Rie Sawamura

Effects of tetrabromobisphenol A, a brominated flame retardant, on the immune response to respiratory syncytial virus infection in mice

Effects of the brominated flame retardants (BFRs), decabrominated diphenyl ether (DBDE), hexabromocyclododecane (HBCD), and tetrabromobisphenol A (TBBPA), on host immunity of mice were evaluated using respiratory syncytial virus (RSV) infection. Five-week-old female mice were fed a diet containing 1% BFRs for 28days, and subsequently infected with RSV. No toxicological sign was observed in BFR-treated mice before infection. TBBPA significantly increased the pulmonary viral titer in the infected mice on day 5 post-infection, but DBDE and HBCD did not. Slight histological changes were observed in lung tissues of TBBPA-treated mice with mock infection. These changes due to TBBPA were much exacerbated by RSV infection. Cytokine analysis of bronchoalveolar lavage fluid (BALF) from RSV-infected mice treated with or without TBBPA revealed that TBBPA significantly increased the levels of tumor necrosis factor (TNF)-alpha, interleukin (IL)-6 and interferon (IFN)-gamma at each time point after virus infection, but no change was observed for IL-1beta and IL-12. The levels of IL-4 and IL-10, Th2 cytokines, significantly decreased. Thus, TBBPA caused unusual production of the various cytokines in RSV-infected mice. Flow cytometry revealed that the percentage of double-positive CD4+CD8+ cells, immature T lymphocytes, in the cell populations in BALF from RSV-infected mice increased due to TBBPA treatment. The change was not observed in spleen cells of TBBPA-treated mice. The response to RSV infection verified that TBBPA treatment affected the host immunity of mice. Irregular changes in cytokine production and immune cell populations due to TBBPA treatment were suggested to cause exacerbation of pneumonia in RSV-infected mice.

Antiviral activities of diarylheptanoids against influenza virus in vitro

The anti-influenza A/PR/8/34 (H1N1) virus activities of ten diarylheptanoids isolated from Alpinia officinarum were examined using the MTT method. The 50% inhibitory concentration of each diarylheptanoid examined was clearly lower than its 50% cytotoxic concentration determined by the MTT assay and/or maximum non-cytotoxic concentration (MNCC) determined by the morphological change of cells. In particular, the influenza virus was more susceptible to 7-(4″-hydroxy-3″-methoxyphenyl)-1-phenyl-4E-hepten-3-one (3) and (5S)-5-hydroxy-7-(4″-hydroxyphenyl)-1-phenyl-3-heptanone (8) than the other diarylheptanoids. Thus, all diarylheptanoids exhibited potential antiviral activity against influenza virus in vitro.

Modulation of cytokine production by 7-hydroxycoumarin in vitro and its efficacy against influenza infection in mice

We previously demonstrated that 7-hydroxycoumarin (7HC) was effective in reducing proinflammatory cytokine production in lipopolysaccharide-exposed macrophage-like P388D1 cells and fever production by suppressing the increase in interleukin (IL)-1alpha production in an influenza virus-intranasal infection model in mice. In this study, we assessed the effects of modulation of cytokine production by 7HC on influenza virus infection in relation to its efficacy in influenza virus-infected mice. 7HC was confirmed to suppress proinflammatory cytokine levels in P388D1 cells due to influenza virus infection. In the murine infection model, oral administration of 7HC (30 mg/kg) was significantly effective in reducing the weight loss of infected mice and virus titers in the bronchoalveolar lavage fluid (BALF) of lungs and in prolonging survival times without toxicity. The rise of proinflammatory and Th1 cytokine (IL-12 and interferon-gamma) production in the BALF from infected mice was significantly suppressed by 7HC at two and four days post-infection, respectively. This suppression correlated with the reduction of virus titers and diminution of lung consolidation. Because 7HC did not exhibit direct anti-influenza virus activity in vitro, 7HC was suggested to suppress pneumonia in influenza virus-infected mice through suppression of the cytokine production induced by infection.

Efficacy of Brazilian Propolis against Herpes Simplex Virus Type 1 Infection in Mice and Their Modes of Antiherpetic Efficacies

Ethanol extracts (AF-06, 07, and 08, 10 mg/kg) of Brazilian propolis were administered orally to cutaneously herpes simplex virus type 1 (HSV-1)-infected mice three times daily on days 0 to 6 after infection to evaluate their efficacies against HSV-1 infection and significantly limited development of herpetic skin lesions. AF-07 and 08 significantly reduced virus titers in brain and/or skin on day 4 without toxicity, but AF-08 had no anti-HSV-1 activity in vitro. AF-06 and 08 significantly enhanced delayed-type hypersensitivity (DTH) to inactivated HSV-1 antigen in infected mice. Oral AF-08-administration significantly augmented interferon (IFN)-γ production by HSV-1 antigen from splenocytes of HSV-1-infected mice, while direct exposure of splenocytes of infected mice to AF-06 significantly elevated IFN-γ production in vitro. Thus, AF-08 might have components that are active in vivo even after oral administration and those of AF-06 might be active only in vitro. Because DTH is a major host defense for intradermal HSV-1 infection, augmentation of DTH response by AF-06 or 08, directly or indirectly, respectively, may contribute to their efficacies against HSV-1 infection. In addition, AF-06 and 07 possibly contain anti-HSV-1 components contributing to their efficacies. Such biological activities of Brazilian propolis may be useful to analyze its pharmacological actions.

