Untying relaxed circular DNA of hepatitis B virus by polymerase reaction provides a new option for accurate quantification and visualization of covalently closed circular DNA.

Hepatitis B virus (HBV) is a small hepatotropic DNA virus that replicates via an RNA intermediate. After entry, the virus capsid carries relaxed circular DNA (rcDNA) into the nucleus where the viral genome is converted into covalently closed circular DNA (cccDNA), which serves as the template for all viral transcripts. To monitor cccDNA levels, preprocessing methods to eliminate rcDNA have emerged for quantitative PCR, although Southern blotting is still the only method to discriminate cccDNA from other DNA intermediates. In this study, we have established a robust method for untying mature rcDNA into double stranded linear DNA using specific polymerases. Untying rcDNA provides not only an alternative method for cccDNA quantification but also a sensitive method for visualizing cccDNA. We combined this method with plasmid-safe DNase and T5 exonuclease preprocessing and revealed that accurate quantification requires cccDNA digestion by a restriction enzyme because heat stability of cccDNA increases after T5 exonuclease treatment. In digital PCR using duplex TaqMan probes, fewer than 1000 copies of cccDNA were successfully visualized as double positive spots that were distinct from single positives derived from untied rcDNA. This method was further applied to the infection model of primary hepatocytes treated with nucleoside analogues and a core protein allosteric modulator to monitor cccDNA levels. Relative quantification of cccDNA by human genome copy demonstrated the possibility of precise evaluation of cccDNA level per nucleus. These results clearly indicate that the sequential reaction from untying rcDNA is useful to investigate cccDNA fates in a small fraction of nuclei.

Association between PM2.5 and primary care visits due to asthma attack in Japan: relation to Beijing's air pollution episode in January 2013.

AIM: In January 2013, extremely high concentrations of fine particles (PM2.5) were observed around Beijing, China. In Japan, the health effects of transboundary air pollution have been a matter of concern. We examined the association between the levels of outdoor PM2.5 and other air pollutants with primary care visits (PCVs) at night due to asthma attack in Himeji City, western Japan. METHODS: A case-crossover study was conducted in a primary care clinic in Himeji City, Japan, involving 112 subjects aged 0-80 years who visited the clinic due to an asthma attack between 9 p.m. and 6 a.m. during the period January-March, 2013. Daily concentrations of particulate matter, ozone, nitrogen dioxide, and some meteorological elements were measured, and a conditional logistic regression model was used to estimate the odds ratios (OR) of PCVs per unit increment in air pollutants or meteorological elements. RESULTS: Of the 112 subjects, 76 (68 %) were aged <15 years. We did not note any association between daily PM2.5 levels and PCVs due to asthma attack at night. A positive relation between ozone and PCVs due to asthma attack was detected. The OR per 10 ppb increment in daily mean ozone the day before the visit was 2.31 (95 % confidence interval 1.16-4.61). CONCLUSION: These findings do not support an association between daily mean concentration of PM2.5 and PCVs at night. However, we did find evidence suggesting that ozone is associated with PCVs.

Association of ambient air pollution and meteorological factors with primary care visits at night due to asthma attack.

AIM: The association of outdoor air pollution and meteorological elements with primary care visits at night due to asthma attack was studied. METHODS: A case-crossover study was conducted in a primary care clinic in Himeji City, Japan. The subjects were 956 children aged 0-14 years who visited the clinic with an asthma attack between the hours of 9 p.m. and 6 a.m. Daily concentrations of particulate matter, ozone, nitrogen dioxide, and a number of meteorological elements were measured, and a conditional logistic regression model was used to estimate odds ratios (ORs) of primary care visits per unit increment of air pollutants or meteorological elements. The analyses took into consideration the effects of seasonality. RESULTS: Of the 956 children, 73 (7.6 %) were aged <2 years and 417 (43.6 %) were aged 2-5 years. No association between daily ozone levels and primary care visits due to asthma attack at night in the spring or summer was found. An inverse relation between suspended particulate matter and primary care visits due to asthma attack was detected in the winter. ORs in the summer per degree increment in daily mean temperature was 1.31 [95 % confidential interval (CI) 1.09-1.56], and ORs in the autumn per hourly increment in daily hours of sunshine was 0.94 (95 % CI 0.90-0.99). CONCLUSION: The findings of our study fail to support any association between daily mean concentration of air pollutant and primary care visits at night. However, we did find evidence indicating that certain meteorological elements may be associated with primary care visits.

Risk factors for wheezing, eczema and rhinoconjunctivitis in the previous 12 months among six-year-old children in Himeji City, Japan: food allergy, older siblings, day-care attendance and parental allergy history.

