Effect of fluid replacement with green tea on body fluid balance and renal responses under mild thermal hypohydration: a randomized crossover study.

PURPOSE: Maintaining an appropriate hydration level by ingesting fluid in a hot environment is a measure to prevent heat-related illness. Caffeine-containing beverages, including green tea (GT), have been avoided as inappropriate rehydration beverages to prevent heat-related illness because caffeine has been assumed to exert diuretic/natriuretic action. However, the influence of caffeine intake on urine output in dehydrated individuals is not well documented. The aim of the present study was to examine the effect of fluid replacement with GT on body fluid balance and renal water and electrolyte handling in mildly dehydrated individuals. METHODS: Subjects were dehydrated by performing three bouts of stepping exercise for 20 min separated by 10 min of rest. They were asked to ingest an amount of water (H2O), GT, or caffeinated H2O (20 mg/100 ml; Caf-H2O) that was equal to the volume of fluid loss during the dehydration protocol; fluid balance was measured for 2 h after fluid ingestion. RESULTS: The dehydration protocol induced hypohydration by ~ 10 g/kg body weight (~ 1% of body weight). Fluid balance 2 h after fluid ingestion was significantly less negative in all trials, and the fluid retention ratio was 52.2 ± 4.2% with H2O, 51.0 ± 5.0% with GT, and 47.9 ± 6.2% with Caf-H2O; those values did not differ among the trials. After rehydration, urine output, urine osmolality, and urinary excretions of osmotically active substances, sodium, potassium and chloride were not different among the trials. CONCLUSION: The data indicate that ingestion of GT or an equivalent caffeine amount does not worsen the hydration level 2 h after ingestion and can be effective in reducing the negative fluid balance for acute recovery from mild hypohydration. TRIAL REGISTRATION: ISRCTN53057185; retrospectively registered.

Hippocampal Inflammation and Gene Expression Changes in Peripheral Lipopolysaccharide Challenged Mice Showing Sickness and Anxiety-Like Behaviors.

Neuroinflammation is often associated with the development of depressive and anxiety disorders. The hippocampus is one of the brain regions affected by inflammation that is associated with these symptoms. However, the mechanism of hippocampal inflammation-induced emotional behavior remains unknown. The aim of this study was to clarify temporal changes in the neuroinflammatory responses in the hippocampus and the response of dentate gyrus (DG) neurons using peripheral lipopolysaccharide (LPS)-challenged mice. LPS administration induced anxiety-like activity in the elevated plus maze test and social interaction test after 24 h, at which time the mice had recovered from sickness behavior. We examined the hippocampal inflammation-related gene expression changes over time. The expression of interleukin-1ß (Il1b) and tumor necrosis factor α (Tnfa) was rapidly enhanced and sustained until 24 h after LPS administration, whereas the expression of Il6 was transiently induced at approx. 6 h. IL-6-dependent downstream signaling of transducer and activator of transcription 3 (STAT3) was also activated approx. 3-6 h after LPS treatment. The expression of innate immune genes including interferon-induced transmembrane proteins such as interferon-induced transmembrane protein 1 (Ifitm1) and Ifitm3 and complement factors such as C1qa and C1qb started to increase approx. 6 h and showed sustained or further increase at 24 h. We also examined changes in the expression of several maturation markers in the DG and found that LPS enhanced the expression of calbindin 1 (Calb1), tryptophan-2,3-dioxigenase 2 (Tdo2), Il1rl, and neurotrophin-3 (Ntf3) at 24 h after LPS treatment. Collectively, these results demonstrate temporal changes of inflammation and gene expression in the hippocampus in LPS-induced sickness and anxiety-like behaviors.

Dynamic changes in insulin requirements with post-operative time using bedside artificial pancreas to maintain normoglycemia without hypoglycemia after cardiac surgery.