In Vitro and in Vivo Anti-Influenza Virus Activity of Diarylheptanoids Isolated from Alpinia Officinarum

Background: Diarylheptanoids (AO-0002 [7-(4″-hydroxy-3″-methoxyphenyl)-1-phenyl-4E-hepten-3-one] and AO-0011 [(5S)-5-hydroxy-7-(4″-hydroxyphenyl)-1-phenyl-3-heptanone]) isolated from Alpinia officinarum have been reported to exhibit anti-influenza virus activity in vitro. Hence, efficacies against influenza virus infection and the mode of antiviral action were evaluated in vivo and in vitro, respectively. Methods: In a murine influenza virus infection model, diarylheptanoids were orally administered three times daily to mice infected with influenza A/PR/8/34 virus for 6 days after infection. AO-0002 was examined for its antiviral activity against the wild types of influenza viruses A/PR/8/34 (H1N1), oseltamivir-resistant A/PR/8/34 (H1N1), A/Bangkok/93/03 (H1N1), A/Ishikawa/7/82 (H3N2), A/Fukushima/13/43 (H3N2), B/Singapore/222/79 and B/Fukushima/15/93 in plaque reduction or yield reduction assays. The mode of anti-influenza virus action was assessed by a virus adsorption assay, immunofluorescence assay of viral antigens, and inhibition of viral messenger RNA synthesis using real-time reverse transcriptase PCR. Results: AO-0002 at 100 mg/kg was significantly effective in reducing the body weight loss and prolonging survival times of infected mice without toxicity, but AO-0011 was not. AO-0002 at 30 and 100 mg/kg significantly reduced virus titres in bronchoalveolar lavage fluids of the lungs on days 3 and 6 after infection. AO-0002 exhibited anti-influenza virus activity against all viruses used, including the oseltamivir-resistant strain in vitro. The compound had no effect on virus adsorption or invasion into cells, but dose-dependently suppressed the expression of viral messenger RNA and antigens. Conclusions: AO-0002 was suggested to have a different anti-influenza virus action to that of oseltamivir and was verified to show anti-influenza activity in vitro and in vivo.

Functional disorder of primary immunity responding to respiratory syncytial virus infection in offspring mice exposed to a flame retardant, decabrominated diphenyl ether, perinatally

Perinatal exposure to a representative flame retardant, decabrominated diphenyl ether (DBDE), was shown previously to increase viral titers in the lungs of respiratory syncytial virus (RSV)‐infected offspring on day 5 post‐infection, resulting in exacerbation of pneumonia. In this study, the significant increase of pulmonary viral titers was confirmed even on day 1 post‐infection and the effect on the primary immune response to RSV infection were examined to assess a mode of DBDE action on developmental immunotoxicity. On day 1 after infection, the secretion of both TNF‐α and IL‐6 decreased significantly in the bronchoalveolar lavage fluid prepared from RSV‐infected offspring exposed to DBDE perinatally, but IL‐1β increased. However, in ex vivo lipopolysaccharide stimulation test, the productivity of TNF‐α in the bronchoalveolar lavage cells, which are mainly primary immune cells responding to RSV infection, prepared from offspring mice exposed to DBDE perinatally was not lower than that in the control. The primary immune cells retained normally the ability of cytokine production after the DBDE exposure. Gene expressions of innate pattern recognition receptors (Toll‐like receptor 3 and 4, melanoma differentiation‐associated gene‐5, and retinoic acid‐inducible gene I) in lung tissues were not affected by DBDE exposure. Because the levels of TNF‐α, IL‐6, and IL‐1β are known to be elevated in the lungs of RSV‐infected mice, these irregular productions due to perinatal DBDE exposure indicate a disorder of the primary immune response to RSV infection. Thus, perinatal exposure to DBDE was suggested to cause a functional disorder of primary immunity responding to RSV infection. J. Med. Virol. 82:1075–1082, 2010.

Antiviral activity of diarylheptanoid stereoisomers against respiratory syncytial virus in vitro and in vivo.

We previously showed that (5S)-5-hydroxy-7-(4-hydroxyphenyl)-1-phenylhept-3-one (AO-0011) and (5S)-5-methoxy-1,7-diphenylhept-3-one (AO-0016) isolated from Alpinia officinarum exhibited stronger anti-influenza virus activity and anti-respiratory syncytial virus (RSV) activity, respectively, than the other isolated diarylheptanoids. In this study, we synthesized an enantiomer (AO-0503) and racemate (AO-0504) of AO-0011 and an enantiomer (AO-0514) of AO-0016. The anti-RSV activities of the three stereoisomers (AO-0503, AO-0504, and AO-0514) and AO-0011 were examined in vitro and in vivo to evaluate the stereoisomeric effect on anti-RSV activity. In a plaque reduction assay using human epidermoid carcinoma cells, all four diarylheptanoids significantly exhibited anti-RSV activity, and AO-0514 and AO-0016 exhibited stronger anti-RSV activity than AO-0503, AO-0504, and AO-0011. In a murine RSV infection model, all four diarylheptanoids with anti-RSV activity in vitro were also significantly effective in reducing virus titers in the lungs of RSV-infected mice. In the histopathological analysis of RSV-infected lungs, the oral administration of even AO-0514, which showed the lowest reduction of virus titers in the lungs, was significantly effective in reducing the infiltration of lymphocytes and in reducing the interferon-γ level, which is a marker of severity of pneumonia due to RSV infection, in bronchoalveolar lavage fluids prepared from RSV-infected mice. Although the stereoisomeric effects of diarylheptanoids on anti-RSV activity varied moderately, all four diarylheptanoids examined were suggested to ameliorate pneumonia and have a potential anti-RSV activity in vivo. They are possibly mother compounds for the development of an anti-RSV drug in the future.