It is well known that low-dose and long-term administration of macrolide antibiotics favourably modify the clinical status of chronic airway inflammatory diseases. However, the therapeutic mode of action of macrolide antibiotics is not well understood. The present study aimed to examine the influence of macrolide antibiotics, roxithromycin (RXM) and josamycin (JM) on matrix metalloproteinase (MMP) production from nasal polyp fibroblasts (NPF)in vitro.NPF, at a concentration of 2.5×10⁵cells·mL⁻¹, were stimulated with tumour necrosis factor (TNF)-α in the presence of various concentrations of RXM or JM for 24 h. MMP‐2 and ‐9 levels in culture supernatants were analysed by ELISA, and MMP mRNA expression was examined by RT-PCR. The influence of RXM on nuclear factor (NF)-κB and activator protein (AP)‐1 activation was also examined.Addition of RXM (but not JM) at 5.0 and 7.5 µg·mL⁻¹significantly suppressed the production of MMP‐2 and ‐9 from NPF induced by TNF-α stimulation. RXM also suppressed MMP mRNA expression through the inhibition of NF-κB and AP-1 activation.The present results suggest that the suppressive activity of roxithromycin on MMP‐2 and ‐9 production is, in part, responsible for the therapeutic action of macrolides on chronic airway inflammatory diseases.

When human myelogenous leukemic cell lines, HL-60 and ML-1, were exposed to 50 Hz electromagnetic fields (EMFs), nucleosome-sized DNA fragmentation (a biochemical marker of apoptosis) was induced as assessed by agarose gel electrophoresis. However, EMFs could not induce detectable DNA fragmentation in either human peripheral blood leukocytes or polymorphonuclear cells. The minimum exposure period required for the onset of DNA fragmentation in leukemic cells was 1.0 h. Although adverse effects of EMFs on human health have been reported, the apoptosis-inducing action of EMFs on leukemic cells may suggest a possible medical application.

Tiotropium bromide inhibits TGF-ß-induced MMP production from lung fibroblasts by interfering with Smad and MAPK pathways in vitro Kazuhito Asano1, Yusuke Shikama2, Naruo Shoji3, Kojiro Hirano3, Harumi Suzaki3, Hiroaki Nakajima21Division of Physiology, School of Nursing and Rehabilitation Sciences, Showa University, Yokohama, Japan; 2Department of Respiratory Diseases, Showa University Northern Yokohamashi Hospital, Yokohama, Japan; 3Department of Otorhinolaryngology, School of Medicine, Showa University, Tokyo, JapanBackground: Chronic obstructive pulmonary disease (COPD) is characterized by chronic inflammation and structural alterations (ie, tissue remodeling) throughout the conducting airways, parenchyma, and pulmonary vasculature. Matrix metalloproteinases (MMPs) are extracellular degrading enzymes that play a critical role in inflammatory cell infiltration and tissue ­remodeling, but the influence of the agents that are used for the treatment of COPD on the production of MMPs is not well understood.Purpose: The present study aimed to examine the influence of tiotropium bromide hydrate (TBH) on the production of MMPs from lung fibroblasts (LFs) induced by transforming growth factor (TGF)-ß in vitro.Methods: LFs, at a concentration of 5 × 105 cells·mL-1, were stimulated with TGF-ß in the p­resence of various concentrations of TBH. MMP-1 and MMP-2 levels in culture ­supernatants were examined by enzyme-linked immunosorbent assay (ELISA), and MMP messenger ­ribonucleic acid (mRNA) expression was examined by real-time polymerase chain reaction (RT-PCR). The influence of TBH on TGF-ß signaling pathways was also analyzed by ­examining Smad activation and signaling protein phosphorylation by ELISA.Results: TBH at more than 15 pg·mL-1 inhibited the production of MMP-1 and MMP-2, but not tissue inhibitor of matrix metalloproteinase (TIMP)-1 and TIMP-2, from LFs, after TGF-ß stimulation. TBH also suppressed MMP mRNA expression through the inhibition of Smad activation and signaling protein, extracellular-signal-regulated kinase (ERK) 1 and 2, and c-Jun N-terminal kinase (JNK), phosphorylation.Conclusion: These results may suggest that TBH suppresses MMP production from LFs, through interference of TGF-ß-mediated signaling pathways and results in favorable ­modification of the clinical status of COPD.Keywords: tiotropium bromide, matrix metalloproteinases, lung fibroblast, TGF-ß, inhibition, in vitro