BACKGROUND: The aim of this study was to clarify whether some environmental and genetic factors (food allergy, older siblings, early day-care attendance and parents' allergy history) are related to the development of allergic symptoms (wheezing in the previous 12 months [WP], eczema symptoms in the previous 12 months [EP], and rhinoconjunctivitis symptoms in the previous 12 months [RP]) in Japanese children. METHODS: Using the modified version of the International Study on Asthma and Allergies in Childhood (ISSAC) questionnaire, we studied the prevalence of WP, EP and RP among six-year-old children attending 72 primary schools throughout Himeji City, Japan, during the two years from 2005 to 2006. RESULTS: Food allergy and parents' history of allergy showed a significant relationship with the prevalence of WP, EP and RP. Day-care attendance in the first two years of life and presence of older siblings showed a significant inverse relationship with the prevalence of RP. However, neither day-care attendance nor presence of older siblings was related to the development of W and ER. CONCLUSIONS: Among Japanese children, food allergy and parents' history of allergy are risk factors for WP, ES or RS. However, early day-care attendance and presence of older siblings might be protective factors against RS. Infections in early life may affect the prevalence of rhinoconjunctivitis in six-year-old children.

Amiselimod, a novel sphingosine 1-phosphate receptor-1 modulator, has potent therapeutic efficacy for autoimmune diseases, with low bradycardia risk.

BACKGROUND AND PURPOSE: We conducted preclinical and clinical studies to examine the pharmacological, particularly cardiac, effects of amiselimod (MT-1303), a second-generation sphingosine 1-phosphate (S1P) receptor modulator, designed to reduce the bradycardia associated with fingolimod and other S1P receptor modulators. EXPERIMENTAL APPROACH: The selectivity of the active metabolite amiselimod phosphate (amiselimod-P) for human S1P receptors and activation of G-protein-coupled inwardly rectifying K+ (GIRK) channels in human atrial myocytes were assessed. Its cardiac distribution was determined in rats, and cardiovascular telemetry was assessed in monkeys. We also examined the pharmacokinetics, pharmacodynamics and safety of amiselimod in healthy humans. KEY RESULTS: Amiselimod-P showed potent selectivity for S1P1 and high selectivity for S1P5 receptors, with minimal agonist activity for S1P4 and no distinct agonist activity for S1P2 or S1P3 receptors and approximately five-fold weaker GIRK activation than fingolimod-P. After oral administration of amiselimod or fingolimod at 1 mg·kg-1 , the concentration of amiselimod-P in rat heart tissue was lower than that of fingolimod-P, potentially contributing to the minimal cardiac effects of amiselimod. A telemetry study in monkeys confirmed that amiselimod did not affect heart rate or ECG parameters. In healthy human subjects, peripheral blood lymphocyte counts gradually reduced over the 21 day dosing period, with similar lymphocyte count profiles with the highest doses by day 21, and no clinically significant bradycardia observed on day 1 or during the study. CONCLUSIONS AND IMPLICATIONS: Amiselimod exhibited potent therapeutic efficacy with minimal cardiac effects at the anticipated clinical dose and is unlikely to require dose titration.

Y-40138, a multiple cytokine production modulator, protects against D-galactosamine and lipopolysaccharide-induced hepatitis.

Acute and fulminant liver failure induced by viral hepatitis, alcohol or other hepatotoxic drugs are associated with tumor necrosis factor (TNF) production. D-Galactosamine (D-GalN) and lipopolysaccharide (LPS)-induced liver injury is an experimental model of fulminant hepatic failure. In this model, TNF-alpha plays a central role in the pathogenesis of D-GalN/LPS-induced liver injury in mice. Y-40138, N-[1-(4-[4-(pyrimidin-2-yl)piperazin-1-yl]methyl phenyl)cyclopropyl] acetamide.HCl inhibits TNF-alpha and augments interleukin (IL)-10 production in LPS-injected mice in plasma. In the present study, we examined the effect of Y-40138 on D-GalN/LPS-induced hepatitis. Y-40138 (10mg/kg, i.v.) significantly suppressed TNF-alpha and monocyte chemoattractant protein-1 (MCP-1) production and augmented IL-10 production in plasma. In addition, Y-40138 significantly inhibited TNF-alpha production induced by direct interaction between human T lymphocytes and macrophages. Y-40138 suppressed plasma alanine transaminase (ALT) elevation and improved survival rate in D-GalN/LPS-injected mice, and it is suggested that the protective effect of Y-40138 on hepatitis may be mediated by inhibition of TNF-alpha and MCP-1, and/or augmentation of IL-10. This compound is expected to be a new candidate for treatment of cytokine and/or chemokine-related liver diseases such as alcoholic hepatitis.

Therapeutic administration of Y-40138, a multiple cytokine modulator, inhibits concanavalin A-induced hepatitis in mice.

Concanavalin A-induced hepatitis is often used as a model of liver injury. In this model, plasma tumor necrosis factor-alpha (TNF-alpha) level increased in concanavalin A-injected mice. Prophylactic treatment with Y-40138, N-[1-(4-[4-(pyrimidin-2-yl)piperazin-1-yl]methyl phenyl)cyclopropyl] acetamide.HCl, significantly suppressed the increase in plasma TNF-alpha level. In this study, we compared the effect of Y-40138 with those of pentoxifylline and anti-TNF-alpha antibody on concanavalin A-induced hepatitis. Prophylactic treatment with pentoxifylline, anti-TNF-alpha antibody and Y-40138 reduced plasma alanine aminotransferase level. Therapeutic treatment with Y-40138 significantly reduced plasma alanine aminotransferase level, but pentoxifylline and anti-TNF-alpha antibody did not. Therapeutic treatment with Y-40138 significantly reduced plasma interferon-gamma (IFN-gamma) and monokine induced by interferon-gamma levels. From these results, Y-40138 may be expected as a new class of therapeutic drug for treatment of TNF-alpha, IFN-gamma and/or chemokine-related liver diseases such as alcoholic liver disease.

