Published papers

OBJECTIVES: Inflammation occurs along with infection during sepsis. 15-Epi-lipoxin A4 has protective and resolving effects in experimental models of infection. In this study, we examined the effects of 15-epi-lipoxin A4 combined with antibiotics on Escherichia coli-induced peritonitis. DESIGN: Prospective experimental study. SETTING: University research laboratory. SUBJECTS: Male C57BL/6 mice. INTERVENTIONS: Mice were injected with E. coli to induce peritonitis and were given either 15-epi-lipoxin A4 (1 mug/mouse) or placebo (saline) with antibiotics (ceftazidime). The effects of 15-epi-lipoxin A4 on peritoneal cell populations, bacterial burden, and cytokine production were assessed. Survival rates were observed for up to 7 days. In addition, we examined the effects of 15-epi-lipoxin A4 on peritoneal macrophages stimulated with lipopolysaccharide, CpG DNA, or live E. coli. MEASUREMENTS AND MAIN RESULTS: Treatment with 15-epi-lipoxin A4 significantly reduced the number of neutrophils in the peritoneum, inhibited production of cytokines and chemokines, and decreased bacterial load in the serum. Combined treatment of 15-epi-lipoxin A4 with antibiotics significantly improved survival in E. coli-infected mice. 15-Epi-lipoxin A4 also attenuated the production of interleukin-6 and tumor necrosis factor-alpha by lipopolysaccharide- or CpG DNA-stimulated peritoneal macrophages. Furthermore, 15-epi-lipoxin A4 combined with antibiotics synergistically reduced the production of interleukin-6 and tumor necrosis factor-alpha by peritoneal macrophages stimulated with live E. coli. CONCLUSIONS: 15-Epi-lipoxin A4 combined with antibiotics attenuated systemic inflammation, inhibited bacteria dissemination, and improved survival in E. coli-infected mice. The reduced production of interleukin-6 and tumor necrosis factor-alpha by peritoneal macrophages suggested that 15-epi-lipoxin A4 blocked the initial proinflammatory response. Taken together, these data suggested that 15-epi-lipoxin A4 combined with antibiotics was beneficial in regulating the proinflammatory response in sepsis without exacerbating infection.

UNLABELLED: Identification of the causative pathogen(s) of pneumonia would allow the selection of effective antibiotics and thus reduce the mortality rate and the emergence of drug-resistant pathogens. To identify such pathogens and to obtain these benefits, it is necessary that a clinical test is rapid, accurate, easily performed, and cost-effective. Here, we devised a PCR-based test, named HIRA-TAN, which is able to discriminate therapeutic targets from commensal organisms (e.g. Streptococcus pneumoniae or Haemophilus influenzae) and to detect foreign organisms (e.g. Mycoplasma pneumoniae or Legionella pneumophila) in the sputum. The utility of this system was validated in a prospective study, using sputum samples from patients with pneumonia. 568 patients were enrolled and the HIRA-TAN assay identified the causative pathogens with an accuracy of 96.7% for H. influenzae; 93.2% for Pseudomonas aeruginosa; 80.6% for Klebsiella pneumoniae; 90.9% for Moraxella catarrhalis; 87.5% for Escherichia coli; 78.1% for MRSA and 91.6% for S. pneumoniae. Overall the HIRA-TAN procedure was able to identify the causative pathogens of pneumonia in 60% of the cases. Additionally, this procedure was able to determine when the pneumonia-causing organism was a commensal organism or a foreign organism in a single assay. The HIRA-TAN approach yielded reproducible results and provided valuable information to plan the course of treatment of pneumonia. Through the rapid identification of the causative pathogens, the HIRA-TAN will promote targeted treatments for pneumonias. CLINICAL TRIALS REGISTRATION: UMIN000001694.

