Details of a Researcher - Fukunaga, Koichi

Papers - Fukunaga, Koichi

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Increased plasma levels of high mobility group box 1 in patients with acute liver failure

Oshima, G., Shinoda, M., Tanabe, M., Ebinuma, H., Nishiyama, R., Takano, K., Yamada, S., Miyasho, T., Masugi, Y., Matsuda, S., Suda, K., Fukunaga, K., Matsubara, K., Hibi, T., Yagi, H., Hayashida, T., Yamagishi, Y., Obara, H., Itano, O., Takeuchi, H., Kawachi, S., Saito, H., Hibi, T., Maruyama, I. and Kitagawa, Y.

Eur Surg Res 48 ( 3 ) 154 - 62 2012

ISSN 1421-9921

BACKGROUND: High-mobility group box 1 (HMGB1) is a monocyte-derived late-acting inflammatory mediator, which is released in conditions such as shock, tissue injury and endotoxin-induced lethality. In this study, we determined the plasma and hepatic tissue levels of HMGB1 in patients with acute liver failure (ALF). PATIENTS AND METHODS: We determined the plasma levels of HMGB1 and aspartate aminotransferase (AST) in 7 healthy volunteers (HVs), 40 patients with liver cirrhosis (LC), 37 patients with chronic hepatitis (CH), 18 patients with severe acute hepatitis (AH), and 14 patients with fulminant hepatitis (FH). The 14 patients with FH were divided into two subgroups depending upon the history of plasma exchange (PE) before their plasma sample collection. The hepatic levels of HMGB1 were measured in tissue samples from 3 patients with FH who underwent living-donor liver transplantation and from 3 healthy living donors. Hepatic tissue samples were also subjected to immunohistochemical examination for HMGB1. RESULTS: The plasma levels of HMGB1 (ng/ml) were higher in patients with liver diseases, especially in FH patients with no history of PE, than in HVs (0.3 +/- 0.3 in HVs, 4.0 +/- 2.0 in LC, 5.2 +/- 2.6 in CH, 8.6 +/- 4.8 in severe AH, 7.8 +/- 2.7 in FH with a history of PE, and 12.5 +/- 2.6 in FH with no history of PE, p < 0.05 in each comparison). There was a strong and statistically significant relationship between the mean plasma HMGB1 level and the logarithm of the mean AST level (R = 0.900, p < 0.05). The hepatic tissue levels of HMGB1 (ng/mg tissue protein) were lower in patients with FH than in healthy donors (539 +/- 116 in FH vs. 874 +/- 81 in healthy donors, p < 0.05). Immunohistochemical staining for HMGB1 was strong and clear in the nuclei of hepatocytes in liver sections from healthy donors, but little staining in either nuclei or cytoplasm was evident in specimens from patients with FH. CONCLUSION: We confirmed that plasma HMGB1 levels were increased in patients with ALF. Based on a comparison between HMGB1 contents in normal and ALF livers, it is very likely that HMGB1 is released from injured liver tissue.

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Comparison of two large earthquakes: the 2008 Sichuan Earthquake and the 2011 East Japan Earthquake

Otani, Y., Ando, T., Atobe, K., Haiden, A., Kao, S. Y., Saito, K., Shimanuki, M., Yoshimoto, N. and Fukunaga, K.

Keio J Med 61 ( 1 ) 35 - 9 2012

ISSN 1880-1293

Between August 15th and 19th, 2011, eight 5th-year medical students from the Keio University School of Medicine had the opportunity to visit the Peking University School of Medicine and hold a discussion session titled "What is the most effective way to educate people for survival in an acute disaster situation (before the mental health care stage)?" During the session, we discussed the following six points: basic information regarding the Sichuan Earthquake and the East Japan Earthquake, differences in preparedness for earthquakes, government actions, acceptance of medical rescue teams, earthquake-induced secondary effects, and media restrictions. Although comparison of the two earthquakes was not simple, we concluded that three major points should be emphasized to facilitate the most effective course of disaster planning and action. First, all relevant agencies should formulate emergency plans and should supply information regarding the emergency to the general public and health professionals on a normal basis. Second, each citizen should be educated and trained in how to minimize the risks from earthquake-induced secondary effects. Finally, the central government should establish a single headquarters responsible for command, control, and coordination during a natural disaster emergency and should centralize all powers in this single authority. We hope this discussion may be of some use in future natural disasters in China, Japan, and worldwide.

