Bakuchiol targets mitochondrial proteins, prohibitins and voltage-dependent anion channels: New insights into developing antiviral agents.

We previously reported that bakuchiol, a phenolic isoprenoid anticancer compound, and its analogs exert anti-influenza activity. However, the proteins targeted by bakuchiol remain unclear. Here, we investigated the chemical structures responsible for the anti-influenza activity of bakuchiol and found that all functional groups and C6 chirality of bakuchiol were required for its anti-influenza activity. Based on these results, we synthesized a molecular probe containing a biotin tag bound to the C1 position of bakuchiol. With this probe, we performed a pulldown assay for Madin-Darby canine kidney cell lysates and purified the specific bakuchiol-binding proteins with SDS-PAGE. Using nanoLC-MS/MS analysis, we identified prohibitin (PHB) 2, voltage-dependent anion channel (VDAC) 1, and VDAC2 as binding proteins of bakuchiol. We confirmed the binding of bakuchiol to PHB1, PHB2, and VDAC2 in vitro using Western blot analysis. Immunofluorescence analysis showed that bakuchiol was bound to PHBs and VDAC2 in cells and colocalized in the mitochondria. The knockdown of PHBs or VDAC2 by transfection with specific siRNAs, along with bakuchiol cotreatment, led to significantly reduced influenza nucleoprotein expression levels and viral titers in the conditioned medium of virus-infected Madin-Darby canine kidney cells, compared to the levels observed with transfection or treatment alone. These findings indicate that reducing PHBs or VDAC2 protein, combined with bakuchiol treatment, additively suppressed the growth of influenza virus. Our findings indicate that bakuchiol exerts anti-influenza activity via a novel mechanism involving these mitochondrial proteins, providing new insight for developing anti-influenza agents.

Clinical Utility of SARS-CoV-2 Antibody Titer Multiplied by Binding Avidity of Receptor-Binding Domain (RBD) in Monitoring Protective Immunity and Clinical Severity.

Conventional serum antibody titer, which expresses antibody level, does not provide antigen binding avidity of the variable region of the antibody, which is essential for the defense response to infection. Here, we quantified anti-SARS-CoV-2 antibody binding avidity to the receptor-binding domain (RBD) by competitive binding-inhibition activity (IC50) between SARS-CoV-2 S1 antigen immobilized on the DCP microarray and various RBD doses added to serum and expressed as 1/IC50 nM. The binding avidity analyzed under equilibrium conditions of antigen-antibody binding reaction is different from the avidity index measured with the chaotropic agent, such as urea, under nonequilibrium and short-time conditions. Quantitative determination of the infection-protection potential of antibodies was assessed by ABAT (antigen binding avidity antibody titer), which was calculated by the quantity (level) × quality (binding avidity) of antibodies. The binding avidity correlated strongly (r = 0.811) with cell-based virus-neutralizing activity. Maturation of the protective antibody induced by repeated vaccinations or SARS-CoV-2 infection was classified into three categories of ABAT, such as an initial, low, and high ABAT. Antibody maturity correlated with the clinical severity of COVID-19. Once a mature high binding avidity was achieved, it was maintained for at least 6-8 months regardless of the subsequent change in the antibody levels.

Anti-influenza Activity of Povidone-Iodine-Integrated Materials.

Personal protective equipment (PPE), including medical masks, should be worn for preventing the transmission of respiratory pathogens via infective droplets and aerosols. In medical masks, the key layer is the filter layer, and the melt-blown nonwoven fabric (NWF) is the most used fabric. However, the NWF filter layer cannot kill or inactivate the pathogens spread via droplets and aerosols. Povidone-iodine (PVP-I) has been used as an antiseptic solution given its potent broad-spectrum activity against pathogens. To develop PPE (e.g., medical masks) with anti-pathogenic activity, we integrated PVP-I into nylon-66 NWF. We then evaluated its antiviral activity against influenza A viruses by examining the viability of Madin-Darby canine kidney (MDCK) cells after inoculation with the virus strains exposed to the PVP-I-integrated nylon-66 NWF. The PVP-I nylon-66 NWF protected the MDCK cells from viral infection in a PVP-I concentration-dependent manner. Subsequently, we found to integrate PVP-I into nylon-66 and polyurethane materials among various materials. These PVP-I materials were also effective against influenza virus infection, and treatment with PVP-I nylon-66 NWF showed the highest cell survival among all the tested materials. PVP-I showed anti-influenza A virus activity when used in conjunction with PPE materials. Moreover, nylon-66 NWF integrated with PVP-I was found to be the best material to ensure anti-influenza activity. Therefore, PVP-I-integrated masks could have the potential to inhibit respiratory virus infection. Our results provide new information for developing multi-functional PPEs with anti-viral activity by integrating them with PVP-I to prevent the potential transmission of respiratory viruses.