It is difficult to manage postoperative blood glucose levels without hyperglycemia and hypoglycemia in cardiac surgery patients even if continuous intravenous insulin infusion is used. Therefore, the insulin requirements for maintaining normoglycemia may be difficult to evaluate and need to be elucidated. In this single-center retrospective study, 30 adult patients (age 71.5 ± 9.0 years old, men 67%, BMI 22.0 ± 3.1 kg/m2, diabetes 33%) who underwent cardiac surgery and used bedside artificial pancreas (STG-55) as a perioperative glycemic control were included. We investigated the insulin and glucose requirements to maintain normoglycemia until the day after surgery. The bedside artificial pancreas achieved intensive glycemic control without hypoglycemia under fasting conditions for 15 h after surgery (mean blood glucose level was 103.3 ± 3.1 mg/dL and percentage of time in range (70-140 mg/dL) was 99.4 ± 2.0%). The total insulin requirement for maintaining normoglycemia differed among surgical procedures, including the use of cardiopulmonary bypass during surgery, while it was not affected by age, body mass index, or the capacity of insulin secretion. Moreover, the mean insulin requirement and the standard deviation of the insulin requirements were variable and high, especially during the first several hours after surgery. Treatment using the bedside artificial pancreas enabled intensive postoperative glycemic control without hypoglycemia. Furthermore, the insulin requirements for maintaining normoglycemia after cardiac surgery vary based on surgical strategies and change dynamically with postoperative time, even in the short term.

Anti-fungal activity of a novel triazole, PC1244, against emerging azole-resistant Aspergillus fumigatus and other species of Aspergillus.

OBJECTIVES: The growing emergence of azole-resistant Aspergillus fumigatus strains worldwide is a major concern for current systemic antifungal treatment. Here we report antifungal activities of a novel inhaled triazole, PC1244, against a collection of multi-azole-resistant A. fumigatus strains. METHODS: MICs of PC1244 were determined for A. fumigatus carrying TR34/L98H (n = 81), TR46/Y121F/T289A (n = 24), M220 (n = 6), G54 (n = 11), TR53 (n = 1), TR463/Y121F/T289A (n = 2), G448S (n = 1), G432C (n = 1) and P216S (n = 1) resistance alleles originating from either India, the Netherlands or France. The effects of PC1244 were confirmed in an in vitro model of the human alveolus and in vivo in temporarily neutropenic, immunocompromised mice. RESULTS: PC1244 exhibited potent inhibition [geometric mean MIC (range), 1.0 mg/L (0.125 to >8 mg/L)] of growth of A. fumigatus strains carrying cyp51A gene mutations, showing much greater potency than voriconazole [15 mg/L (0.5 to >16 mg/L)], and an effect similar to those on other azole-susceptible Aspergillus spp. (Aspergillus flavus, Aspergillus terreus, Aspergillus tubingensis, Aspergillus nidulans, Aspergillus niger, Aspergillus nomius, Aspergillus tamarii) (0.18-1 mg/L). In TR34/L98H and TR46/Y121F/T289A A. fumigatus-infected in vitro human alveolus models, PC1244 achieved superior inhibition (IC50, 0.25 and 0.34 mg/L, respectively) compared with that of voriconazole (IC90, >3 mg/L and >10 mg/L, respectively). In vivo, once-daily intranasal administration of PC1244 (0.56-70 µg/mouse) to the A. fumigatus (AF91 with M220V)-infected mice reduced pulmonary fungal load and serum galactomannan more than intranasal posaconazole. CONCLUSIONS: PC1244 has the potential to become a novel topical treatment of azole-resistant pulmonary aspergillosis.

Cell death-inducing DNA fragmentation factor A-like effector A and fat-specific protein 27ß coordinately control lipid droplet size in brown adipocytes.