Induction of mucin and MUC5AC expression by the protease activity of Aspergillus fumigatus in airway epithelial cells

Oguma, T., Asano, K., Tomomatsu, K., Kodama, M., Fukunaga, K., Shiomi, T., Ohmori, N., Ueda, S., Takihara, T., Shiraishi, Y., Sayama, K., Kagawa, S., Natori, Y., Lilly, C. M., Satoh, K., Makimura, K. and Ishizaka, A.

J Immunol 187 ( 2 ) 999 - 1005 2011.07

ISSN 1550-6606

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-alpha. We also found that A. fumigatus extracts activated the TNF-alpha-converting enzyme (TACE), critical for the cleavage of membrane-bound pro-TGF-alpha, 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-alpha/epidermal growth factor receptor pathway, may be a pathogenetic mechanism of allergic bronchopulmonary mycosis.

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Role of prostaglandin D2 receptor CRTH2 in sustained eosinophil accumulation in the airways of mice with chronic asthma

Kagawa, S., Fukunaga, K., Oguma, T., Suzuki, Y., Shiomi, T., Sayama, K., Kimura, T., Hirai, H., Nagata, K., Nakamura, M. and Asano, K.

Int Arch Allergy Immunol 155 Suppl 1 6 - 11 2011

ISSN 1423-0097

The prostaglandin D(2) (PGD(2))/CRTH2 pathway is important for eosinophil trafficking in vitro; however, genetic deficiency of CRTH2 does not suppress in vivo eosinophilic airway inflammation in acute models of asthma, and the role of CRTH2 in the pathogenesis of asthma is still ambiguous. Therefore, in the present study we explored whether the PGD(2)/CRTH2 pathway could affect the phenotypes of chronic asthma. Either CRTH2-deficient (CRTH2-/-) or wild-type mice were sensitized and exposed to ovalbumin (OVA) for 3 days (acute model) or 6 weeks (chronic model). While the magnitude of the acute eosinophilic inflammation was equivalent between CRTH2-/- and wild-type mice, the number of inflammatory cells and eosinophils in bronchoalveolar lavage fluid after chronic OVA exposure was significantly reduced in CRTH2-/- mice (18.0 +/- 2.6 x 10(4) cells and 2.0 +/- 0.5 x 10(4) cells) compared to wild-type mice (27.9 +/- 2.5 x 10(4) cells and 6.8 +/- 1.1 x 10(4) cells, p < 0.001). On the contrary, no difference was observed between CRTH2-/- and wild-type mice in terms of airway hyperresponsiveness or remodeling (goblet cell hyperplasia) in the chronic model of asthma. In conclusion, CRTH2 that mediates PGD(2) activity is essential for sustained eosinophilic inflammation in the airways, and its antagonists could exert an anti-inflammatory effect in chronic asthma.

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Prediction of the pathogens that are the cause of pneumonia by the battlefield hypothesis

Hirama, T., Yamaguchi, T., Miyazawa, H., Tanaka, T., Hashikita, G., Kishi, E., Tachi, Y., Takahashi, S., Kodama, K., Egashira, H., Yokote, A., Kobayashi, K., Nagata, M., Ishii, T., Nemoto, M., Tanaka, M., Fukunaga, K., Morita, S., Kanazawa, M. and Hagiwara, K.

PLoS One 6 ( 9 ) e24474 2011

ISSN 1932-6203

Commensal organisms are frequent causes of pneumonia. However, the detection of these organisms in the airway does not mean that they are the causative pathogens; they may exist merely as colonizers. In up to 50% cases of pneumonia, the causative pathogens remain unidentified, thereby hampering targeting therapies. In speculating on the role of a commensal organism in pneumonia, we devised the battlefield hypothesis. In the "pneumonia battlefield," the organism-to-human cell number ratio may be an index for the pathogenic role of the organism. Using real-time PCR reactions for sputum samples, we tested whether the hypothesis predicts the results of bacteriological clinical tests for 4 representative commensal organisms: Streptococcus pneumoniae, Haemophilus influenzae, Pseudomonas spp., and Moraxella catarrhalis. The cutoff value for the organism-to-human cell number ratio, above which the pathogenic role of the organism was suspected, was set up for each organism using 224 sputum samples. The validity of the cutoff value was then tested in a prospective study that included 153 samples; the samples were classified into 3 groups, and each group contained 93%, 7%, and 0% of the samples from pneumonia, in which the pathogenic role of Streptococcus pneumoniae was suggested by the clinical tests. The results for Haemophilus influenzae, Pseudomonas spp., and Moraxella catarrhalis were 100%, 0%, and 0%, respectively. The battlefield hypothesis enabled legitimate interpretation of the PCR results and predicted pneumonia in which the pathogenic role of the organism was suggested by the clinical test. The PCR reactions based on the battlefield hypothesis may help to promote targeted therapies for pneumonia. The prospective observatory study described in the current report had been registered to the University Hospital Medical Information Network (UMIN) registry before its initiation, where the UMIN is a registry approved by the International Committee of Medical Journal Editors (ICMJE). The UMIN registry number was UMIN000001118: A prospective study for the investigation of the validity of cutoff values established for the HIRA-TAN system (April 9, 2008).