Induction of systemic, mucosal, and cellular immunity against SARS-CoV-2 in mice vaccinated by trans-airway with a S1 protein combined with a pulmonary surfactant-derived adjuvant SF-10.

Background: There is a need for vaccines that can induce effective systemic, respiratory mucosal, and cellular immunity to control the COVID-19 pandemic. We reported previously that a synthetic mucosal adjuvant SF-10 derived from human pulmonary surfactant works as an efficient antigen delivery vehicle to antigen presenting cells in the respiratory and gastrointestinal tracts and promotes induction of influenza virus antigen-specific serum IgG, mucosal IgA, and cellular immunity. Methods: The aim of the present study was to determine the effectiveness of a new administration route of trans-airway (TA) vaccine comprising recombinant SARS-CoV-2 spike protein 1 (S1) combined with SF-10 (S1-SF-10 vaccine) on systemic, local, and cellular immunity in mice, compared with intramuscular injection (IM) of S1 with a potent adjuvant AddaS03™ (S1-AddaS03™ vaccine). Results: S1-SF-10-TA vaccine induced S1-specific IgG and IgA in serum and lung mucosae. These IgG and IgA induced by S1-SF-10-TA showed significant protective immunity in a receptor binding inhibition test of S1 and angiotensin converting enzyme 2, a receptor of SARS-CoV-2, which were more potent and faster achievement than S1-AddaS03™-IM. Enzyme-linked immunospot assay showed high numbers of S1-specific IgA and IgG secreting cells (ASCs) and S1-responsive IFN-γ, IL-4, IL-17A cytokine secreting cells (CSCs) in the spleen and lungs. S1-AddaS03™-IM induced IgG ASCs and IL-4 CSCs in spleen higher than S1-SF-10-TA, but the numbers of ASCs and CSCs in lungs were low and hardly detected. Conclusions: Based on the need for effective systemic, respiratory, and cellular immunity, the S1-SF-10-TA vaccine seems promising mucosal vaccine against respiratory infection of SARS-CoV-2.

A Murine Model of Food Allergy by Epicutaneous Adjuvant-Free Allergen Sensitization Followed by Oral Allergen Challenge Combined with Aspirin for Enhanced Detection of Hypersensitivity Manifestations and Immunotherapy Monitoring.

Food allergy is one of the major existing health problems, but no effective treatment is available. In the current work, a murine model that closely mimics pathogenesis of human food allergy and its quantifiable diagnostic parameter design, even for mild hypersensitivity reactions, were established. BALB/c mice were epicutaneously sensitized with 1 mg chicken egg ovomucoid (OVM) or cow's milk casein, free of adjuvants, five times a week for two consecutive weeks. Eleven days later, allergen-specific IgG1 and IgE in serum were measured by ELISA. On day 25, 20 mg OVM or 12 mg α-casein was administered orally, and allergic reactions such as the fall in rectal temperature, symptom scores during 90-120 min, serum mast cell protease-1 and cytokine levels were monitored. The detection of mild allergic reactions due to adjuvant-free allergen sensitization and oral allergen challenge routes was amplified by the combination of oral allergen and aspirin administration simultaneously or aspirin administration within 15-30 min before an allergen challenge. Quantification of the maximum symptom score and the frequency of symptoms during the monitoring period improved evaluation accuracy of food allergy signals. Based on these results, efficacy of casein oral immunotherapy for cow's milk allergies, which are generally difficult to detect, was monitored adequately.

