Influence of Epinastine Hydrochloride, an H1-Receptor Antagonist, on the Function of Mite Allergen-Pulsed Murine Bone Marrow-Derived Dendritic Cells In Vitro and In Vivo

There is established concept that dendritic cells (DCs) play essential roles in the development of allergic immune responses. However, the influence of H1 receptor antagonists on DC functions is not well defined. The aim of the present study was to examine the effect of epinastine hydrochloride (EP), the most notable histamine H1 receptor antagonists in Japan, on Dermatophagoides farinae (Der f)-pulsed mouse bone marrow-derived DCs in vitro and in vivo. EP at more than 25 ng/mL could significantly inhibit the production of IL-6, TNF-α and IL-10 from Der f-pulsed DCs, which was increased by Der f challenge in vitro. On the other hand, EP increased the ability of Der f-pulsed DCs to produce IL-12. Intranasal instillation of Der f-pulsed DCs resulted in nasal eosinophilia associated with a significant increase in IL-5 levels in nasal lavage fluids. Der f-pulsed and EP-treated DCs significantly inhibited nasal eosinophila and reduced IL-5. These results indicate that EP inhibits the development of Th2 immune responses through the modulation of DC functions and results in favorable modification of clinical status of allergic diseases

Suppression of matrix metalloproteinase-9 production from neutrophils by a macrolide antibiotic, roxithromycin, in vitro.

BACKGROUND: Macrolide antibiotics such as erythromycin and roxithromycin (RXM) have an anti-inflammatory effect that may account for their clinical benefit in the treatment of chronic airway inflammatory diseases. However, the precise mechanism of this anti-inflammatory effect is not well understood. PURPOSE: The influence of RXM on matrix metalloproteinase (MMP)-9 production from neutrophils in response to lipopolysaccharide (LPS) stimulation was examined in vitro. METHODS: Neutrophils prepared from normal human peripheral blood (1 x 10(5) cells/ml) were treated with various concentrations of RXM for 1 h, and then stimulated with 1.0 microg/ml of LPS in the presence of the agent for 24 h. MMP-9 and tissue inhibitor of metalloproteinase (TIMP)-1 levels in culture supernatants were examined by enzyme-linked immunosorbent assay. RESULTS: Addition of RXM at more than 5.0 microg/ml into cell cultures caused significant suppression of MMP-9 production, which was increased by LPS stimulation. However, the ability of cells to produce TIMP-1 was not affected by RXM treatment, even when the cells were cultured in the presence of agent at 10.0 microg/mL We then examined the influence of RXM on transcriptional factor, nuclear factor-kappaB and activator protein (AP)-1 activation by LPS stimulation. RXM exerted suppressive action on NF-kappaB (P50 and P65) activation when the cells were cultured for 4 h at more than 5.0 microg/ml of the agent. RXM at more than 2.5 microg/ml also suppressed AP-1 (Fra 1 and Jun B) activation in 4-h cultured cells. We finally examined the influence of RXM on MMP-9 mRNA expression in neutrophils. Addition of RXM into cell cultures at more than 5.0 microg/ml caused significant inhibition of mRNA expression, which was enhanced by LPS stimulation for 12 h. CONCLUSION: These results strongly suggest that RXM inhibits neutrophil transmigration into inflammatory sites and results in favorable modification of the clinical status of inflammatory diseases

Suppressive activity of tiotropium bromide on matrix metalloproteinase production from lung fibroblasts in vitro