Adipose tissue stores neutral lipids and is a major metabolic organ involved in regulating whole-body energy homeostasis. Triacylglycerol is stored as unilocular large lipid droplets (LDs) in white adipocytes and as multilocular small LDs in brown adipocytes. Proteins of the cell death-inducing DNA fragmentation factor A-like effector (Cide) family include CideA, CideB, and fat-specific protein of 27 (FSP27). Of these, FSP27 has been shown to play a crucial role in the formation of unilocular large LDs in white adipocytes. However, the mechanisms by which brown adipocytes store small and multilocular LDs remain unclear. An FSP27 isoform, FSP27ß, was recently identified. We herein report that CideA and FSP27ß are mainly expressed in brown adipose tissue and that FSP27ß overexpression inhibits CideA-induced LD enlargements in a dose-dependent manner in COS cells. Furthermore, RNAi-mediated FSP27ß depletion resulted in enlarged LDs in HB2 adipocytes, which possess the characteristics of brown adipocytes. Brown adipocytes in FSP27-knock-out mice that express CideA, but not FSP27ß, had larger and fewer LDs. Moreover, we confirmed that FSP27ß and CideA form a complex in brown adipose tissue. Our results suggest that FSP27ß negatively regulates CideA-promoted enlargement of LD size in brown adipocytes. FSP27ß appears to be responsible for the formation of small and multilocular LDs in brown adipose tissue, a morphology facilitating free fatty acid transport to mitochondria adjacent to LDs for oxidation in brown adipocytes.

In Vitro and In Vivo Efficacy of a Novel and Long-Acting Fungicidal Azole, PC1244, on Aspergillus fumigatus Infection.

The antifungal effects of the novel triazole PC1244, designed for topical or inhaled administration, against Aspergillus fumigatus were tested in a range of in vitro and in vivo studies. PC1244 demonstrated potent antifungal activities against clinical A. fumigatus isolates (n = 96) with a MIC range of 0.016 to 0.25 µg/ml, whereas the MIC range for voriconazole was 0.25 to 0.5 µg/ml. PC1244 was a strong tight-binding inhibitor of recombinant A. fumigatus CYP51A and CYP51B (sterol 14α-demethylase) enzymes and strongly inhibited ergosterol synthesis in A. fumigatus with a 50% inhibitory concentration of 8 nM. PC1244 was effective against a broad spectrum of pathogenic fungi (MIC range, <0.0078 to 2 µg/ml), especially Aspergillus terreus, Trichophyton rubrum, Candida albicans, Candida glabrata, Candida krusei, Cryptococcus gattii, Cryptococcus neoformans, and Rhizopus oryzae PC1244 also proved to be quickly absorbed into both A. fumigatus hyphae and bronchial epithelial cells, producing persistent antifungal effects. In addition, PC1244 showed fungicidal activity (minimum fungicidal concentration, 2 µg/ml) which indicated that it was 8-fold more potent than voriconazole. In vivo, once-daily intranasal administration of PC1244 (3.2 to 80 µg/ml) to temporarily neutropenic, immunocompromised mice 24 h after inoculation with itraconazole-susceptible A. fumigatus substantially reduced the fungal load in the lung, the galactomannan concentration in serum, and circulating inflammatory cytokine levels. Furthermore, 7 days of extended prophylaxis with PC1244 showed in vivo effects superior to those of 1 day of prophylactic treatment, suggesting accumulation of the effects of PC1244. Thus, PC1244 has the potential to be a novel therapy for the treatment of A. fumigatus infection in the lungs of humans.

Decreased carbonyl reductase 1 expression promotes tumor growth via epithelial mesenchymal transition in uterine cervical squamous cell carcinomas.

PURPOSE: Carbonyl reductase 1 (CBR1) is involved in cancer progression. Recently, the authors reported that the loss of CBR1 expression is associated with a poor prognosis in uterine cervical cancer. Here, we investigated whether the decreased CBR1 expression promotes cancer progression by inducing the epithelial mesenchymal transition (EMT). METHODS: Antisense constructs of CBR1 complementary DNA (antisense clones) and the empty vectors (control clones) were transfected into human uterine cervical squamous cell carcinoma cell lines (SKG II and SiHa) and the proliferation and EMT marker expression of these clones were analyzed in vitro. In an in vivo study, 107 cells of the antisense and control clones were subcutaneously injected into nude mice and the tumorigenesis was observed for 8 weeks. RESULTS: With the decreased CBR1 expression, the proliferation of the antisense clones increased, accompanied by a decrease in epithelial markers (E-cadherin and cytokeratin) and an increase in mesenchymal markers (fibronectin, alpha-smooth muscle actin, and N-cadherin), which suggests EMT induction. In the in vivo study, the tumor volume in the antisense group was significantly larger than that in the control group. CONCLUSION: Decreased CBR1 expression promotes tumor growth by inducing EMT in uterine cervical squamous cell carcinomas.