Exposure to air pollution and meteorological factors associated with children's primary care visits at night due to asthma attack: case-crossover design for 3-year pooled patients.

OBJECTIVE: We examined the association of outdoor air pollution and meteorological parameters with primary care visits (PCVs) at night due to asthma attack. SETTING: A case-crossover study was conducted in a primary care clinic in Himeji City, Japan. PARTICIPANTS: Participants were 1447 children aged 0-14 years who visited the clinic with an asthma attack from April 2010 until March 2013. EXPOSURE: Daily concentrations of air pollutants and meteorological parameters were measured. PRIMARY OUTCOME: PCVs at night due to asthma attack. A conditional logistic regression model was used to estimate ORs of PCVs per unit increment of air pollutants or meteorological parameters (the per-unit increments of particulate matter with an aerodynamic diameter ≤2.5 µm (PM2.5) and ozone were 10 µg/m(3) and 10 ppb, respectively). Analyses took into consideration the effects of seasonality. RESULTS: We noted an association between PCVs and daily ozone levels on the day before a PCV (OR=1.17; 95% CI 1.01 to 1.35; p=0.04), as well as between PCVs and 3-day mean ozone levels before a PCV (OR=1.29; 95% CI 1.00 to 1.46; p=0.04), from April until June. We also observed an association between PCVs and daily PM2.5 levels on the day before a PCV from December until March (OR=1.16; 95% CI 1.01 to 1.33; p=0.05). Meteorological parameters, such as hours of sunshine from September until November, atmospheric pressure from April until June, and temperature from April until August, were also found to be associated with PCVs. CONCLUSIONS: The findings in the present study supported an association between ozone and PCVs and suggest that certain meteorological items may be associated with PCVs.

Small GTPase Rho signaling is involved in beta1 integrin-mediated up-regulation of intercellular adhesion molecule 1 and receptor activator of nuclear factor kappaB ligand on osteoblasts and osteoclast maturation.

We assessed the characteristics of human osteoblasts, focusing on small GTPase Rho signaling. Beta1 Integrin were highly expressed on osteoblasts. Engagement of beta1 integrins by type I collagen augmented expression of intercellular adhesion molecule 1 (ICAM-1) and receptor activator of nuclear factor kappaB ligand (RANKL) on osteoblasts. Rho was activated by beta1 stimulation in osteoblasts. Beta1 Integrin-induced up-regulation of ICAM-1 and RANKL was inhibited by transfection with adenoviruses encoding C3 transferase or pretreated with Y-27632, specific Rho and Rho-kinase inhibitors. Engagement of beta1 integrin on osteoblasts induced formation of tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells (MNC) in a coculture system of osteoblasts and peripheral monocytes, but this action was completely abrogated by transfection of C3 transferase. Our results indicate the direct involvement of Rho-mediated signaling in beta1 integrin-induced up-regulation of ICAM-1 and RANKL and RANKL-dependent osteoclast maturation. Thus, Rho-mediated signaling in osteoblasts seems to introduce major biases to bone resorption.

Small GTP-binding protein Rho-mediated signaling promotes proliferation of rheumatoid synovial fibroblasts.

Rho is a major small GTP-binding protein that is involved in the regulation of various cell functions, including proliferation and cell migration, through activation of multiple signaling molecules in various types of cells. We studied its roles in synovial fibroblasts (SFs) in patients with rheumatoid arthritis (RA) and clarified its relevance to RA synovitis, with the following results. 1)We found that the thrombin receptor was overexpressed on RA synovial fibroblasts (RA SFs) and that thrombin induced a marked proliferation and progression of the cell cycle to the S phase in these cells. 2)We also found that thrombin efficiently activated Rho. 3)Rho activation and proliferation and the progression of the cell cycle to the S phase were completely blocked by p115RGS (an N-terminal regulator of the G-protein signaling domain of p115RhoGEF) and by the C-terminal fragments of Galpha13 (an inhibitor of the interaction of receptors with G13). 4)Thrombin induced the secretion of IL-6 by RA SFs, but this action was blocked by p115RGS or Galpha13. Our findings show that the actions of thrombin on the proliferation of RA SFs, cell-cycle progression to the S phase, and IL-6 secretion were mainly mediated by the G13 and RhoGEF pathways. These results suggest that p115RGS and Galpha13 could be potent inhibitors of such functions. A rational design of future therapeutic strategies for RA synovitis could perhaps include the exploitation of the Rho pathway to directly reduce the growth of synovial cells.