Kazuhito Asano1, Yusuke Shikama2, Yasuhiro Shibuya2, Hiroaki Nakajima2, Ken-ichi Kanai3, Naohiro Yamada3, Harumi Suzaki31Division of Physiology, School of Nursing and Rehabilitation Sciences; 2Respiratory Disease Center, Showa University Northern Yokohama Hospital, Yokohama, Japan; 3Department of Otolaryngology, School of Medicine, Showa University, Tokyo, JapanBackground: Chronic obstructive pulmonary disease (COPD) is characterized by airway remodeling with an accumulation of inflammatory cells. There is also increasing evidence that metalloproteinases (MMPs) may contribute to the pathogenesis of COPD, but the influence of agents that used for the treatment of COPD is not well understood.Objective: We evaluated whether tiotropium bromide hydrate (TBH), a M3 muscarinic receptor antagonist, could inhibit MMP production from lung fibroblasts (LFs) in response to tumor necrosis factor (TNF)-α stimulation.Methods: LFs were established from normal lung tissues taken from patients with lung tumors. LFs (5 × 105 cells/ml) were stimulated with TNF-α in the presence of various concentrations of TBH. After 24 h, culture supernatants were obtained and assayed for the levels of MMPs and tissue inhibitor of metalloproteinases (TIMPs) by ELISA. The influence of TBH on mRNA expression of MMPs and TIMPs in 4 h-cultured cells was also examined by real-time RT-PCR. Furthermore, nuclear factor-κB (NF-κB) and activator protein-1 (AP-1) in LFs treated with TBH for 4 h was examined by ELISA.Results: TBH at more than 15 pg/ml inhibited the production of MMP-2 from LFs after TNF-α stimulation, whereas TIMP-1 and TIMP-2 production was scarcely affected by TBH through the suppression of both mRNA expression and transcription factor, NF-κB, activation in LFs induced by TNF-α stimulation.Conclusion: These results suggest that the attenuating effect of TBH on MMP-2 production from LFs induced by inflammatory stimulation may be additional beneficial therapeutic effects not directly relating to its bronchodilatory effects.Keywords: tiotropium, lung fibroblasts, matrix metalloproteinases, suppression, in vitr

Suppressive activity of macrolide antibiotics on nitric oxide production by lipopolysaccharide stimulation in mice.

BACKGROUND: Low-dose and long-term administration of macrolide antibiotics into patients with chronic airway inflammatory diseases could favorably modify their clinical conditions. However, the therapeutic mode of action of macrolides is not well understood. Free oxygen radicals, including nitric oxide (NO), are well recognized as the important final effector molecules in the development and the maintenance of inflammatory diseases. PURPOSE: The influence of macrolide antibiotics on NO generation was examined in vivo. METHODS: Male ICR mice, 5 weeks of age, were orally administered with either roxithromycin, clarithromycin, azithromycin or josamycin once a day for 2-4 weeks. The mice were then injected intraperitoneally with 5.0 mg/kg lipopolysaccharide (LPS) and the plasma NO level was examined 6 h later. RESULTS: Although pre-treatment of mice with macrolide antibiotics for 2 weeks scarcely affected NO generation by LPS injection, the administration of macrolide antibiotics, except for josamycin, for 4 weeks significantly inhibited LPS-induced NO generation. The data in the present study also showed that pre-treatment of mice with macrolide antibiotics for 4 weeks significantly suppresses not only production of pro-inflammatory cytokines interleukin-1beta, interleukin-6, and tumor necrosis factor-alpha, but also inducible nitric oxide synthase mRNA expressions, which are enhanced by LPS injection. CONCLUSION: These results strongly suggest that suppressive activity of macrolide antibiotics on NO generation in response to LPS stimulation in vivo may, in part, account for the clinical efficacy of macrolides on chronic inflammatory diseases

Age-dependent plasticity in the superior temporal sulcus in deaf humans: a functional MRI study

BACKGROUND: Sign-language comprehension activates the auditory cortex in deaf subjects. It is not known whether this functional plasticity in the temporal cortex is age dependent. We conducted functional magnetic-resonance imaging in six deaf signers who lost their hearing before the age of 2 years, five deaf signers who were >5 years of age at the time of hearing loss and six signers with normal hearing. The task was sentence comprehension in Japanese sign language. RESULTS: The sign-comprehension tasks activated the planum temporale of both early- and late-deaf subjects, but not that of hearing signers. In early-deaf subjects, the middle superior temporal sulcus was more prominently activated than in late-deaf subjects. CONCLUSIONS: As the middle superior temporal sulcus is known to respond selectively to human voices, our findings suggest that this subregion of the auditory-association cortex, when deprived of its proper input, might make a functional shift from human voice processing to visual processing in an age-dependent manner

