Yasuhito Higashiyama

Azithromycin Inhibits MUC5AC Production Induced by the Pseudomonas aeruginosa Autoinducer N-(3-Oxododecanoyl) Homoserine Lactone in NCI-H292 Cells

ABSTRACT The features of chronic airway diseases, including chronic bronchitis, cystic fibrosis, bronchiectasis, and diffuse panbronchiolitis, include chronic bacterial infection and airway obstruction by mucus. Pseudomonas aeruginosa is one of the most common pathogens in chronic lung infection, and quorum-sensing systems contribute to the pathogenesis of this disease. The quorum-sensing signal molecule [N-(3-oxododecanoyl) homoserine lactone (3O-C12-HSL)] not only regulates bacterial virulence but also is associated with the immune response. In this study, we investigated whether 3O-C12-HSL could stimulate the production of a major mucin core protein, MUC5AC. The effect of a macrolide on MUC5AC production was also studied. 3O-C12-HSL induced NCI-H292 cells to express MUC5AC at both the mRNA and the protein levels in time- and dose-dependent manners. A 15-membered macrolide, azithromycin, inhibited MUC5AC production that was activated by 3O-C12-HSL. 3O-C12-HSL induced extracellular signal-regulated kinase (ERK) 1/2 and I-κB phosphorylation in cells, and this induction was suppressed by azithromycin. 3O-C12-HSL-induced MUC5AC production was blocked by the ERK pathway inhibitor PD98059. Our findings suggest that the P. aeruginosa autoinducer 3O-C12-HSL contributes to excessive mucin production in chronic bacterial infection. Azithromycin seems to reduce this mucin production by interfering with intracellular signal transduction.

Immunokinetics in severe pneumonia due to influenza virus and bacteria coinfection in mice

Coinfections of bacteria and influenza are a major cause of excessive mortality during influenza epidemics. However, the mechanism of the synergy between influenza virus and bacteria are poorly understood. In this study, mice were inoculated with influenza virus, followed 2 days later by inoculation with Streptococcus pneumoniae. The kinetics of viral titres, bacterial numbers and the immune response (cytokine and chemokine production) were also analysed. Short-term survival correlated with pathological changes in the lungs of infected mice. Influenza virus or S. pneumoniae infection alone induced moderate pneumonia; however, severe bronchopneumonia with massive haemorrhage in coinfected mice, which caused death of these mice ∼2 days after inoculation with S. pneumoniae, was noted. Intrapulmonary levels of inflammatory cytokines/chemokines, type‐1 T‐helper cell cytokines and Toll-like receptors, and the related mitogen-activated protein kinase signalling molecules (phosphorylated extracellular signal-regulated kinase ‐1 and ‐2, p38 and c‐Jun N‐terminal kinase), were increased in coinfected mice. These results suggest that immune mediators, including cytokines and chemokines, through Toll-like receptors/mitogen-activated protein kinase pathways, play important roles in the pathology of coinfection caused by influenza virus and Streptococcus pneumoniae.

Azithromycin Exhibits Bactericidal Effects on Pseudomonas aeruginosa through Interaction with the Outer Membrane

ABSTRACT The aim of the present study was to elucidate the effect of the macrolide antibiotic azithromycin on Pseudomonas aeruginosa. We studied the susceptibility to azithromycin in P. aeruginosa PAO1 using a killing assay. PAO1 cells at the exponential growth phase were resistant to azithromycin. In contrast, PAO1 cells at the stationary growth phase were sensitive to azithromycin. The divalent cations Mg2+ and Ca2+ inhibited this activity, suggesting that the action of azithromycin is mediated by interaction with the outer membranes of the cells, since the divalent cations exist between adjacent lipopolysaccharides (LPSs) and stabilize the outer membrane. The divalent cation chelator EDTA behaved in a manner resembling that of azithromycin; EDTA killed more PAO1 in the stationary growth phase than in the exponential growth phase. A 1-N-phenylnaphthylamine assay showed that azithromycin interacted with the outer membrane of P. aeruginosa PAO1 and increased its permeability while Mg2+ and Ca2+ antagonized this action. Our results indicate that azithromycin directly interacts with the outer membrane of P. aeruginosa PAO1 by displacement of divalent cations from their binding sites on LPS. This action explains, at least in part, the effectiveness of sub-MICs of macrolide antibiotics in pseudomonal chronic airway infection.