Effects of intranasally dosed posaconazole on fungal load and biomarkers in Aspergillus fumigatus infected immunocompromised mice.

Although anti-fungal triazoles are dosed orally or systemically for Aspergillus fumigatus infection, systemic adverse events and limited exposure of the lung cavity would make a topical treatment for the lung an attractive option. In this study, we examined the effects of intranasally dosed posaconazole on survival rates and biomarkers in A. fumigatus (itraconazole susceptible: ATCC13073 [Af]; or resistant: NCPF7100 [AfR]) infected, temporarily neutropenic A/J mice. Once daily treatment produced a dose-dependent improvement of survival of Af-infected mice (ED50 : 0.019 mg/mouse [approx. 0.755 mg/kg, in]), similar to its potency (ED50 : 0.775 mg/kg, po) after once daily oral dosing. For AfR infection, either intranasal or oral posaconazole was largely ineffective on survival, although the highest dose of intranasal treatment (0.35 mg/mouse) achieved 75% survival rate. Early intervention (treated on days 0, 1, 2 and 3 postinfection) and late intervention (treated on days 1, 2 and 3) with intranasal posaconazole (0.014-0.35 mg/mouse) demonstrated potent inhibition of lung fungal load and galactomannan levels in both bronchoalveolar lavage fluid (BALF) and serum as well as inflammatory cells, IFN-γ, IL-17 and malondialdehyde (MDA) in BALF. Thus, posaconazole when dosed intranasally once daily showed an improvement of survival equivalent to or better than oral treatment, and produced potent inhibition of fungal load and biomarkers.

Nitric Oxide Production-Inhibitory Activity of Limonoids from Azadirachta indica and Melia azedarach.

Seventy-three limonoids isolated from three Meliaceae plants, Azadirachta indica, A. indica var. siamensis, and Melia azedarach, or semi-synthesized from the Meliaceae limonoids, were evaluated for their inhibitory activity against nitric oxide (NO) production in mouse macrophage RAW 264.7 cells induced by lipopolysaccharide (LPS), as a primary screening test for anti-inflammatory agents. Of the compounds tested, 21 compounds exhibited inhibitory activity (IC50 4.6 - 58.6 µm) without any significant toxicity (IC50 > 100 µm) which were more potent than l-NMMA (NO-production inhibitory activity, IC50 65.6 µm; cytotoxicity, IC50 > 100 µm), and among which, nine compounds, i.e., 17-hydroxy-15-methoxynimbocinol (6), ohchinin (20), 1-cis-cinnamoyl-1-decinnamoylohchinin (24), salannin (27), methyl nimbidate (32), isosalannin (55), nimbolinin D (58), mesendanin E (69), and 7-deacetylgedunin (73) exhibited potent inhibitory activity (IC50 4.6 - 29.3 µm). In particular, compounds 6 (IC50 7.3 µm), an azadirone-type limonoid, and 73 (IC50 4.6 µm), a gedunin-type limonoid, exhibited remarkable activity. Western blot analysis revealed that 27 and 73 reduced the expression levels of the inducible NO synthase and cyclooxygenase-2 proteins in a concentration-dependent manner. These findings suggest that limonoids of A. indica, A. indica var. siamensis, and M. azedarach, and their semi-synthetic derivatives may be effective against inflammation.

Functional significance of transgelin-2 in uterine cervical squamous cell carcinoma.