Enantioselective disposition of clenbuterol in rats

Clenbuterol is a long-acting β2-adrenoceptor agonist and bronchodilator that is used for the treatment of asthma, but the desired activities reside almost exclusively in the (-)-R-enantiomer. This study examined enantioselectivity in the disposition of clenbuterol following administration of clenbuterol racemate to rats. Concentrations of clenbuterol enantiomers in plasma, urine and bile were determined by LC-MS/MS assay with a Chirobiotic T column. This method was confirmed to show high sensitivity, specificity and precision, and clenbuterol enantiomers in 0.1ml volumes of plasma were precisely quantified at concentrations as low as 0.25ng/ml. The pharmacokinetic profiles of clenbuterol enantiomers following intravenous and intraduodenal administration of clenbuterol racemate (2mg/kg) in rats were significantly different. The distribution volume of (-)-R-clenbuterol (9.17l/kg) was significantly higher than that of (+)-S-clenbuterol (4.14l/kg). The total body clearance of (-)-R-clenbuterol (13.5ml/min/kg) was significantly higher than that of the (+)-S-enantiomer (11.5ml/min/kg). An in situ absorption study in jejunal loops showed no difference in the residual amount between the (-)-R- and (+)-S-enantiomers. Urinary clearance was the same for the two enantiomers, but biliary excretion of (-)-R-clenbuterol was higher than that of the (+)-S-enantiomer. The fractions of free (non-protein-bound) (-)-R- and (+)-S-clenbuterol in rat plasma were 48.8% and 33.1%, respectively. These results indicated that there are differences in the distribution and excretion of the clenbuterol enantiomers, and these may be predominantly due to enantioselective protein binding. © 2013 John Wiley & Sons, Ltd.発行後1年より全文公

Clinical Aspect of Peripheral Cholangiocarcinoma: A Study of 7 Hepatectomy Cases

To clarify the features and problems presented by a peripheral cholangiocarcinoma (CCC), seven patients with hepatectomy from the First Department of Surgery, Nagasaki University School of Medicine (6 patients), and from Department of Surgery, National Ureshino Hospital (one patient) were reviewed. Men predominate with ratio of 5:2, and an average age was 65.4 years. Tumor location was left lateral segment in 4 patients, right lobe, middle lobe and posterior segment in one, respectively. Three patients were associated with hepatolithiasis. Underlying liver disease was found in 4 patients (57%); cirrhosis in 3 patients, and chronic hepatitis in one. Initial symptoms were abdominal pain, fever and palpable abdominal mass. In imaging modalities available, the detection rates of tumor were 100% in CT and 67% in US and angiography, respectively. Combination of MRI and CT clearly showed tumor characteristics. The serum CEA was slightly elevated in 5 patients (83%), but serum CA19-9 rose strikingly in 3 patients. Most tumors showed an infiltrating growth along intrahepatic bile duct, with a portal vein thrombus and/or satellite tumors frequently. In 3 patients, early recurrence with intrahepatic metastasis occured within the first 6 months. The patient of poorly differentiated adenocarcinoma containing a squamous or signet ring cell carinoma showed an extremely poor prognosis. This study suggests that early detection of small CCC and an extended resection are the most important factors for the survival of patient

Gene Organization in Rice Revealed by Full-Length cDNA Mapping and Gene Expression Analysis through Microarray

Rice (Oryza sativa L.) is a model organism for the functional genomics of monocotyledonous plants since the genome size is considerably smaller than those of other monocotyledonous plants. Although highly accurate genome sequences of indica and japonica rice are available, additional resources such as full-length complementary DNA (FL-cDNA) sequences are also indispensable for comprehensive analyses of gene structure and function. We cross-referenced 28.5K individual loci in the rice genome defined by mapping of 578K FL-cDNA clones with the 56K loci predicted in the TIGR genome assembly. Based on the annotation status and the presence of corresponding cDNA clones, genes were classified into 23K annotated expressed (AE) genes, 33K annotated non-expressed (ANE) genes, and 5.5K non-annotated expressed (NAE) genes. We developed a 60mer oligo-array for analysis of gene expression from each locus. Analysis of gene structures and expression levels revealed that the general features of gene structure and expression of NAE and ANE genes were considerably different from those of AE genes. The results also suggested that the cloning efficiency of rice FL-cDNA is associated with the transcription activity of the corresponding genetic locus, although other factors may also have an effect. Comparison of the coverage of FL-cDNA among gene families suggested that FL-cDNA from genes encoding rice- or eukaryote-specific domains, and those involved in regulatory functions were difficult to produce in bacterial cells. Collectively, these results indicate that rice genes can be divided into distinct groups based on transcription activity and gene structure, and that the coverage bias of FL-cDNA clones exists due to the incompatibility of certain eukaryotic genes in bacteria