Type-2 innate immune responses that occur in airways and are accompanied by goblet-cell hyperplasia and mucus production are largely driven by interleukin-33 (IL-33) and natural helper (NH) cells, a member of group 2 innate lymphoid cells (ILC2s) and the major target of IL-33. Here we report that the corticosteroid resistance observed as a result of airway inflammation triggered by sensitization and exposure to allergen is induced via the IL-33/NH-cell axis. Thymic stromal lymphopoietin (TSLP) synthesized during airway inflammation plays a pivotal role in the induction of NH-cell corticosteroid resistance in vitro and in vivo, by controlling phosphorylation of STAT5 and expression of Bcl-xL in NH cells. Blockade of TSLP with a neutralizing antibody or blocking the TSLP/STAT5 signalling pathway with low molecular-weight STAT5 inhibitors such as pimozide restores corticosteroid sensitivity. Thus, the TSLP-STAT5 pathway could be a new therapeutic target in severe, corticosteroid-resistant asthma.

Acute lung injury (ALI) is a severe illness with excess mortality and no specific therapy. In its early exudative phase, neutrophil activation and accumulation in the lung lead to hypoxemia, widespread tissue damage, and respiratory failure. In clinical trials, inhibition of proinflammatory mediators has not proven effective. In this study, we pursued a new investigative strategy that emphasizes mediators promoting resolution from lung injury. A new spontaneously resolving experimental murine model of ALI from acid aspiration was developed to identify endogenous proresolving mechanisms. ALI increased cyclooxygenase 2 (COX-2) expression in murine lung. Selective pharmacologic inhibition or gene disruption of COX-2 blocked resolution of ALI. COX-2-derived products increased levels of the proresolving lipid mediators lipoxin A4 (LXA4) and, in the presence of aspirin, 15-epi-LXA4. Both LXA4 and 15-epi-LXA4 interact with the LXA4 receptor (ALX) to mediate anti-inflammatory actions. ALX expression was markedly induced by acid injury and transgenic mice with increased ALX expression displayed dramatic protection from ALI. Together, these findings indicate a protective role in ALI for COX-2-derived mediators, in part via enhanced lipoxin signaling, and carry potential therapeutic implications for this devastating clinical disorder.

Thromboxane A2 receptor (TP) mediates bronchial smooth muscle cell (BSMC) contraction, airway hyperresponsiveness, and airway inflammation in patients with asthma. In the present study, a pathogenic role of TP activation in airway remodeling was examined using primary cultures of human BSMC. A TP agonist, I-BOP, concentration-dependently enhanced not only bromodeoxyuridine (BrdU) uptake but also cell proliferation of BSMC. A TP-selective antagonist, AA-2414, blocked the effects of I-BOP on both BrdU uptake and cell proliferation. I-BOP-induced BrdU uptake was significantly blocked by two non-selective tyrosine kinase inhibitors, genistein and herbimycin A, or a Src family tyrosine kinase inhibitor, PP2, but not by an inhibitor of epidermal growth factor (EGF) receptor-associated tyrosine kinase, AG1478. In conclusion, TP receptor activation causes DNA synthesis and cell proliferation of human BSMC by activating tyrosine kinases including Src, but not by EGF receptor transactivation.

Our understanding of asthma severity was advanced by the identification of biomarkers which account for differences in lung function impairment. We tried to examine the effects of corticosteroid treatment on known correlates of asthma severity including peripheral eosinophil counts, total IgE, IL-5, and eotaxin Levels in plasma. We compared these biomarkers among groups of stable asthmatics categorized by the dose of corticosteroid (N: steroid-free, n = 25; L: low-dose inhaled, n = 27; MH: medium or high-dose inhaled, n= 19; O: inhaled plus oral, n= 8). Next we compared these markers and peak expiratory flow rate (PEFR) in unstable asthmatics before and after treatment with steroids (n = 22). Eotaxin levels in the O group were higher than those in the N and MH groups (P < 0.05). Logistic regression analysis demonstrated that plasma eotaxin level was correlated with the severity of asthma defined by treatment intensity (P = 0.01) and % FEV1 (P = 0.04) while the other markers were not. Eotaxin levels did not change after steroid treatment in unstable patients, whereas eosinophil counts decreased in parallel with PEFR. Among biomarkers of asthma severity studied, plasma eotaxin level was not significantly affected by corticosteroid treatment, and was associated with the severity even in the presence of steroid therapy.

