Antimalarial activity of vitamin D3 (VD3) does not result from VD3-induced antimicrobial agents including nitric oxide or cathelicidin.

Recent evidence suggests that 1α,25-dihydroxyvitamin D3 (VD3), the active form of vitamin D, inhibits microbial proliferation. Previously, we used in vivo murine models to investigate the antimalarial activity of VD3 and confirmed potent antimalarial activity in the acute phase. This study aimed to clarify the mechanisms underlying the antimalarial activity of VD3 in vivo, particularly extensive inhibition of parasitemia in the acute phase, focusing on nitric oxide (NO), a potent antimalarial molecule. VD3 is a good NO inducer. When most Plasmodium chabaudi AS (PcAS)-infected mice treated with VD3 survived, NO was present in blood samples obtained from VD3-treated mice at a significantly higher rate at 2 and/or 3 days post-infection than that in vehicle-treated control mice. To verify the involvement of NO in the antimalarial activity of VD3, we used aminoguanidine (AG), an inducible NO synthase (iNOS) inhibitor, to abrogate the antimalarial activity of VD3. However, despite AG-induced reductions in NO levels, parasitemia remained inhibited during the acute phase, even in the presence of AG, and the antiplasmodial faculty of VD3 was not ablated. VD3-mediated antimalarial activity irrelevant of NO compelled us to consider another candidate. In a pilot experiment, we used cathelicidin (CAMP), an antimicrobial peptide, since it is known that VD3 induces CAMP synthesis. Serum CAMP levels increased on days 4 or 5 post-infection with or without VD3 administration, but experiments using exogenous CAMP did not display curative effects in PcAS-infected mice. The present study using VD3 to target the malarial parasite thus suggests a potential novel approach to treat malarial infections.

Contribution of neutrophil-derived myeloperoxidase in the early phase of fulminant acute respiratory distress syndrome induced by influenza virus infection.

Because the pathogenesis of acute respiratory distress syndrome (ARDS) induced by influenza virus infection remains unknown, we can only improve on existing therapeutic interventions. To approach the subject, we investigated immunological etiology focused on cytokines and an acute lung damage factor in influenza-induced ARDS by using a PR-8 (A/H1N1)-infected mouse model. The infected mouse showed fulminant severe pneumonia with leukocyte infiltration, claudin alteration on tight junctions, and formation of hyaline membranes. In addition to interferon (IFN)-α, plenty of keratinocyte-derived chemokines (KC), macrophage inflammatory protein 2 (MIP-2), regulated on activation normal T-cell expressed and secreted (RANTES), and monocyte chemotactic protein 1 (MCP-1) were significantly released into bronchoalveolar lavage fluid (BALF) of the model. We focused on neutrophil myeloperoxidase (MPO) as a potent tissue damage factor and examined its contribution in influenza pneumonia by using mice genetically lacking in MPO. The absence of MPO reduced inflammatory damage with suppression of leakage of total BALF proteins associated with alteration of claudins in the lung. MPO(-/-) mice also suppressed viral load in the lung. The present study suggests that MPO-mediated OCl(-) generation affects claudin molecules and leads to protein leakage and viral spread as a damage factor in influenza-induced ARDS.

Formalin-treated UV-inactivated SARS coronavirus vaccine retains its immunogenicity and promotes Th2-type immune responses.

The demand for rapid and simple development of a vaccine against a newly emerging infectious disease is increasing worldwide. We previously revealed that UV-inactivated severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) virions (UV-V) elicited high levels of humoral immunity and a weak Th0 response in mice immunized subcutaneously. To ensure the safety of such a whole inactivated SARS-CoV vaccine, we additionally treated the UV-V vaccine with formalin, resulting in the UV-F-V vaccine. Analysis of the immunogenicity of the UV-F-V+alum vaccine in mice revealed that it generated comparable neutralizing serum anti-SARS-CoV IgG antibody levels as the UV-V+alum vaccine. Moreover, both vaccines induced similar frequencies of anti-SARS-CoV IgG antibody-producing cells in bone marrow. Interestingly, the UV-F-V vaccine induced fewer IgG(2a) subtype antibodies and higher interleukin-4 production in vaccinated mice than did UV-V. Thus, UV-F-V imposes a Th2-type bias on the immune response, unlike UV-V. We propose here that doubly-inactivated SARS-CoV virions by UV and formalin constitute a safe vaccine that may effectively induce neutralizing antibodies in humans.