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Protective effect of high-mobility group box 1 blockade on acute liver failure in rats

Takano, K., Shinoda, M., Tanabe, M., Miyasho, T., Yamada, S., Ono, S., Masugi, Y., Suda, K., Fukunaga, K., Hayashida, T., Hibi, T., Obara, H., Takeuchi, H., Kawachi, S., Kawasako, K., Okamoto, M., Yokota, H., Maruyama, I. and Kitagawa, Y.

Shock 34 ( 6 ) 573 - 9 2010.12

ISSN 1540-0514

High-mobility group box 1 (HMGB1) is a monocyte-derived inflammatory mediator that is released in some conditions including shock, tissue injury, and endotoxin-induced lethality. In this study, we determined the plasma and hepatic tissue levels of HMGB1 in a drug-induced rat acute liver failure (ALF) model and investigated the effect of HMGB1 blockade on ALF. Adult male Sprague-Dawley rats, weighing 250 to 300 g, were used for this study. d-galactosamine was injected into the penile vein to induce ALF. To determine HMGB1 levels, plasma and hepatic tissue samples were serially collected after the d-galactosamine injection. To test the effect of HMGB1 blockade, anti-HMGB1 polyclonal antibodies or control antibodies were injected into the penile vein right after injection of d-galactosamine. Levels of HMGB1 were increased in plasma and decreased in hepatic tissue after induction of ALF. Immunohistochemical examination for HMGB1 showed that liver from animals with ALF had little staining, whereas normal liver had strong staining in the nuclei. Injection of anti-HMGB1 antibodies resulted in significant suppression of plasma HMGB1 and hepatic enzymes, marked suppression of plasma inflammatory cytokines, marked improvement of histological findings, and significant improvement of survival. The decrease of hepatic HMGB1 was also significantly suppressed in the group injected with anti-HMGB1 antibodies. The present study suggests that in ALF, the liver may release HMGB1 into the plasma, and that neutralizing the released HMGB1 has a protective effect against injury.

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Effect of Toll-like receptor 4 inhibitor on LPS-induced lung injury

Seki, H., Tasaka, S., Fukunaga, K., Shiraishi, Y., Moriyama, K., Miyamoto, K., Nakano, Y., Matsunaga, N., Takashima, K., Matsumoto, T., Ii, M., Ishizaka, A. and Takeda, J.

Inflamm Res 59 ( 10 ) 837 - 45 2010.10

ISSN 1420-908X

OBJECTIVE AND DESIGN: Toll-like receptor 4 (TLR4) plays important roles in the recognition of lipopolysaccharide (LPS) and the activation of inflammatory cascade. In this study, we evaluated the effect of TAK-242, a selective TLR4 signal transduction inhibitor, on acute lung injury (ALI). MATERIALS AND METHODS: C57BL/6J mice were intravenously treated with TAK-242 15 min before the intratracheal administration of LPS or Pam3CSK4, a synthetic lipopeptide. Six hours after the challenge, bronchoalveolar lavage fluid was obtained for a differential cell count and the measurement of cytokine and myeloperoxidase levels. Lung permeability and nuclear factor-kappaB (NF-kappaB) DNA binding activity were also evaluated. RESULTS: TAK-242 effectively attenuated the neutrophil accumulation and activation in the lungs, the increase in lung permeability, production of inflammatory mediators, and NF-kappaB DNA-binding activity induced by the LPS challenge. In contrast, TAK-242 did not suppress inflammatory changes induced by Pam3CSK4. CONCLUSION: TAK-242 may be a promising therapeutic agent for ALI, especially injuries associated with pneumonia caused by Gram-negative bacteria.