Neurotropic influenza A virus infection causes prion protein misfolding into infectious prions in neuroblastoma cells.

Misfolding of the cellular prion protein, PrPC, into the amyloidogenic isoform, PrPSc, which forms infectious protein aggregates, the so-called prions, is a key pathogenic event in prion diseases. No pathogens other than prions have been identified to induce misfolding of PrPC into PrPSc and propagate infectious prions in infected cells. Here, we found that infection with a neurotropic influenza A virus strain (IAV/WSN) caused misfolding of PrPC into PrPSc and generated infectious prions in mouse neuroblastoma cells through a hit-and-run mechanism. The structural and biochemical characteristics of IAV/WSN-induced PrPSc were different from those of RML and 22L laboratory prions-evoked PrPSc, and the pathogenicity of IAV/WSN-induced prions were also different from that of RML and 22L prions, suggesting IAV/WSN-specific formation of PrPSc and infectious prions. Our current results may open a new avenue for the role of viral infection in misfolding of PrPC into PrPSc and formation of infectious prions.

Organic synthesis and anti-influenza A virus activity of cyclobakuchiols A, B, C, and D.

Novel antiviral agents for influenza, which poses a substantial threat to humans, are required. Cyclobakuchiols A and B have been isolated from Psoralea glandulosa, and cyclobakuchiol C has been isolated from P. corylifolia. The structural differences between cyclobakuchiol A and C arise due to the oxidation state of isopropyl group, and these compounds can be derived from (+)-(S)-bakuchiol, a phenolic isoprenoid compound present in P. corylifolia seeds. We previously reported that bakuchiol induces enantiospecific anti-influenza A virus activity involving nuclear factor erythroid 2-related factor 2 (Nrf2) activation. However, it remains unclear whether cyclobakuchiols A-C induce anti-influenza A virus activity. In this study, cyclobakuchiols A, B, and C along with cyclobakuchiol D, a new artificial compound derived from cyclobakuchiol B, were synthesized and examined for their anti-influenza A virus activities using Madin-Darby canine kidney cells. As a result, cyclobakuchiols A-D were found to inhibit influenza A viral infection, growth, and the reduction of expression of viral mRNAs and proteins in influenza A virus-infected cells. Additionally, these compounds markedly reduced the mRNA expression of the host cell influenza A virus-induced immune response genes, interferon-ß and myxovirus-resistant protein 1. In addition, cyclobakuchiols A-D upregulated the mRNA levels of NAD(P)H quinone oxidoreductase 1, an Nrf2-induced gene, in influenza A virus-infected cells. Notably, cyclobakuchiols A, B, and C, but not D, induced the Nrf2 activation pathway. These findings demonstrate that cyclobakuchiols have anti-influenza viral activity involving host cell oxidative stress response. In addition, our results suggest that the suitably spatial configuration between oxidized isopropyl group and phenol moiety in the structure of cyclobakuchiols is required for their effect.

A novel aqueous extract from rice fermented with Aspergillus oryzae and Saccharomyces cerevisiae possesses an anti-influenza A virus activity.

Human influenza virus infections occur annually worldwide and are associated with high morbidity and mortality. Hence, development of novel anti-influenza drugs is urgently required. Rice Power® extract developed by the Yushin Brewer Co. Ltd. is a novel aqueous extract of rice obtained via saccharization and fermentation with various microorganisms, such as Aspergillus oryzae, yeast [such as Saccharomyces cerevisiae], and lactic acid bacteria, possessing various biological and pharmacological properties. In our previous experimental screening with thirty types of Rice Power® extracts, we observed that the 30th Rice Power® (Y30) extract promoted the survival of influenza A virus-infected Madin-Darby canine kidney (MDCK) cells. Therefore, to identify compounds for the development of novel anti-influenza drugs, we aimed to investigate whether the Y30 extract exhibits anti-influenza A virus activity. In the present study, we demonstrated that the Y30 extract strongly promoted the survival of influenza A H1N1 Puerto Rico 8/34 (A/PR/8/34), California 7/09, or H3N2 Aichi 2/68 (A/Aichi/2/68) viruses-infected MDCK cells and inhibited A/PR/8/34 or A/Aichi/2/68 viruses infection and growth in the co-treatment and pre-infection experiments. The pre-treatment of Y30 extract on MDCK cells did not induce anti-influenza activity in the cell. The Y30 extract did not significantly affect influenza A virus hemagglutination, and neuraminidase and RNA-dependent RNA polymerase activities. Interestingly, the electron microscopy experiment revealed that the Y30 extract disrupts the integrity of influenza A virus particles by permeabilizing the viral membrane envelope, suggesting that Y30 extract has a direct virucidal effect against influenza A virus. Furthermore, we observed that compared to the ethyl acetate (EtOAc) extract, the water extract of Y30 extract considerably promoted the survival of cells infected with A/PR/8/34 virus. These results indicated that more anti-influenza components were present in the water extract of Y30 extract than in the EtOAc extract. Our results highlight the potential of a rice extract fermented with A. oryzae and S. cerevisiae as an anti-influenza medicine and a drug source for the development of anti-influenza compounds.

