意識調査では、NSAIDを単独で処方する場合には、患者の全身状態や胃腸障害の既往歴が胃酸分泌抑制薬を処方するかどうかを判断する要因となっていた。ステロイド薬を処方する場合には、それらの要因にかかわらず、意識的に胃酸分泌抑制薬を処方することが示唆された。今後、緩和治療における胃酸分泌抑制薬の効果についても調査し、その適正使用について検討していきたい。(著者抄録)