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Association between beta-adrenoceptor gene polymorphisms and relative response to beta 2-agonists and anticholinergic drugs in Japanese asthmatic patients

Asano, K., Yamada-Yamasawa, W., Kudoh, H., Matsuzaki, T., Nakajima, T., Hakuno, H., Hiraoka, R., Fukunaga, K., Oguma, T., Sayama, K., Yamaguchi, K., Nagabukuro, A., Harada, Y. and Ishizaka, A.

Respirology 15 ( 5 ) 849 - 54 2010.07

ISSN 1440-1843

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.

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Spherical sulfated cellulose adsorbs high-mobility-group box chromosomal protein 1 in vitro and in vivo

Suda, K., Takeuchi, H., Hagiwara, T., Miyasho, T., Yamada, S., Inoue, T., Okamoto, M., Wada, N., Saikawa, Y., Fukunaga, K., Hashimoto, S., Yokota, H., Maruyama, I. and Kitagawa, Y.

ASAIO J 56 ( 3 ) 210 - 4 2010.05

ISSN 1538-943X

High-mobility-group box chromosomal protein 1 (HMGB1) has recently been identified as a late mediator of various kinds of acute and chronic inflammation. A method for efficiently removing HMGB1 from systemic circulation could be a promising therapy for HMGB1-mediated inflammatory diseases. It is well known that the cationic portion of HMGB1 binds to heparin, which has abundant sulfates in its structure. In this study, we determined whether spherical sulfated cellulose (SC) efficiently adsorbed HMGB1, as well as other inflammatory mediators, in vitro. Then, we investigated the efficacy of hemoperfusion with the SC (SC group) or cellulose beads (control group) at adsorbing endogenous mediators, including HMGB1, in vivo. We have demonstrated that the SC adsorbed significantly larger amounts of HMGB1, interleukin (IL)-4, and IL-8 when compared with cellulose beads, in vitro. Hemoperfusion with the SC for 30 minute, starting 2 hour after an abdominal opening and closure operation, significantly reduced serum HMGB1 levels (p = 0.004) and consistently increased serum IL-10 levels, in vivo. These data suggest the potential benefits of hemoperfusion using the SC in treating HMGB1-mediated inflammatory diseases.

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Neutrophil elastase inhibitor improves survival of rats with clinically relevant sepsis

Suda, K., Takeuchi, H., Hagiwara, T., Miyasho, T., Okamoto, M., Kawasako, K., Yamada, S., Suganuma, K., Wada, N., Saikawa, Y., Fukunaga, K., Funakoshi, Y., Hashimoto, S., Yokota, H., Maruyama, I., Ishizaka, A. and Kitagawa, Y.

Shock 33 ( 5 ) 526 - 31 2010.05

ISSN 1540-0514

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.

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The anti-inflammatory and proresolving mediator resolvin E1 protects mice from bacterial pneumonia and acute lung injury

Seki, H., Fukunaga, K., Arita, M., Arai, H., Nakanishi, H., Taguchi, R., Miyasho, T., Takamiya, R., Asano, K., Ishizaka, A., Takeda, J. and Levy, B. D.

J Immunol 184 ( 2 ) 836 - 43 2010.01

ISSN 1550-6606

Whereas pneumonia is the most common cause of death and disability worldwide, most cases of pneumonia spontaneously resolve. Mechanisms that promote pneumonia resolution remain to be determined. Resolvin E1 (RvE1) is an endogenous mediator that displays proresolving actions in sterile inflammation. In this study, we developed a new model of aspiration pneumonia to evaluate the effect of RvE1 on acute lung injury caused by acid aspiration and subsequent bacterial challenge. Mice received hydrochloric acid into the left lung followed by the enteric pathogen Escherichia coli. I.v. administration of RvE1 (approximately 0.005 mg/kg) prior to acid injury selectively decreased lung neutrophil accumulation by 55% and enhanced clearance of E. coli. RvE1 significantly decreased lung tissue levels of several proinflammatory chemokines and cytokines, including IL-1beta, IL-6, HMGB-1, MIP-1alpha, MIP-1beta, keratinocyte-derived chemokine, and MCP-1, in a manner independent of the anti-inflammatory mediators IL-10 and lipoxin A4. In addition, animals treated with RvE1 had a marked improvement in survival. These findings in experimental aspiration pneumonia have uncovered protective roles for RvE1 in pathogen-mediated inflammation that are both anti-inflammatory for neutrophils and protective for host defense, suggesting that RvE1 represents the first candidate for a novel therapeutic strategy for acute lung injury and pneumonia that harnesses natural resolution mechanisms.