AIM: Transgelin-2 (TAGLN2) has previously been found to be highly expressed in uterine cervical squamous cell carcinoma (SCC) tissues by proteomic analyses. The present study investigated the role of TAGLN2 in the malignant behaviors of cervical SCC cells in vitro and in vivo, and the clinical significance of TAGLN2 using immunohistochemistry for human cervical SCC tissues. METHODS: Antisense (AS) constructs of TAGLN2 cDNA (AS clones) and the empty vector (control clone) were transfected into a human uterine SCC cell line (SKG IIIa), and malignant behaviors were analyzed in vitro. In an in vivo experiment, 10(7) cells of the AS and control clones were subcutaneously inoculated into female BALB/c nude mice. In immunohistochemistry with anti-TAGLN2 antibodies for human cervical SCC, FIGO stage IA and IB (n = 75), the expression patterns of TAGLN2 were divided into two groups: weak and strong. The relation between expression pattern and prognosis was analyzed. RESULTS: Suppression of TAGLN2 inhibited cancer cell migration and secretion of matrix metalloproteinases. Tumors in the control clone group continued to grow, whereas those in the AS clone group clearly stopped growing. Six weeks after injection, the tumor size was significantly smaller in the AS clone group than in the control clone group. Immunohistochemistry revealed that the strong pattern was associated with poor overall survival compared with the weak pattern by the Kaplan-Meier method. CONCLUSION: TAGLN2 plays functional roles in the progression of cervical SCC. Suppression of TAGLN2 may be a new strategy for the treatment of cervical SCC.

Cytotoxic and nitric oxide production-inhibitory activities of limonoids and other compounds from the leaves and bark of Melia azedarach.

Nine limonoids, 1-9, one apocarotenoid, 11, one alkaloid, 12, and one steroid, 13, from the leaf extract; and one triterpenoid, 10, five steroids, 14-18, and two flavonoids, 19 and 20, from the bark extract of Melia azedarach L. (Chinaberry tree; Meliaceae) were isolated. Among these compounds, three compounds, 4-6, were new, and their structures were established as 3-deacetyl-28-oxosalannolactone, 3-deacetyl-28-oxosalanninolide, and 3-deacetyl-17-defurano-17,28-dioxosalannin, respectively, on the basis of extensive spectroscopic analyses and comparison with literature data. All of the isolated compounds were evaluated for their cytotoxic activities against leukemia (HL60), lung (A549), stomach (AZ521), and breast (SK-BR-3) cancer cell lines. 3-Deacetyl-4'-demethyl-28-oxosalannin (3) against HL60 and AZ521 cells, and methyl kulonate (10) against HL60 cells exhibited potent cytotoxicities with IC50 values in the range of 2.8-5.8 µM. In addition, upon evaluation of compounds 1-13 against production of nitric oxide (NO) in mouse macrophage RAW 264.7 cells induced by lipopolysaccharide (LPS), seven, i.e., trichilinin B (1), 4, ohchinin (7), 23-hydroxyohchininolide (8), 21-hydroxyisoohchininolide (9), 10, and methyl indole 3-carboxylate (12), inhibited production of NO with IC50 values in the range of 4.6-87.3 µM with no, or almost no, toxicity to the cells (IC50 93.2-100 µM). Western blot analysis revealed that compound 7 reduced the expression levels of the inducible NO synthase (iNOS) and COX-2 proteins in a concentration-dependent manner. Furthermore, compounds 5, 6, 13, and 18-20 exhibited potent inhibitory effects (IC50 299-381 molar ratio/32 pmol TPA) against Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cell line.

Cancer chemopreventive effect of bergenin from Peltophorum pterocarpum wood.

The aqueous extract of Peltophorum pterocarpum (Fabaceae) wood exhibited potent inhibitory effects against EpsteinBarr virus early antigen (EBV-EA) activation induced with 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cells and against melanogenesis in α-melanocyte-stimulating hormone (α-MSH)-stimulated B16 melanoma cells, as well as potent 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging activity. Two phenolic acid derivatives, bergenin (1) and gallic acid (2), were isolated from the ethyl acetate (AcOEt)-soluble fraction obtained from the extract. Compound 1 exhibited potent inhibitory effect against EBV-EA activation and against skin tumor promotion in an in vivo two-stage mouse skin carcinogenesis test based on 7,12-dimethylbenz[a]anthracene (DMBA) as initiator, and with TPA as promoter. Both compounds 1 and 2 exhibited melanogenesis-inhibitory activities in α-MSH-stimulated B16 melanoma cells, and, in addition, compound 2 showed strong DPPH radical-scavenging activity.