Approximately 4% of the Japanese population genetically lack plasma platelet activating factor acetylhydrolase (PAF-AH) and show a higher prevalence of thromboembolic disease, but whether they are susceptible to another PAF-related disease, asthma, remains controversial. To determine the role of plasma PAF-AH in airway physiology, we performed PAF bronchoprovocation tests in 8 plasma PAF-AH-deficient subjects and 16 control subjects. Serial inhalation of PAF (1-1000 microg/ml) concentration-dependently induced acute bronchoconstriction, but there was no significant difference between PAF-AH-deficient and control subjects (11.7 +/- 4.6% vs. 9.6 +/- 2.8% decrease in forced expiratory volume in 1 s). Transient neutropenia after single inhalation of PAF (1000 microg/ml) showed no significant difference between the groups either in its magnitude (72 +/- 11% vs. 65 +/- 9% decrease) or duration (4.1 +/- 1.0 vs. 3.3 +/- 0.8 min). In conclusion, a lack of plasma PAF-AH activity alone does not augment physiological responses to PAF in the airway.

We retrospectively analyzed the effects of dosages of docetaxel (DOC) in combination chemotherapy with carboplatin (CBDCA) against advanced non-small cell lung cancer (NSCLC). Twenty-three cases of previously untreated NSCLC (stage III and IV, performance status: 0-1) that were pathologically or cytologically confirmed were divided into 2 groups (DOC-60, DOC-70) based on their received DOC doses (60 and 70 mg/m2, respectively). Both groups had similar patient characteristics and received similar treatment except for their DOC doses. Although not statistically significant, the response rates after the completion of whole cycles of the treatment (mean: 4 cycles) were 18.2% in DOC-60 and 41.6% in DOC-70. Leukocytopenia, neutropenia and anemia were observed similarly in both groups. However, while thrombocytopenia was not observed in DOC-70, it was found in 36.4% of the cases in DOC-60. In combination chemotherapy with carboplatin against advanced NSCLC, the use of DOC at 70 mg/m2 was rather protective against thrombocytopenia, keeping the response equivalent or possibly superior to that obtained in the patients treated with DOC at 60 mg/m2.

CysLT(1) antagonists are effective for a subset of patients with asthma; however, there has been no good way to predict a given patient's response. We examined the interaction between the clinical response to a cysLT(1) antagonist, pranlukast, and DNA sequence variant A(-444)C in leukotriene C(4) synthase (LTC(4) S) gene in Japanese patients with moderate asthma. The frequency of LTC(4) S C(-444) allele was 21.6% in the Japanese general population (n = 171) and 19.4% in the asthmatic subjects ( n= 349). A 4-week prospective, open trial of pranlukast (225 mg twice daily) was performed in 50 patients with moderate asthma who had been well controlled with inhaled corticosteroid (beclomethasone 400-800 microg/day or fluticasone 200-400 microg/day). The C(-444) allele carriers (n = 16) responded better to pranlukast compared to the A(-444) allele homozygotes ( n= 31) [14.3 5.3% vs. 3.1 2.4% improvement of forced expiratory volume in one second (FEV(1) ), 0.01], while LTC(4) S genotype-stratified response to inhaled beta-agonist salbutamol (200 microg) was not observed (17.5 2.1% vs. 18.7 2.2% improvement of FEV(1) ). Univariate analysis demonstrated that the better response to pranlukast (more than 10% improvement of FEV(1) ) was correlated with LTC(4) S genotype (P < 0.01) and pretreatment airway reversibility to salbutamol (P < 0.01), but not with sex, age, atopic status, urinary leukotriene E(4) excretion rate, or daily dose of inhaled corticosteroid. Furthermore, multivariate regression analysis suggested that LTC(4) S genotype and the bronchodilatory effect of salbutamol were independent variables to predict the clinical response to pranlukast (P < 0.05). We conclude that LTC(4) S genotype is predictive of the clinical response to a cysLT(1) antagonist, pranlukast, in Japanese patients with moderate asthma.