Introduction. Medical care for Japanese cancer patients includes Western and Kampo medicines, and treatments with juzentaihoto (JTT) reportedly prevent cancer metastasis and recurrence. In this study, we examined the effects of JTT on natural killer (NK) cell activity and metastasis in combined treatments with anti‐PD‐1 antibody in a mouse model of melanoma metastasis. Methods. C57BL/6 male mice were intravenously injected with B16 melanoma cells (B16 cell) and were given chow containing 3% JTT. In subsequent in vivo experiments, we assessed serum cytokine levels and tumor colony formation in the lungs. Additionally, we assessed NK cell activity in ex vivo experiments. Results. JTT significantly suppressed B16 cell metastasis, whereas injection of anti‐asialo‐GM1 antibody into mice abrogated the inhibitory actions of JTT. JTT significantly increased interleukin‐ (IL‐) 12 and interferon‐ (IFN‐) γ levels in serum and induced NK cell activity. It increased the inhibitory actions of the anti‐PD‐1 antibody on B16 cell metastasis. Discussion. These data suggest that JTT inhibits B16 cell metastasis by inducing NK cell activity. Additionally, combination therapy with JTT and anti‐PD‐1 antibody increased treatment response rates for B16 melanoma.

Quercetin, a dietary flavonoid found in many fruits, red wine and onion, among others, has been reported to have potent anti-oxidant, anti-viral and anti-cancer effects. Although quercetin is also reported to have anti-inflammatory and anti-allergic effects, the precise mechanisms by which quercetin favorably modify the clinical conditions of allergic diseases such as allergic rhinitis (AR). The present study was designed to examine the influence of quercetin on the development of AR by using AR model rats. Sprague-Dawley (SD) rats were sensitized with toluene 2,4-diisocyanate (TDI) by intranasal instillation of a 10 % TDI in ethyl acetate in a volume of 5 μl once a day for 5 consecutive days. This sensitization procedure was repeated after a 2-day interval. After 5 days of the second sensitization, rats were treated with various doses of quercetin once a day for 2 to 7 days. Nasal allergy-like symptoms, which were induced by bilateral application of 5 μl of 10 % TDI in ethyl acetate, were assessed by counting sneezing and nasal rubbing behaviors for 10 min just after TDI nasal challenge. The levels of substance P (SP), calcitonin gene-related peptide (CGRP) and nerve growth factor (NGF) in nasal lavage fluids obtained 6 h after TDI nasal challenge was examined by ELISA. Oral administration of quercetin for 5 and 7 days, but not 2 and 3 days, could inhibit sneezing and nasal rubbing movements, which were increased by TDI nasal challenge. The minimum dose that caused significant inhibition was 25 mg/kg. Oral administration of quercetin at more than 25 mg/kg for 5 days significantly inhibited the increase in SP, CGRP and NGF contents in nasal lavage fluids induced by TDI nasal challenge. The present results strongly suggested that quercetin will be a good candidate for the supplement on the management and treatment of allergic diseases, especially AR.

Nitric oxide (NO) is known to play pivotal roles as one of the final effector molecules in the development of allergic diseases, including allergic rhinitis (AR). Although quercetin has been reported to attenuate the clinical conditions of AR, its influence on NO production is not well defined. The present study aimed to examine the influence of quercetin onin vitroNO production from nasal epithelial cells after interleukin‐ (IL‐) 4 stimulation. Human nasal epithelial cells (HNEpCs) at a concentration of 1 x 10⁵cells/ml were stimulated with 10.0 ng/ml of IL‐4 in the presence and absence of quercetin. After 48 hours, the culture supernatants were collected and assayed for NO (NO₂and NO₃) using the Griess method. The influences of quercetin on the transcription factor, STAT6, activation, and iNOS mRNA expression were also examined using ELISA and real‐time quantitative RT‐PCR, respectively. Addition of quercetin to cell cultures caused suppression of NO production from HNEpCs after IL‐4 stimulation. The minimum concentration of quercetin that caused significant suppression was 1.0 nM. Treatment of cells with quercetin at more than 1.0 nM suppressed STAT6 activation and iNOS mRNA expression induced by IL‐4 stimulation. The present results strongly suggested that quercetin favorably modified the clinical condition of AR through the suppression of NO production from nasal epithelial cells after IL‐4 stimulation.