[Anti-inflammatory Effects of a Src Inhibitor on the Murine Model of Asthma Exacerbation Induced by Ovalbumin and Lipopolysaccharide].

Asthma is often exacerbated by airway infection, and some patients with severe asthma may be unresponsive to conventional corticosteroid treatment. Src family kinases (SFKs) were recently implicated in the inflammatory responses of mice induced by allergen and bacterial toxin lipopolysaccharide (LPS). Therefore, we examined the effects of dasatinib (DAS), a Src inhibitor, on airway inflammation in mice induced by ovalbumin (OVA) and LPS. Male A/J mice were sensitized to OVA Day -14 and -7, challenged with intranasal OVA on Day 0, 2, 4, 6 and 8, and on Day 10, mice were also challenged with OVA via inhalation. Mice were treated intranasally with DAS or fluticasone propionate (FP), a glucocorticoid, twice daily for 3 d starting 1 d after OVA inhalation. Moreover, some mice were also administrated LPS 2 h after DAS or FP treatment to model of asthma exacerbation. One day after the last intervention, lung tissue and bronchoalveolar lavage fluid (BALF) were collected. DAS attenuated the accumulation of inflammatory cells and cytokines/chemokines in BALF induced by both OVA and OVA+LPS, while FP did not reduce accumulations induced by OVA+LPS. Therefore, targeting SFKs may be a superior therapeutic approach for asthma exacerbation by infection.

Dasatinib attenuates airway inflammation of asthma exacerbation in mice induced by house dust mites and dsRNA.

Asthma exacerbation is a significant clinical problem that causes resistance to corticosteroid therapy and elevated hospitalization risk. Src family kinases (SFKs) contribute to various steps of the immune response, such as airway inflammation in viral or bacterial infections and allergic asthma. Therefore, we determined the effects of dasatinib (DAS), a typical Src inhibitor, on a murine asthma exacerbation model induced by house dust mites (HDM) and synthetic analog of double-stranded RNA, poly(I:C). A/J mice were sensitized to intrapreneurial HDM twice every seven days and challenged with intranasal HDM once every second day for a total of six exposures, and/or exposed to poly(I:C) twice daily for three consecutive days. Drug treatments were performed twice daily for three days, starting one day after the last HDM challenge or 2 h before each poly(I:C) exposure. DAS improved poly(I:C)-induced acute inflammation dose-dependently. Both DAS and fluticasone propionate (FP) attenuated HDM-induced allergic airway inflammation. However, in HDM and poly(I:C) induced-asthma exacerbated mice, DAS significantly improved inflammatory cells in bronchoalveolar lavage fluid and histological changes in the lungs, whereas FP did not. Therefore, SFKs are important targets for controlling severe asthma refractory to conventional therapies.

Relationship between anti-fungal effects and lung exposure of PC945, a novel inhaled antifungal agent, in Aspergillus fumigatus infected mice: Pulmonary PK-PD analysis of anti-fungal PC945.