Prostaglandins and thromboxanes are important modulators of airway physiology. The synthesis of these mediators depends on two isoforms of cyclooxygenase (COX), constitutive COX-1 and inducible COX-2. COX-2 expression has been observed in various inflammatory diseases, but not all aspects of the expression and the role of COX-2 in conditions of allergic inflammation such as asthma are clear. In the present study, we examined the 72-h kinetics of the expression of COX-isoform mRNA in ovalbumin-sensitized and -challenged guinea-pig lungs. The sensitized animals showed a robust and transient induction of COX-2 mRNA expression within 1 h after ovalbumin challenge, whereas their COX-1 mRNA levels remained unchanged. Upregulation of the level and activity of COX-2 protein followed the induction of COX-2 mRNA. Lung slices harvested from ovalbumin-challenged animals released more prostaglandin D(2) and prostaglandin E(2) spontaneously or in response to A23187 (10 microM) ex vivo than did those from unchallenged animals. This response was significantly blocked by the COX-2 selective inhibitors, NS-398 and JTE-522. In vivo administration of NS-398 significantly inhibited the accumulation of eosinophils and neutrophils in the lungs. In conclusion, de novo COX-2 expression during allergic inflammation modifies prostanoid synthesis in the lung and airway pathophysiology.

OBJECTIVE AND DESIGN: The gene expression profile of CCR3 ligands, eotaxin, RANTES, and monocyte chemoattractant protein-3 (MCP-3), was examined in normal and inflamed guinea pig lungs. MATERIAL: Male Hartley guinea pigs (n = 49). METHODS: Pulmonary mRNA was obtained from naive animals, animals treated with intravenous lipopolysaccharide administration, and animals repeatedly exposed to aerosolized allergen (ovalbumin). Northern analysis was performed to quantify pulmonary expression of eotaxin, RANTES, and MCP-3 mRNA. Pulmonary eosinophil peroxidase (EPO) activity was measured to quantify eosinophil accumulation. RESULTS: Eotaxin and RANTES mRNAs, but not MCP-3 mRNA, were constitutively expressed in guinea pig lungs. Lipopolysaccharide treatment increased MCP-3 mRNA expression, but not eotaxin or RANTES mRNA. In contrast, allergen exposure in sensitized animals caused an increase in eotaxin mRNA, which demonstrated good temporal and quantitative correlation with pulmonary EPO activity, but not in MCP-3 or RANTES mRNA. CONCLUSIONS: Guinea pig CCR3 ligands demonstrated different gene expression profiles in normal and inflamed airways, suggesting that they play different physiological and pathophysiological roles in the airway.

Various proinflammatory and vasoactive actions of platelet-activating factor (PAF) are mediated through a specific G-protein-coupled PAF receptor (PAFR). We identified a novel DNA variant in the human PAFR gene, which substitutes an aspartic acid for an alanine residue at position 224 (A224D) in the putative third cytoplasmic loop. This mutation was observed in a Japanese population at an allele frequency of 7.8%. To delineate the functional consequences of this structural alteration, Chinese hamster ovary cells were stably transfected with constructs encoding either wild-type or A224D mutated PAFR. No significant difference was observed in the expression level of the receptor or the affinity to PAF or to an antagonist, WEB2086, between the cells transfected with wild-type and mutant PAFR. Chinese hamster ovary cells expressing A224D mutant PAFR displayed partial but significant reduction of PAF-induced intracellular signals such as calcium mobilization, inositol phosphate production, inhibition of adenylyl cyclase, and chemotaxis. These findings suggest that this variant receptor produced by a naturally occurring mutation exhibits impaired coupling to G-proteins and may be a basis for interindividual variation in PAF-related physiological responses, disease predisposition or phenotypes, and drug responsiveness.