Regulation of γ-glutamycysteine synthetase expression in response to oxidative stress

Glutathione (GSH) is synthesized by the activity of two ATP-requiring GSH synthesizing enzymes. Gammaglutamylcysteine synthetase (γ-GCS) is the rate limiting enzyme for the GSH synthesis. γ-GCS is a heterodimer of heavy, catalytic subunit and light, regulatory subunit and responsive to many stresses, such as heat shock, oxidative stress or cytokines. To know the regulation of the expression of γ-GCS gene, in the present study, we show evidences that γ-GCS heavy subunit is upregulated by oxidative stress by ionizing radiation and TNF-α mediated by nuclear factor-κB (NF-κB), and impairment of the expression of γ-GCS by TNF-α in diabetic condition. Furthermore we describe the importance of GSH in the regulation of NF-κB subunits.

Acute infection with influenza virus enhances susceptibility to fatal pneumonia following Streptococcus pneumoniae infection in mice with chronic pulmonary colonization with Pseudomonas aeruginosa

We established a mouse model in which fatal pneumonia was induced by pneumococcal superinfection following influenza virus infection in chronic Pseudomonas aeruginosa infected mice. In this mouse model, influenza virus infection caused a significant increase in inflammatory cells, cytokines and severe tissue damage in the lungs of these P. aeruginosa infected mice, before pneumococcal infection. Intrapulmonary virus titres were significantly increased in mice with chronic P. aeruginosa infection, compared with control mice. Neutrophil function analysis showed significant reduction of myeloperoxidase (MPO) activity and lysozyme secretion by influenza virus infection in these mice. Our results suggest that influenza virus infection may play an important role in inducing pneumococcal pneumonia in chronic P. aeruginosa infected mice. Our results suggested that our mouse model is useful for investigating the pathogenesis of influenza virus infection in patients with chronic lung infection.

Reactive oxygen species accelerate production of vascular endothelial growth factor by advanced glycation end products in RAW264.7 mouse macrophages.

Advanced glycation end products (AGEs) are believed to play an important role in the development of angiopathy in diabetes mellitus. Previous reports suggested a correlation between accumulation of AGEs and production of vascular endothelial growth factor (VEGF) in human diabetic retina. However, the mechanisms involved were not revealed. In this study, we investigated the transcriptional regulation of the expression of vascular endothelial growth factor (VEGF) by AGEs, and possible involvement of reactive oxygen species (ROS) in the induction. We employed an AGE of bovine serum albumin (BSA) prepared by an incubation of BSA with D-glucose for 40 weeks and N(epsilon)-(carboxymethyl)lysine (CML), a major AGE. The expression of VEGF was induced by CML-BSA in RAW264.7 mouse macrophage-like cells. CML-BSA stimulated the DNA-binding activity of activator protein-1 (AP-1). Promoter assay showed that the induction of VEGF was dependent on AP-1. The activity of Ras/Raf-1/MEK/ERK1/2 was involved in the CML-BSA-stimulated signaling pathways to activate the AP-1 transcription with a peak at 1 h. AGE-BSA also induced VEGF mediated by AP-1, however, there was a difference of effect between AGE-BSA and CML-BSA in the activation of AP-1. AGE-BSA-stimulated AP-1 activity showed a peak at 5 h, which paralleled the formation of ROS. Reduction of AGE-BSA with NaBH(4) or addition of vitamin E attenuated the AGE-BSA-stimulated signaling pathways leading to the same pattern as for CML-BSA-stimulated signals. These results suggest an important role for AGEs in stimulation of the development of angiogenesis observed in diabetic complications, and that ROS accelerates the AGE-stimulated VEGF expression.

Intrapulmonary concentrations of inflammatory cytokines in a mouse model of chronic respiratory infection caused by Pseudomonas aeruginosa

We investigated the role of inflammatory cytokines in a mouse model of chronic Pseudomonas aeruginosa infection mimicking diffuse panbronchiolitis (DPB), and determined the effects of clarithromycin therapy on the production of these cytokines. The concentrations of IL‐1β, IL‐2, IL‐4, IL‐5, interferon‐gamma (IFN‐γ) and tumour necrosis factor‐alpha (TNF‐α) were measured serially in the lungs of mice with experimentally induced chronic respiratory P. aeruginosa infection until 60 days after inoculation. The concentrations of these cytokines during the course of the disease were significantly higher than baseline (before inoculation, P < 0·01 for all cytokines). Clarithromycin significantly inhibited the production of IL‐1β and TNF‐α in the lung (P < 0·01). The same treatment also reduced the levels of other cytokines, albeit insignificantly. Treatment with anti‐TNF‐α antibody significantly reduced the number of pulmonary lymphocytes and concentration of IL‐1β in the lung (P < 0·01), but did not change the number of viable bacteria. Our findings resemble those detected in bronchoalveolar lavage fluid of patients with DPB and indicate that inflammatory cytokines play an important role in chronic P. aeruginosa lung infection. Our results also show that macrolides modulated the production of these cytokines, ultimately reducing lymphocyte accumulation in the lung. Our data suggest that anti‐TNF‐α antibody might be a useful new strategy for the treatment of chronic respiratory P. aeruginosa infection.