PC945 is a novel antifungal agent, optimised for inhaled treatment. In this study, the relationship between antifungal effects of PC945 and its exposure in the lungs was investigated in Aspergillus fumigatus intranasally infected, temporarily neutropenic mice. Mice were given prophylactic PC945 intranasally once daily (0.56 µg/mouse) on either Day -7 to 0 (8 doses) or Day -1 to 0 (2 doses). Lung tissue, plasma and bronchoalveolar lavage (BAL) fluid were collected 24 or 72 h post A. fumigatus inoculation for biomarker and pharmacokinetic analyses. BAL cell pellets and supernatants were prepared separately by centrifugation. 8 prophylactic doses of PC945 were found to demonstrate significantly stronger antifungal effects (lung fungal burden and galactomannan (GM) in BAL and plasma) than prophylaxis with 2 doses. PC945 concentrations were below the limit of detection in plasma but readily measured in lung extracts. The concentrations were much higher after extended prophylaxis (709 and 312 ng/g of lung) than short prophylaxis (301 and 195 ng/g of lung) at 24 and 72 h post last dose, respectively, suggesting PC945 accumulation in whole lung after repeat dosing although it was likely to be a mixture of dissolved and undissolved PC945, meaning that the data should be interpreted with caution. Interestingly, low concentrations of PC945 were detected in BAL supernatant (6.6 and 1.9 ng/ml) whereas high levels of PC945 were measured in BAL cell pellets (626 and 406 ng/ml) at 24 and 72 h post last dose, respectively, in extended prophylaxis. In addition, the PC945 concentrations in BAL cells showed a statistically significant correlation with measured anti-fungal activities. These observations will be pursued, and it is intended that BAL cell concentrations of PC945 be measured in future clinical studies rather than standard measurement in BAL itself. Thus, PC945's profile makes it an attractive potential prophylactic agent for the prevention of pulmonary fungal infections.

Glycyrrhizin attenuates mucus production by inhibition of MUC5AC mRNA expression in vivo and in vitro.

We have previously indicated that glycyrrhizin (GL), a major component of licorice, has glucocorticoid-like anti-inflammatory effects in cultured airway epithelial cells and suggested its usefulness in the treatment of inflammatory respiratory diseases. On the other hand, mucus hypersecretion in the respiratory tract and goblet cell hyperplasia in the airway epithelium contribute to the morbidity and mortality associated with airway inflammatory diseases. This study, therefore, aimed to examine the effects of GL on airway mucus hyperproduction and define the mechanisms behind these effects. In an in vivo model, GL significantly attenuated goblet cell hyperplasia and MUC5AC mRNA expression in mice treated with lipopolysaccharide or interleukin-4. In addition, GL significantly attenuated MUC5AC protein and mRNA expression by tumor growth factor (TGF)-alpha in cultured NCI-H292 cells. GL also attenuated TGF-alpha-stimulated MUC5AC promoter activity in a luciferase reporter gene assay, but did not affect the stability of MUC5AC mRNA. Taken together, we concluded that GL has an inhibitory effect on mucus hyperproduction both in vivo and in vitro and that GL-mediated inhibition may be mediated through the inhibition of MUC5AC gene transcription.

[Inhibitory Effects of Dabigatran on Airway Inflammation Induced by Lipopolysaccharide in Mice].

Asthma and chronic obstructive pulmonary disease (COPD) are characterised by chronic inflammation in the lung that is associated with airway obstruction. Inhaled therapy with a combination of corticosteroid and a long-acting ß2-agonist is an effective anti-inflammatory medicine for asthma, but in patients with severe asthma and COPD fails to completely control these symptoms with current therapies. The inflammatory process in these diseases, which involves activation of the coagulation and fibrinolytic system in the lung, offers the opportunity for alternative anti-inflammatory therapies. In this study, we investigated the effects of anti-coagulants on lipopolysaccharide (LPS)-induced airway inflammation in mice. A/J mice were exposed to LPS, a bacterial endotoxin, intranasally and accumulation of inflammatory cells, TNF-α, C-X-C motif chemokine (CXCL) 1, and osteopontin in bronchoalveolar lavage fluid (BALF) was monitored by flow cytometry and an enzyme-linked immunosorbent assay. LPS exposure induced airway neutrophilia and accumulation of TNF-α, CXCL1, and osteopontin in BALF. This LPS-induced airway inflammation was not relieved using a corticosteroid, fluticasone propionate (FP), or a direct inhibitor of Factor Xa, rivaroxaban. In contrast, a direct thrombin inhibitor, dabigatran, inhibited LPS-induced airway neutrophilia and decreased inflammatory cytokine production in a dose dependent manner. Furthermore, combination of dabigatran and FP elicited stronger inhibition of LPS-induced airway inflammation. Therefore, these results suggest that dabigatran could be an effective new therapy for severe respiratory diseases.