Prion protein signaling induces M2 macrophage polarization and protects from lethal influenza infection in mice.

The cellular prion protein, PrPC, is a glycosylphosphatidylinositol anchored-membrane glycoprotein expressed most abundantly in neuronal and to a lesser extent in non-neuronal cells. Its conformational conversion into the amyloidogenic isoform in neurons is a key pathogenic event in prion diseases, including Creutzfeldt-Jakob disease in humans and scrapie and bovine spongiform encephalopathy in animals. However, the normal functions of PrPC remain largely unknown, particularly in non-neuronal cells. Here we show that stimulation of PrPC with anti-PrP monoclonal antibodies (mAbs) protected mice from lethal infection with influenza A viruses (IAVs), with abundant accumulation of anti-inflammatory M2 macrophages with activated Src family kinases (SFKs) in infected lungs. A SFK inhibitor dasatinib inhibited M2 macrophage accumulation in IAV-infected lungs after treatment with anti-PrP mAbs and abolished the anti-PrP mAb-induced protective activity against lethal influenza infection in mice. We also show that stimulation of PrPC with anti-PrP mAbs induced M2 polarization in peritoneal macrophages through SFK activation in vitro and in vivo. These results indicate that PrPC could activate SFK in macrophages and induce macrophage polarization to an anti-inflammatory M2 phenotype after stimulation with anti-PrP mAbs, thereby eliciting protective activity against lethal infection with IAVs in mice after treatment with anti-PrP mAbs. These results also highlight PrPC as a novel therapeutic target for IAV infection.

Role of host trypsin-type serine proteases and influenza virus-cytokine-trypsin cycle in influenza viral pathogenesis. Pathogenesis-based therapeutic options.

Influenza A virus (IAV) is one of the most common infectious pathogen and associated with significant morbidity and mortality. Although processing the IAV hemagglutinin (HA) envelope glycoprotein precursor is a pre-requisite for viral membrane fusion activity, viral entry and transmission, HA-processing protease is not encoded in the IAV genome and thus the cellular trypsin-type serine HA-processing proteases determine viral infectious tropism and viral pathogenicity. The initial process of IAV infection of the airway is followed by marked upregulation of ectopic trypsin in various organs and endothelial cells through the induction of various proinflammatory cytokines, and this process has been termed the "influenza virus-cytokine-trypsin" cycle. In the advanced stage of IAV infection, the cytokine storm induces disorders of glucose and lipid metabolism and the "metabolic disorders-cytokine" cycle is then linked with the "influenza virus-cytokine-trypsin" cycle, to advance the pathogenic process into energy crisis and multiple organ failure. Application of protease inhibitors and treatment of metabolic disorders that break these cycles and their interconnection is therefore a promising therapeutic approach against influenza. This review discusses IAV pathogenicity on trypsin type serine HA-processing proteases, cytokines, metabolites and therapeutic options.

Oral vaccination with influenza hemagglutinin combined with human pulmonary surfactant-mimicking synthetic adjuvant SF-10 induces efficient local and systemic immunity compared with nasal and subcutaneous vaccination and provides protective immunity in mice.