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Strain-specific phenotypes of airway inflammation and bronchial hyperresponsiveness induced by epicutaneous allergen sensitization in BALB/c and C57BL/6 mice

Kodama, M., Asano, K., Oguma, T., Kagawa, S., Tomomatsu, K., Wakaki, M., Takihara, T., Ueda, S., Ohmori, N., Ogura, H., Miyata, J., Tanaka, K., Kamiishi, N., Fukunaga, K., Sayama, K., Ikeda, E., Miyasho, T. and Ishizaka, A.

Int Arch Allergy Immunol 152 Suppl 1 67 - 74 2010

ISSN 1423-0097

BACKGROUND: Allergen sensitization through a disrupted skin barrier appears to play a prominent role in the development of atopic diseases, including allergic asthma. The role of the genetic background in immunological and physiological phenotypes induced by epicutaneous sensitization is undetermined. METHODS: BALB/c and C57BL/6 mice were sensitized either epicutaneously by patch application of ovalbumin (OVA) or systemically by intraperitoneal injection of OVA with alum before exposure to aerosolized OVA. The concentrations of OVA-specific immunoglobulin in serum and cytokines in bronchoalveolar lavage fluid (BALF) were measured by enzyme-linked immunosorbent assay. The severity of airway inflammation was evaluated by cell counts in BALF, and bronchial responsiveness to methacholine was measured by the flexiVent system. RESULTS: The production of OVA-specific IgG1 and IgE was greater in the epicutaneously sensitized BALB/c than C57BL/6 mice. In contrast, both eosinophilic airway inflammation and bronchial responsiveness to methacholine were more prominent in the C57BL/6 than in the BALB/c mice. The concentrations of interleukin-4 increased significantly in the BALF from C57BL/6 mice only. No between-strain differences were observed after intraperitoneal sensitization. CONCLUSIONS: The C57BL/6 mouse is a more appropriate model than the BALB/c mouse to study the relationship between skin barrier dysfunction and the pathogenesis of allergic asthma.

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Therapeutic effects of various initial combinations of chemotherapy including clarithromycin against Mycobacterium avium complex pulmonary disease

Hasegawa, N., Nishimura, T., Ohtani, S., Takeshita, K., Fukunaga, K., Tasaka, S., Urano, T., Ishii, K., Miyairi, M. and Ishizaka, A.

Chest 136 ( 6 ) 1569 - 1575 2009.12

ISSN 1931-3543

BACKGROUND: The objective of this study was to find an optimal initial combination chemotherapy that includes clarithromycin (CAM) for treatment-naive patients with Mycobacterium avium complex (MAC) pulmonary disease, as assessed by microbiological conversion using a Mycobacterium growth indicator tube (MGIT). METHODS: Thirty-four patients with treatment-naive MAC pulmonary disease (determined using 1997 American Thoracic Society criteria) were evaluated retrospectively. They demonstrated a nodular and bronchiectatic pattern without cavity on high-resolution CT (HRCT) scans. The following three regimens were administered: regimen A (n = 9) consisted of CAM (400 mg/d), ethambutol (EB) [750 mg/d], and rifampicin (RFP) [450 mg/d]; regimen B (n = 12) consisted of CAM (800 mg/d), EB (750 mg/d), and RFP (450 mg/d); and regimen C (n = 13) consisted of CAM (800 mg/d), EB (1,000 mg/d), and RFP (600 mg/d) during the first 2 months followed by a reduction of the dosage of EB from 1,000 to 750 mg/d. Gender, age, BMI, and HRCT scan finding scores were not significantly different among the three groups. Chemotherapy was continued for 18 months. Sputum culture was periodically assessed by MGIT. RESULTS: Culture conversion at 18 months in regimen A (55.6%), which included a daily dosage of 400 mg of CAM (9.5 mg/kg), was significantly inferior to that in regimen B (91.7%), which included daily 800 mg of CAM (17.6 mg/kg; p < 0.05), but regimen B and C (92.3%) showed no between-group difference after > 18 months of chemotherapy. CONCLUSIONS: The higher dose of CAM allowed for better culture conversion. Daily combination chemotherapy that includes CAM (800 mg) seems appropriate as an initial treatment against treatment-naive patients with nodular and bronchiectatic MAC pulmonary disease.