In Vitro and In Vivo Activities of Novel Fluoroquinolones Alone and in Combination with Clarithromycin against Clinically Isolated Mycobacterium avium Complex Strains in Japan

ABSTRACT The recommended treatments for Mycobacterium avium complex (MAC) infectious disease are combination regimens of clarithromycin (CLR) or azithromycin with ethambutol and rifamycin. However, these chemotherapy regimens are sometimes unsuccessful. Recently developed antimicrobial agents, such as newer fluoroquinolones (FQs) containing C-8 methoxy quinolone (moxifloxacin [MXF] and gatifloxacin [GAT]), are expected to be novel antimycobacterial agents. Here, we evaluated the in vitro and in vivo antimycobacterial activities of three FQs (MXF, GAT, and levofloxacin) and CLR against clinically isolated MAC strains. Subsequently, the in vitro and in vivo synergic activities of FQ-CLR combinations against MAC strains were investigated. CLR and the individual FQs alone showed promising activity against MAC strains in vitro, and the bacterial counts in organs (lungs, liver, and spleen) of MAC-infected mice treated with single agents were significantly reduced compared to control mice. CLR showed the best anti-MAC effect in vivo. When the three FQs were individually combined with CLR in vitro, mild antagonism was observed for 53 to 57% of the tested isolates. Moreover, mice were infected with MAC strains showing mild antagonism for FQ-CLR combinations in vitro, and the anti-MAC effects of the FQ-CLR combinations were evaluated by counting the viable bacteria in their organs and by histopathological examination after 28 days of treatment. Several FQ-CLR combinations exhibited bacterial counts in organs significantly higher than those in mice treated with CLR alone. Our results indicate that the activity of CLR is occasionally attenuated by combination with an FQ both in vitro and in vivo and that this effect seems to be MAC strain dependent. Careful combination chemotherapy using these agents against MAC infectious disease may be required.

Effects of macrolides on pneumolysin of macrolide-resistant Streptococcus pneumoniae

To clarify the discrepancy between increasing resistance and conservative clinical effects of macrolides on macrolide-resistant Streptococcus pneumoniae, the authors evaluated the effects of sub-minimum inhibitory concentrations of macrolides on pneumolysin. In vitro, S. pneumoniae was incubated with 1, 2 and 4 µg·mL−1 of clarithromycin (CLR) and azithromycin (AZM) for 8 h. Western blot analysis and haemolytic assay were performed to examine the production and activities of pneumolysin. In vivo, mice were infected with S. pneumoniae intra-nasally and treated with CLR (40 or 200 mg·kg−1 twice daily) or AZM (40 or 200 mg·kg−1 once daily) orally for 7 days. After 72 h post-infection, western blot analysis was performed to examine pneumolysin production in lungs. Survival rates were observed for 10 days. In vitro, every concentration of macrolide inhibited pneumolysin production more than the control. CLR (2 and 4 µg·mL−1) and AZM (4 µg·mL−1) reduced the pneumolysin activities more than the control. In vivo, macrolides (200 mg·kg−1) reduced pneumolysin in murine lungs more than the control. CLR (40 and 200 mg·kg−1) and AZM (200 mg·kg−1) improved the survival rates more than the control. The study results show that sub-minimum inhibitory concentrations of macrolides reduced pneumolysin. This might be related to the effectiveness of macrolides against pneumonia caused by high-level macrolide-resistant Streptococcus pneumoniae. Further investigations are necessary to evaluate the effects of macrolides on macrolide-resistant Streptococcus pneumoniae.

Environmental isolation of Cryptococcus neoformans from an endemic region of HIV-associated cryptococcosis in Thailand

We successfully isolated Cryptococcus neoformans from chicken faeces in suburban areas of Thailand. C. neoformans was isolated from 36/150 houses (24.0%) in the dry season and 6/150 (4.0%) in the rainy season. All environmental isolates were of serotype A. The high isolation rate of 24% from chicken faeces has never been reported previously. Our environmental study could probably explain the high incidence of cryptococcal meningitis in HIV patients in Thailand. Copyright