頭頸部領域および食道領域の各種症状に対する食道運動機能の関与について : High Resolution Manometry を用いた検討

頭頸部領域および食道領域における各種症状に対する食道運動機能の関与を検討した.2007年9月から2012年6月までに,咽喉頭異常感などの頭頸部領域の症状および嚥下困難感,胸痛,胸やけなどの食道領域に関連した症状を主訴に,当科を受診した261例(男性138例,女性123例,平均年齢56.8±17.1才)を対象とし,健康関連QOL(Health Related Quality of Life: HRQL)の測定と食道内圧検査を施行した.健康関連QOLの検討では,咽喉頭違和感,嚥下困難感,胸やけ等の各症状を訴えた患者で,身体的QOL,精神的QOL を表すPCS(physical component summary)あるいはMCS(mental component summary)が低下し,健常者と比較して有意にQOLの低下を認めた.食道内圧検査による食道運動機能異常は,全対象患者中62.0%に認めた.各症状別に食道運動機能障害の内訳を見ると,咽喉頭違和感ではIEM(ineffective esophageal motility)(31.8%),嚥下困難感は食道アカラシア(56.6%),喉のつかえ感は食道アカラシア(35.5%),胸やけはIEM(39.4%),胸痛は食道アカラシア(50.0%),噫気はIEM(50.0%)を最も多く認めた.咽喉頭違和感,嚥下困難感,喉のつかえ感,胸やけ,胸痛などの頭頚部および食道症状を有するものの,器質的疾患を認めない患者のQOLは障害されており,その病態の一つとして食道運動機能異常の存在を念頭に置き,診療にあたることが重要である.There are many cases of patients presenting at the hospital complaining of dysphagia or chest pain. At that time organic diseases are not always detected in these patients;previously several studies showed esophageal motility dysfunction was one of the important factors in the development of these symptoms. However, there have been few reports investigating the relationship between esophageal motility dysfunction and these symptoms. Between September 2007 and June 2012, 216 patients (138 men,123 women;mean age 56.8±17.1 years old) were admitted complaining of the following symptoms:dysphagia, globus sensation, or heart burn and non-cardiac chest pain,but without any organic diseases related symptoms. They all underwent high-resolution esophageal manometry (HRM) and completed a questionnaire regarding their health-related quality of life (HRQOL). The aim of this study was to evaluate the relationship between these symptoms and esophageal motility dysfunction and QOL. In our series, 62% of patients with non-obstructive dysphagia, globus sensation, heartburn and unexplained (non-cardiac) chest pain have an esophageal motility disorder (Achalasia 31.5%, IEM 26.4%, Diffuse esophageal spasm 4.6%;nutcracker esophagus 2.8% and Impaired ECJ relaxation 0.5%). SF-8 score of PCS(Physical Component Summary) in all of the symptoms were significantly lower in comparison with the matched healthy control. SF-8 score of MCS(Mental Component Summary) with globus sensation and esophageal dysphagia were significantly lower in comparison with the matched healthy control. The patients complaining of several symptoms located in the pharynx or esophagus have impaired quality of life and esophageal motility dysfunctions.It is important to consider these issues while examining these patients

Suppressive Activity of Vitamin D3 on Matrix Metalloproteinase Production From Cholesteatoma Keratinocytes In Vitro

There is much evidence that degradation of the extracellular matrix is essential for the development of cholesteatomas and that this is induced by activation of matrix metalloproteinases (MMPs). Vitamin D3 (VD3) has several well-recognised biological activities, including suppression of MMP production. The present study, therefore, was undertaken to examine whether VD3 could suppress MMP production from cholesteatoma keratinocytes in vitro. Keratinocytes (2.5×105 cells/mL) induced from cholesteatoma tissue specimens were cultured with various concentrations of VD3. After one hour, lipopolysaccharide was added to the cell cultures at 100 μg/mL. The culture supernatants were then collected and assayed for MMP-1 and MMP-3 by ELISA. We also used ELISA to measure the levels of both TIMP (tissue inhibitor of metalloproteinase)-1 and TIMP-2 in culture supernatants. Addition of VD3 into keratinocyte cultures caused the suppression of MMP and TIMP production, which was increased by LPS stimulation. This was dose-dependent. The present results showing the suppressive activity of VD3 on the production of MMPs, which are responsible for tissue remodeling, strongly suggest that VD3 would be a good candidate for an agent in the medical treatment of, or prophylaxis for, cholesteatomas