Whole genome scan analyses have revealed that chromosomal region 3p21-24, which contains a gene cluster of CC chemokine receptors such as CCR3, is possibly linked to asthma. Because CCR3 ligands play a pivotal role in the selective recruitment and activation of inflammatory cells in the asthmatic airway, the authors examined whether there is any association between asthma and the CCR3 gene polymorphisms. Three polymorphisms were identified using the single stranded conformational polymorphism method in Japanese (Asian) and British (Caucasian) subjects; one silent mutation T51C and two missense mutations G824A and T971C. These polymorphisms were examined in 391 Japanese subjects (210 asthmatics and 181 nonasthmatic controls) and 234 British subjects (142 asthmatics and 92 nonasthmatic controls). Asthma diagnosis was based on episodic symptoms, documented wheeze, and the presence of reversible airflow limitation. CCR3 T51C demonstrated a significant association with the diagnosis of asthma in the British population (odds ratio 2.35, p<0.01), but not in the Japanese population. Multiple logistic regression analysis also showed that CCR3 T51C was associated with asthma (odds ratio 2.83, p < 0.02), independent of atopic phenotypes such as high levels of total or house dust mite-specific immunoglobulin-E in serum. In conclusion, a significant association between asthma and CCR3 T51C polymorphism localized on chromosome 3p21 was found.

Platelet activating factor (PAF) is immediately degraded and inactivated in the bloodstream by plasma PAF acetylhydrolase (PAF-AH). Although plasma PAF-AH-like activity was secreted in vitro from various cell types such as macrophages and hepatocytes, the exact cellular source(s) of this enzyme activity in vivo remains unclear. There is a naturally-occurring missense mutation (V279F) in the plasma PAF-AH gene in the Japanese population which results in complete loss of the enzyme activity. We analyzed 52 Japanese who had received an allogeneic bone marrow transplant and maintained donor-derived hematopoiesis. Ten recipients had chimeric plasma PAF-AH genotypes between the donor-derived peripheral blood leukocytes and the recipient-derived epithelial cells of buccal mucosa. Multiple regression analysis demonstrated that PAF-AH activity in plasma depended on the donor's genotype (standardized regression coefficient = 0.68, P < 0.0001), but not on the recipient's genotype (p = 0.48). One recipient who was a V279F homozygote in leukocytes and wild type homozygote in buccal mucosa had undetectable PAF-AH activity in plasma. We conclude that most of the PAF-AH activity in human plasma originates from hematopoietic lineage cells.

Platelet-activating factor (PAF), a phospholipid with a wide range of proinflammatory actions, is immediately degraded and inactivated in vivo by PAF acetylhydrolase (PAF-AH). Surprisingly, 4% of the Japanese population lacks the extracellular isoform of this enzyme, plasma PAF-AH, due to a genetic missense (V279F) mutation. We studied the association of this mutation with asthma prevalence and phenotypes in the Japanese adult population. The allele frequency of V279F mutation was 18.6% in 279 patients with asthma (28.7% heterozygotes and 4.3% homozygotes) and 21.7% in 217 healthy subjects (32.3% heterozygotes and 5.5% homozygotes). V279F mutant allele prevalence was consistent regardless of asthma type (16.3% in atopic [n = 156] and 21.6% in nonatopic [n = 123]), or the severity of disease (21.7% in patients with mild [n = 97], 17.5% in those with moderate [n = 131], and 15.8% in those with severe [n = 51] asthma). Plasma PAF-AH activity was inversely proportional to the number of mutant alleles, and did not correlate with asthma prevalence, type, or severity. We concluded that plasma PAF-AH deficiency due to V279F mutation is not essential to the pathophysiology of asthma in the Japanese adult population.