Tomomi Shimizu

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.

Anti-Influenza Virus Activity of Propolis in Vitro and its Efficacy against Influenza Infection in Mice

Background: Propolis has been used worldwide as a dietary supplement to maintain and improve human health. We examined whether ethanol extracts of Brazilian propolis exhibit antiviral activity against influenza virus in vitro and in vivo. Methods: Among 13 ethanol extracts screened in a plaque reduction assay, four showed anti-influenza virus activity. The anti-influenza efficacy of the four extracts was further examined in a murine influenza virus infection model. The mice were infected intranasally with influenza virus, and the four extracts were orally administered at 10 mg/kg three times daily for seven successive days after infection. Results: In this infection model, only one extract, AF-08, was significantly effective at 10 mg/kg in reducing the body weight loss of infected mice. The doses of 2 and 10 mg/kg were also effective in prolonging the survival times of infected mice significantly, but 0.4 mg/kg was not. The anti-influenza efficacy of AF-08 at 10 mg/kg was confirmed in a dose-dependent manner in mice. AF-08 at 10 mg/kg significantly reduced virus yields in the bronchoalveolar lavage fluids of lungs in infected mice as compared with the control. The reduction of virus yields by AF-08 at 10 mg/kg significantly corresponded to those induced by oseltamivir at 1 mg/kg twice daily from day 1 to day 4 after infection. Conclusion: The Brazilian propolis AF-08 was indicated to possess anti-influenza virus activity and to ameliorate influenza symptoms in mice. AF-08 may be a possible candidate for an anti-influenza dietary supplement for humans.

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.

Effects of decabrominated diphenyl ether (DBDE) on developmental immunotoxicity in offspring mice.

Decabrominated diphenyl ether (DBDE), a representative brominated flame retardant ubiquitous in the environment, is suspected of being hazardous to humans. We evaluated the developmental immunotoxicity of DBDE by an assay system using respiratory syncytial virus (RSV) infection in offspring mice. Pregnant mice were continuously exposed to DBDE (10, 100, 1000, or 10,000ppm) in the diet from gestation day 10 to weaning on postnatal day 21. Offspring mice born to these dams were intranasally infected with RSV. Virus titers in the lungs of RSV-infected offspring exposed perinatally to DBDE increased dose-dependently compared with the control. The level of interferon-γ in the bronchoalveolar lavage fluids and gene expression of the chemokine RANTES in the lungs were also significantly elevated in offspring mice exposed to DBDE. Histopathological analysis revealed that pneumonia in the lungs of offspring mice exposed to 10,000ppm of DBDE was exacerbated compared with the control. These results indicate that DBDE is a developmental immunotoxic agent.

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.

A new assay system for evaluation of developmental immunotoxicity of chemical compounds using respiratory syncytial virus infection to offspring mice

We evaluated the effect of 6-propyl-2-thiouracil (PTU), an anti-thyroid agent, on developmental immunity using respiratory syncytial virus (RSV) infection to offspring mice as a new risk assessment for brominated flame retardants (BFRs), because some BFRs are suspected of affecting the thyroid system. Pregnant mice were exposed to PTU in drinking water from gestation day 10 to weaning on postnatal day 21. Their offspring mice were infected intranasally with RSV. Exposure of 100ppm PTU significantly increased virus titers in the lungs of RSV-infected offspring compared with the control, and the 10ppm also elevated levels of interferon-γ, a marker of pneumonia, in the bronchoalveolar lavage fluids of offspring. Histopathological analysis revealed that PTU-exposure exacerbated pneumonia in RSV-infected offspring. Thus, exacerbation of RSV infection suggested PTU-exposure of dams elicited developmental immune disorder in the offspring. The murine RSV infection model may be useful to evaluate the developmental immunotoxicity of BFRs.

Development of New Antiviral Agents from Natural Products~!2010-01-17~!2010-04-12~!2010-08-27~!

The recent great advances in medical treatment and scientific technology include the many antiviral agents that have been developed and are used for treatment of infectious diseases, but such advances have also provoked the appear- ance of resistant virus strains. Therefore, the development of new antiviral agents with diverse kinds of antiviral actions is required. The search for new antiviral agents focuses on not only synthetic compounds but also natural products such as traditional medicines, dietary supplements, and functional foods, including plants, insects, animal organs, and their com- ponents. Natural products have their own metabolites, and some of the metabolites may recognize the differences between viral and host metabolisms, resulting in antiviral activity. In general, they can be obtained cheaply and may be useful re- sources to develop new antiviral agents with different antiviral actions from those of known antiviral agents. Also, natural products and their components have been demonstrated to modify immunological activities and are candidates for biological response modifiers that are effective in alleviating symptoms and reducing mortality in virus infection. The first half of this chapter introduces natural products and purified compounds that were confirmed by in vitro experiments and animal infection models to have direct antiviral activity against herpes simplex virus type 1 (HSV-1) or influenza virus. However, even if a natural product or purified component has strong antiviral activity in vitro, if it has no therapeu- tic efficacy against virus infection in an animal infection model, it is merely an inhibitor and not a medicine. The search for antiviral agents should be based on the demonstration of prophylactic and/or therapeutic efficacy at the proper dosage in animals. In the second half, we introduce a Kampo medicine, Kakkon-to, which is a biological response modifier rather than a direct antiviral agent. It is the most common cold medicine used in traditional therapy and prescribed to about 20 million people annually in Japan. We also introduce the mode of immunomodulating activity of Kakkon-to on influenza virus and HSV-1 infection and its components, which can modulate cytokine production. We hope that this chapter will be useful in verifying the antiviral therapeutic efficacy of natural products against influenza infection and helpful in encouraging development of anti-influenza virus medicines from natural products.