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High-mobility group box 1 contributes to lethality of endotoxemia in heme oxygenase-1-deficient mice

Takamiya, R., Hung, C. C., Hall, S. R., Fukunaga, K., Nagaishi, T., Maeno, T., Owen, C., Macias, A. A., Fredenburgh, L. E., Ishizaka, A., Blumberg, R. S., Baron, R. M. and Perrella, M. A.

Am J Respir Cell Mol Biol 41 ( 2 ) 129 - 35 2009.08

ISSN 1535-4989

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.

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Impact of pharmacokinetics and pharmacogenetics on the efficacy of pranlukast in Japanese asthmatics

Asano, K., Nakade, S., Shiomi, T., Nakajima, T., Suzuki, Y., Fukunaga, K., Oguma, T., Sayama, K., Fujita, H., Tanigawara, Y. and Ishizaka, A.

Respirology 14 ( 6 ) 822 - 7 2009.08

ISSN 1440-1843

BACKGROUND AND OBJECTIVE: Wide inter-individual variability in therapeutic effects limits the efficacy of leukotriene (LT) receptor antagonists in the treatment of asthma. We have reported that genetic variability in the expression of LTC(4) synthase is associated with responsiveness to pranlukast in Japanese asthmatic patients. However, the effects of pharmacokinetic variability are less well known. This was an analysis of the pharmacokinetics of pranlukast in a population of adult asthmatics, and its effect on clinical responses. Other factors that may be related to the therapeutic effects of pranlukast, including LTC(4) synthase gene polymorphisms, were also investigated. METHODS: The population pharmacokinetics of pranlukast was analysed in a one-compartment model, using data collected in 50 Japanese adults with moderate to severe asthma, who were treated with pranlukast, 225 mg bd for 4 days. In 32 of these patients, in whom the clinical response to pranlukast (increase in FEV(1) after 4 weeks of treatment) was measured in a previous study, a combined pharmacokinetic and pharmacogenetic analysis was performed. RESULTS: Using the population pharmacokinetic model, the estimated the mean oral clearance (CL/F) of pranlukast was 16.4 L/h, and the inter-individual variability was 30.1%. Univariate and multivariate analyses showed that LTC(4) synthase polymorphisms, but not the CL/F of the drug, predicted an improvement in pulmonary function with pranlukast treatment (P < 0.05). CONCLUSIONS: There was marked inter-individual variability in the pharmacokinetics of pranlukast among adult asthmatics, but this had little impact on the clinical effectiveness of the drug.

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Exacerbation of bleomycin-induced injury and fibrosis by pneumonectomy in the residual lung of mice

Kakizaki, T., Kohno, M., Watanabe, M., Tajima, A., Izumi, Y., Miyasho, T., Tasaka, S., Fukunaga, K., Maruyama, I., Ishizaka, A. and Kobayashi, K.

J Surg Res 154 ( 2 ) 336 - 44 2009.06

ISSN 1095-8673

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-1beta, 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.

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TP receptor-mediated release of eosinophil chemotactic activity from human bronchial smooth muscle cells

Suzuki, Y., Asano, K., Niimi, K., Miyata, J., Shiraishi, Y., Fukunaga, K., Shiomi, T., Nakajima, T., Oguma, T., Sayama, K. and Ishizaka, A.

Eur J Pharmacol 600 ( 1-3 ) 133 - 9 2008.12

ISSN 1879-0712

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.

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Role of soluble receptor for advanced glycation end products on endotoxin-induced lung injury

Zhang, H., Tasaka, S., Shiraishi, Y., Fukunaga, K., Yamada, W., Seki, H., Ogawa, Y., Miyamoto, K., Nakano, Y., Hasegawa, N., Miyasho, T., Maruyama, I. and Ishizaka, A.

