Fabrication of nanofibrous inhaler device.

It has been proven that administration by inhalation is one of the alternative and effective approaches to dose biomedicines such as insulin for diabetics. Here, we introduce a new approach to fabricate the inhaler, posing the merits of not using volatile solvent, having selectable size of drug particles, and being simple and low cost compared with conventional devices. Creatine particles were prepared by spray-drying, and simultaneously a nanofiber mat of crosslinked poly(vinyl pyrrolidone) (PVP) was used to collect the graduated creatine nanoparticles. The scanning electron microscopy (SEM) photos indicated that the nanoparticles were individually dispersed without any agglomerate on the PVP nanofibers and released after the nitrogen gas flow (10 L/min) passed through the PVP nanofiber mat. This approach is unique and universal for nonagglomerate dispersing of nanoparticles and releasing without using any dispersing solvent.

Promoter activity of human tissue inhibitor of metalloproteinase 2 gene with novel single nucleotide polymorphisms.

OBJECTIVE: The single nucleotide polymorphism (SNP) -418G > C in the TIMP2 gene promoter region has been shown to be associated with in chronic obstructive pulmonary disease (COPD). The purpose of this study was to search for novel single nucleotide polymorphism (SNP) in the TIMP2 promoter region around the -418G > C locus, and to investigate whether any of these SNP, including -418G > C, had an influence on TIMP2 transcription activity. METHODOLOGY: DNA sequencing was performed on a 689 base-pair polymerase chain reaction fragment of the promoter region. The novel SNP were characterized and genotype analysis was performed for COPD and control subjects. A reporter gene assay was performed using the wild-type promoter (-418G/-177C/+34C) and the mutant-type promoter (-418C/-177T/+34A). RESULTS: Nine novel SNP were identified. The SNP -177C > T and +34C > A were in complete linkage disequilibrium with -418G > C. The other seven SNP were not associated with COPD. No significant difference was detected in the reporter gene assay between the activities of the wild-type and the mutant-type promoters. CONCLUSIONS: The SNP -418G > C, -177C > T and +34C > A, might not themselves be functional from a transcriptional point of view in the development of COPD, but may be in linkage disequilibrium with other functional polymorphisms.

Anti-inflammatory activity of creatine supplementation in endothelial cells in vitro.

1 Creatine (CR) supplementation augments muscle strength in skeletal muscle cells by increasing intracellular energy pools. However, the effect of CR supplementation on endothelial cells remains to be clarified. 2 In this study, we investigated whether CR supplementation had any anti-inflammatory activity against human pulmonary endothelial cells in culture. 3 We confirmed that supplementation with 0.5 mM CR significantly increased both intracellular CR and phosphocreatine (PC) through a CR transporter while keeping intracellular ATP levels constant independent of CR supplementation and a CR transporter antagonist. 4 In the assay system of endothelial permeability, supplementation with 5 mM CR significantly suppressed the endothelial permeability induced by serotonin and H(2)O(2). 5 In cell adhesion experiments, supplementation with 5 mM CR significantly suppressed neutrophil adhesion to endothelial cells. 6 In the measurement of adhesion molecules, CR supplementation with more than 0.5 mM CR significantly inhibited the expressions of ICAM-1 and E-selectin on endothelial cells, and the inhibition was significantly suppressed by an adenosine A(2A) receptor antagonist. 7 The present study suggests that CR supplementation has anti-inflammatory activities against endothelial cells.

S-carboxymethylcysteine inhibits neutrophil activation mediated by N-formyl-methionyl-leucyl-phenylalanine.

In this study, the possible mechanisms of action for the inhibitory effects of S-carboxymethylcysteine on the activation of human neutrophils by N-formyl-methionyl-leucyl-phenylalanine (FMLP) were investigated. Preincubation of neutrophils with more than 10 microg/ml of S-carboxymethylcysteine was found to impair neutrophil chemotactic activity toward FMLP, and to inhibit FMLP-mediated neutrophil adherence to pulmonary vascular endothelial cells. Preincubation of neutrophils with 10 and 100 microg/ml of S-carboxymethylcysteine decreased in the production of inositol 1,4,5-triphosphate (IP(3)) and diacylglycerol in neutrophils stimulated with FMLP, respectively. Preincubation of neutrophils with S-carboxymethylcysteine did not affect the cellular cyclic AMP (cAMP) levels in neutrophils stimulated with FMLP. S-carboxymethylcysteine inhibited the enzymatic activity of phosphatidyl inositol-specific phospholipase C in vitro in a concentration-dependent manner. These findings indicate that S-carboxymethylcysteine attenuates FMLP-stimulated neutrophil activation at least in part by inhibiting phosphatidyl inositol-specific phospholipase C-mediated signal transduction.

Ebselen suppresses late airway responses and airway inflammation in guinea pigs.

Although ebselen, a seleno-organic compound, inhibits inflammation in various animal models, its efficacy as an anti-asthma drug remains to be clarified. In this study, we investigated the inhibitory effect of ebselen on a guinea pig asthma model. Ebselen was orally administered at dosages of 1-20 mg/kg 2 h before an ovalbumin (OA) challenge, and then airway responses, airway inflammation, the generation of superoxide, H(2)O(2), and nitrotyrosine, and the induction of inducible nitric oxide synthase (iNOS) were evaluated. Sensitized animals challenged with OA aerosol showed dual airflow limitations, i.e., immediate and late airway responses (IAR and LAR). Ebselen significantly inhibited LAR at dosages greater than 10 mg/kg, but did not inhibit IAR at any dosage. Bronchoalveolar lavage (BAL) examination showed that airway inflammation was significantly suppressed by ebselen at 10 mg/kg. The generation of superoxide and H(2)O(2) occurred on endothelial cells of LAR bronchi, and was inhibited by 10 mg/kg of ebselen. Superoxide generation was inhibited by diphenyleneiodonium chloride (DPI), a NAD(P)H oxidase inhibitor, but not by allopurinol, a xanthine oxidase inhibitor. Immunoreactivities for iNOS and nitrotyrosine were also observed on endothelial cells of LAR bronchi and were abolished in ebselen-treated animals. The present findings suggest that ebselen can be applied as a new therapeutic agent for asthma. The possible mechanisms by which ebselen inhibits LAR likely involve suppression of oxidant formation and iNOS induction in endothelial cells.

Involvement of thromboxane A(2) in airway mucous cells in asthma-related cough.

The aim of this study was to elucidate the role of thromboxane A(2) (TxA(2)) on asthma-related cough in guinea pigs. Animals were immunosensitized and repeatedly challenged with ovalbumin as an antigen. Coughs were induced by the inhalation of 10(-5) M capsaicin solution for 10 min. Thromboxane synthetase (TxS) inhibitor OKY-046 and thromboxane-receptor antagonist AA-2414 significantly inhibited cough responses in repeatedly challenged animals. Inhalation of TxA(2) mimic STA-2- potentiated cough responses in normal and immunosensitized animals but not in repeatedly challenged ones. Moreover, STA-2-potentiated coughs were inhibited by administration of neurokinin-receptor antagonist FK-224. In repeatedly challenged animals, concentration of TxB(2) in airway lavage fluid, expression of TxS mRNA in tracheal epithelia, and the immunostaining intensity against TxS in mucous cells of the epithelium significantly increased compared with normal and sensitized animals. These results suggest that TxA(2) derived from mucous cells potentiated cough responses to capsaicin in allergic airway inflammation.