[Influenza telephone consultation target the general public--2003-2004, 2004-2005].

The NPO Biomedical Science Association provided telephone consultation, including contacts by fax and email, targeting the general public within the framework of influenza control measures worked out by the Japanese Ministry of Health, Labor and Welfare (MHLW). We received 2,813 inquiries during the 2003-2004 flu season and 2,444 inquiries during the 2004-2005 season. By month, the highest number was in October-November, accounting for 42.6%. The preceding season showed a similar trend. By gender, 72.5% of those seeking advice were women. By area of residence, the highest number was living in metropolitan Tokyo, and the remainder lived in the prefectures of Kanagawa, Chiba, Saitama, Nagano, Shizuoka, and Ibaraki in this order. We received no inquiries from the prefectures of Shimane or Saga. By occupation, housewives accounted for 1,114 inquiries (45.6%), followed by private companies with 447 inquiries (18.3%) and health-care providers with 227 inquiries (9.3%), similar to the 2003-2004 flu season. By subject, 1,545 inquiries concerned vaccines (62.2%) mainly, the pros and cons of vaccination, adverse reactions, and the number of inoculations required. Inquiries about pregnancy, infants and young children, and breast-feeding accounted for 19.2%. Inquiries on vaccine shortages during the 2004-2005 flu season (7), SARS (22), and bird flu (22) decreased compared to the previous season, while the number of consultations on antiviral agents increased (209). In discussing how information on influenza should be communicated to the public, we propose that "Influenza Q & A" provided by the Infectious Diseases Surveillance Center of the NIID, MHLW, should include information on influenza specifically addressing pregnant woman and breast-feeding or child-rearing mothers.

Dual antiplasmodial activity of vitamin D3 and its analog, 22-oxacalcitriol, by direct and indirect mechanisms.

Recent evidence suggests that 1α,25-dihydroxyvitamin D3 (calcitriol, VD3), the active form of vitamin D (VD), can inhibit the proliferation of microorganisms. In the present study, we conducted in vitro experiments and utilized in vivo murine models to investigate the antimalarial activity of VD3 and its analog, 22-oxacalcitriol (22-OCT), which was designed to cause less hypercalcemia than VD3. VD3 and 22-OCT treatments effectively resolved a Plasmodium chabaudi (Pc) infection in wild-type mice. Reduced parasitemia was observed during the acute phase of infection in the presence of VD3 and 22-OCT, followed by a delayed peak during the chronic stage of infection. Some anti-Pc activity was observed in VD receptor knockout (KO) mice. VD3 and 22-OCT also completely inhibited the proliferation of P. falciparum (Pf) in human red blood cells in vitro. Plasma levels of interferon (IFN)-γ in VD3-treated B10 and B6 mice were lower than those in vehicle-treated animals, and VD3 resolved a Pc infection in IFN-γ-KO mice, which greatly improved survival. These data suggest that the protective effects of VD3 are elicited through an IFN-γ-independent mechanism. Effective antiplasmodial doses of VD3 and 22-OCT resulted in a loss of body weight in mice. This loss in body weight occurred concomitantly with the development of hypercalcemia. Zoledronic acid partially attenuated VD3-induced hypercalcemia and abrogated the antiparasitic effects of VD3. This study highlights a potential therapeutic role for VD3 in the treatment of malarial infections and shows that hypercalcemia is excellent indicator of the antiplasmodial activity of VD3.

Anti-trypanosome effects of nutritional supplements and vitamin D3: in vitro and in vivo efficacy against Trypanosoma brucei brucei.