Am J Respir Crit Care Med 178 ( 4 ) 356 - 62 2008.08

ISSN 1535-4970

RATIONALE: The interaction of receptor for advanced glycation end products (RAGE) and its ligands often leads to inflammatory processes or tissue injury, although the effect of the blockade of RAGE signaling on lung injury remains to be investigated. OBJECTIVES: Using a murine model of lung injury induced by intratracheal lipopolysaccharide (LPS), we evaluated RAGE expression in the airspace and the effect of recombinant soluble RAGE (sRAGE) on LPS-induced lung injury. METHODS: First, the expression of sRAGE in bronchoalveolar lavage (BAL) fluid was determined at 24 hours after intratracheal instillation of LPS or phosphate-buffered saline. Next, to evaluate the effect of sRAGE, BAL fluid was collected for cell counting and measurements of lung permeability and cytokine concentrations 24 hours after intratracheal LPS in the mice with or without intraperitoneal administration of sRAGE 1 hour after the instillation. In another series, lungs were sampled for histopathology and detection of apoptotic cells. The activation of nuclear factor (NF)-kappaB was analyzed 4 hours after LPS instillation. MEASUREMENTS AND MAIN RESULTS: In response to LPS challenge, a RAGE isoform of 48 kD was detected in the BAL fluid. Treatment with sRAGE significantly attenuated the increases in neutrophil infiltration, lung permeability, production of inflammatory cytokines, NF-kappaB activation, and apoptotic cells in the lung as well as development of pathologic changes after LPS instillation. CONCLUSIONS: RAGE plays an important role in the pathogenesis of LPS-induced lung injury in mice. It was suggested that sRAGE should be tested as a treatment modality in other models of acute lung injury.

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Cyclooxygenase-2/prostaglandin D2/CRTH2 pathway mediates double-stranded RNA-induced enhancement of allergic airway inflammation

Shiraishi, Y., Asano, K., Niimi, K., Fukunaga, K., Wakaki, M., Kagyo, J., Takihara, T., Ueda, S., Nakajima, T., Oguma, T., Suzuki, Y., Shiomi, T., Sayama, K., Kagawa, S., Ikeda, E., Hirai, H., Nagata, K., Nakamura, M., Miyasho, T. and Ishizaka, A.

J Immunol 180 ( 1 ) 541 - 9 2008.01

ISSN 0022-1767

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.

TLR3-mediated synthesis and release of eotaxin-1/CCL11 from human bronchial smooth muscle cells stimulated with double-stranded RNA

Niimi, K., Asano, K., Shiraishi, Y., Nakajima, T., Wakaki, M., Kagyo, J., Takihara, T., Suzuki, Y., Fukunaga, K., Shiomi, T., Oguma, T., Sayama, K., Yamaguchi, K., Natori, Y., Matsumoto, M., Seya, T., Yamaya, M. and Ishizaka, A.

J Immunol 178 ( 1 ) 489 - 95 2007.01

ISSN 0022-1767

Respiratory infections with RNA viruses, such as rhinovirus or respiratory syncytial virus, are a major cause of asthma exacerbation, accompanied by enhanced neutrophilic and/or eosinophilic inflammation of the airways. We studied the effects of dsRNA synthesized during RNA virus replication, and of its receptor, TLR3, on the synthesis of eosinophilic chemokines in bronchial smooth muscle cells (BSMC). Synthetic dsRNA, polyinosinic-cystidic acid (poly(I:C)), induced the synthesis of eosinophilic chemokines, eotaxin-1/CCL11 and RANTES/CCL5, from primary cultures of human BSMC, and IL-4 increased synergistically the synthesis of poly(I:C)-induced CCL11. A robust eosinophil chemotactic activity was released from BSMC stimulated with poly(I:C) and IL-4, which was mostly inhibited by preincubation with an anti-CCL11, but not with an anti-CCL5 Ab. Although the immunoreactivity of TLR3 was detectable on the cellular surface of BSMC by flow cytometric analysis, pretreatment with an anti-TLR3-neutralizing Ab failed to block the poly(I:C)-induced synthesis of CCL11. We have determined by confocal laser-scanning microscopy that the immunoreactivity of TLR3 was aggregated intracellularly in poly(I:C)-stimulated BSMC, colocalizing with fluorescein-labeled poly(I:C). The synthesis of CCL11 was prominently inhibited by the transfection of TLR3-specific small interfering RNA or by bafilomycin A1, an endosomal acidification inhibitor, further supporting the essential role played by intracellular TLR3 in the synthesis of poly(I:C)-induced CCL11 in BSMC. In conclusion, these observations suggest that, by activating intracellular TLR3 in BSMC, respiratory RNA virus infections stimulate the production of CCL11 and enhance eosinophilic inflammation of the airways in the Th2-dominant microenvironment.

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