Age-related pulmonary emphysema in mice lacking alpha/beta hydrolase domain containing 2 gene.

The alpha/beta hydrolase family genes have been identified as down-regulated genes in human emphysematous lungs. Although proteins in the alpha/beta hydrolase family generally act as enzymes, such as lipases, the specific functions of the Abhd2 protein are unknown. To examine the role of Abhd2 in the lung, we analyzed Abhd2 deficient mice obtained by gene trap mutagenesis. Abhd2 was expressed in the alveolar type II cells. Abhd2 deficiency resulted in a decreased level of phosphatidylcholine in the bronchoalveolar lavage. These mice developed spontaneous gradual progression of emphysema, due to increased macrophage infiltration, increased inflammatory cytokines, a protease/anti-protease imbalance and enhanced apoptosis. This phenotype is more akin to the pace of emphysema that develops in humans. Our findings suggest that derangement in alveolar phospholipid metabolism can induce emphysema, and that Abhd2 plays a critical role in maintaining lung structural integrity.

[TNF-α Decreased Corticosteroid Responsiveness in Mice Models of Airway Inflammation Induced by Double Strand RNA and/or Tobacco Smoke Exposure].

Reduction of corticosteroid responsiveness is one of the important clinical problems in chronic obstructive pulmonary disease (COPD). In this study, we determined the effects of neutralization of tumor necrosis factor-α (TNF-α) on corticosteroid insensitivity in mice models of airway inflammation induced by poly(I:C) and tobacco smoke (TS) exposure. Mice (male A/J strain, 5 weeks old) were exposed to TS for 10 d, or TS for 11 d and poly(I:C) for 3 d. Anti-TNF-α antibody was intranasally treated once every other day 2 h before the TS exposure, and dexamethasone 21-phosphate (DEX) was treated 30 min before the TS or poly(I:C) exposure. On the next day of the last stimulation, mice were sacrificed. The combination treatment of DEX and TNF-α neutralization was significantly attenuated the increase of the numbers of inflammatory cells in BALF and the TNF-α mRNA expression levels induced by TS and poly(I:C) exposure, even though TNF-α neutralization alone had little effect. These data indicated that neutralization of TNF-α restores corticosteroid responsiveness. Therefore, our study suggests that targeting TNF-α signaling pathway provides a new therapeutic approach to corticosteroid refractory airway diseases.

Fat-specific protein 27α inhibits autophagy-dependent lipid droplet breakdown in white adipocytes.

AIMS/INTRODUCTION: Fat-specific protein 27 (FSP27) α is the major isoform of FSP27 in white adipose tissue (WAT), and is essential for large unilocular lipid droplet (LD) formation in white adipocytes. In contrast, FSP27ß is abundantly expressed in brown adipose tissue (BAT), and plays an important role in small multilocular LD formation. In FSP27 KO mice in which FSP27α and ß are both depleted, WAT is characterized by multilocular LD formation, and by increased mitochondrial abundance and energy expenditure, whereas BAT conversely manifests large oligolocular LDs and reduced energy expenditure. MATERIALS AND METHODS: We investigated the effects of autophagy in WAT and BAT of wild type (WT) and FSP27 knockout (KO) mice. In addition, we examined the effects of FSP27α and FSP27ß to the induction of autophagy in COS cells. RESULTS: Food deprivation induced autophagy in BAT of WT mice, as well as in WAT of FSP27 KO mice, suggesting that enhanced autophagy is characteristic of adipocytes with small multilocular LDs. Pharmacological inhibition of autophagy attenuated the fasting-induced loss of LD area in adipocytes with small multilocular LDs (BAT of WT mice and WAT of FSP27 KO mice), without affecting that in adipocytes with large unilocular or oligolocular LDs (WAT of WT mice or in BAT of FSP27 KO mice). Overexpression of FSP27α inhibited autophagy induction by serum deprivation in COS cells, whereas that of FSP27ß had no such effect. CONCLUSIONS: The present results thus showed that FSP27α inhibits autophagy and might thereby contribute to the energy-storage function of WAT.