BACKGROUND: Previous publications suggest that nutritional supplements have anti-trypanosome activity in vitro, although apparent efficacy was not noted in vivo. This study was conducted by experimentally infecting mice with Trypanosoma brucei brucei to assess the anti-trypanosome activity of various nutritional supplements with the hope of finding possible application in the treatment of African trypanosomiasis. METHODS: Activities of nutritional supplements were screened in vitro against bloodstream forms of T. b. brucei. To evaluate selectivity, we used two mammalian cells, Jurkat cells and Vero cells. The IC50 values and selectivity index values were calculated, and supplements with promising efficacy in vitro were selected for further testing in vivo. Mice were infected intraperitoneally with 1 × 10(3) T. b. brucei. We observed parameters for disease progression such as parasitemia, red blood cell count, white blood cell count, survivability, and splenomegaly. Morphological profiles after the treatment were analyzed by scanning electron microscopy. RESULTS: Vitamin D3 showed anti-trypanosome efficacies both in vitro and in vivo. It seemed to have suppressive effects on parasitemia, and spleen weight was also significantly lower in vitamin D3-treated mice when compared to non-treated control mice. There was, however, no significant prolonged survivability of infected mice treated with vitamin D3. Among green tea extracts, polyphenon-60 and epigallocatechin gallate had suppressive effects against T. b. brucei in vitro, but in vivo efficacies were marginal. CONCLUSIONS: Treatment with nutritional supplements, vitamin D3, and polyphenon-60 seemed to have anti-trypanosome activity in vitro and protective activity to some extent in vivo, respectively, although those supplements themselves did not have curable effects. The exact mechanisms of action are not clear, but the significant efficacy in vitro suggested direct effects of supplements against African trypanosome parasites.

Immunological detection of severe acute respiratory syndrome coronavirus by monoclonal antibodies.

In order to establish immunological detection methods for severe acute respiratory syndrome coronavirus (SARS-CoV), we established monoclonal antibodies directed against structural components of the virus. B cell hybridomas were generated from mice that were hyper-immunized with inactivated SARS-CoV virion. By screening 2,880 generated hybridomas, we established three hybridoma clones that secreted antibodies specific for nucleocapsid protein (N) and 27 clones that secreted antibodies specific for spike protein (S). Among these, four S-protein specific antibodies had in vitro neutralization activity against SARS-CoV infection. These monoclonal antibodies enabled the immunological detection of SARS-CoV by immunofluorescence staining, Western blot or immunohistology. Furthermore, a combination of monoclonal antibodies with different specificities allowed the establishment of a highly sensitive antigen-capture sandwich ELISA system. These monoclonal antibodies would be a useful tool for rapid and specific diagnosis of SARS and also for possible antibody-based treatment of the disease.

Leucomycin A3, a 16-membered macrolide antibiotic, inhibits influenza A virus infection and disease progression.

Severe respiratory disease arising from influenza virus infection has a high fatality rate. Neutrophil myeloperoxidase (MPO) has been implicated in the pathogenesis of severe influenza-induced pneumonia because extracellularly released MPO mediates the production of hypochlorous acid, a potent tissue injury factor. To search for candidate anti-influenza compounds, we screened leucomycin A3 (LM-A3), spiramycin (SPM), an erythromycin derivative (EM900, in which anti-bacterial activity has been eliminated), and clarithromycin (CAM), by analyzing their ability to inhibit MPO release in neutrophils from mice and humans. When each candidate was injected into mice infected with a lethal dose of A/H1N1 influenza virus (PR-8), LM-A3 produced the highest survival rate (80.9%). We found that LM-A3 induced beneficial effects on lung pathology and viral proliferation involved in the regulatory activity of MPO release, pro-inflammatory cytokines and interferon-α production in the lung. SPM and EM900 also induced positive survival effects in the infected mice, whereas CAM did not. We further found that these compounds inhibit virus proliferation in human pneumonia epithelial A549 cells in vitro. LM-A3 showed effective action against influenza A virus infection with high anti-viral activity in human host cells, indicating the possibility that LM-A3 is a prospective lead compound for the development of a drug for human influenza. The positive survival effect induced by EM900 suggests that pharmacological architectures between anti-bacterial and anti-influenza virus activities can be dissociated in macrolide derivatives. These observations provide valuable evidence for the potential development of novel macrolide derivatives that have strong anti-viral but no anti-bacterial activity.