We reported previously that a synthetic mucosal adjuvant SF-10, which mimics human pulmonary surfactant, delivers antigen to mucosal dendritic cells in the nasal cavity and promotes induction of humoral and cellular immunity. The aim of the present study was to determine the effects of oral administration of antigen combined with SF-10 (antigen-SF-10) on systemic and local immunity. Oral administration of ovalbumin, a model antigen, combined with SF-10 enhanced ovalbumin uptake into intestinal antigen presenting MHC II+CD11c+ cells and their CD11b+CD103+ and CD11b+CD103- subtype dendritic cells, which are the major antigen presenting subsets of the intestinal tract, more efficiently compared to without SF-10. Oral vaccination with influenza hemagglutinin vaccine (HAv)-SF-10 induced HAv-specific IgA and IgG in the serum, and HAv-specific secretory IgA and IgG in bronchoalveolar lavage fluid, nasal washes, gastric extracts and fecal material; their levels were significantly higher than those induced by subcutaneous HAv or intranasal HAv and HAv-SF-10 vaccinations. Enzyme-linked immunospot assay showed high numbers of HAv-specific IgA and IgG antibody secreting cells in the gastrointestinal and respiratory mucosal lymphoid tissues after oral vaccination with HAv-SF-10, but no or very low induction following oral vaccination with HAv alone. Oral vaccination with HAv-SF-10 provided protective immunity against severe influenza A virus infection, which was significantly higher than that induced by HAv combined with cholera toxin. Oral vaccination with HAv-SF-10 was associated with unique cytokine production patterns in the spleen after HAv stimulation; including marked induction of HAv-responsive Th17 cytokines (e.g., IL-17A and IL-22), high induction of Th1 cytokines (e.g., IL-2 and IFN-γ) and moderate induction of Th2 cytokines (e.g., IL-4 and IL-5). These results indicate that oral vaccination with HAv-SF-10 induces more efficient systemic and local immunity than nasal or subcutaneous vaccination with characteristically high levels of secretory HAv-specific IgA in various mucosal organs and protective immunity.

Prion protein protects mice from lethal infection with influenza A viruses.

The cellular prion protein, designated PrPC, is a membrane glycoprotein expressed abundantly in brains and to a lesser extent in other tissues. Conformational conversion of PrPC into the amyloidogenic isoform is a key pathogenic event in prion diseases. However, the physiological functions of PrPC remain largely unknown, particularly in non-neuronal tissues. Here, we show that PrPC is expressed in lung epithelial cells, including alveolar type 1 and 2 cells and bronchiolar Clara cells. Compared with wild-type (WT) mice, PrPC-null mice (Prnp0/0) were highly susceptible to influenza A viruses (IAVs), with higher mortality. Infected Prnp0/0 lungs were severely injured, with higher inflammation and higher apoptosis of epithelial cells, and contained higher reactive oxygen species (ROS) than control WT lungs. Treatment with a ROS scavenger or an inhibitor of xanthine oxidase (XO), a major ROS-generating enzyme in IAV-infected lungs, rescued Prnp0/0 mice from the lethal infection with IAV. Moreover, Prnp0/0 mice transgenic for PrP with a deletion of the Cu-binding octapeptide repeat (OR) region, Tg(PrPΔOR)/Prnp0/0 mice, were also highly susceptible to IAV infection. These results indicate that PrPC has a protective role against lethal infection with IAVs through the Cu-binding OR region by reducing ROS in infected lungs. Cu content and the activity of anti-oxidant enzyme Cu/Zn-dependent superoxide dismutase, SOD1, were lower in Prnp0/0 and Tg(PrPΔOR)/Prnp0/0 lungs than in WT lungs. It is thus conceivable that PrPC functions to maintain Cu content and regulate SOD1 through the OR region in lungs, thereby reducing ROS in IAV-infected lungs and eventually protecting them from lethal infection with IAVs. Our current results highlight the role of PrPC in protection against IAV infection, and suggest that PrPC might be a novel target molecule for anti-influenza therapeutics.

Enterokinase Enhances Influenza A Virus Infection by Activating Trypsinogen in Human Cell Lines.

Cleavage and activation of hemagglutinin (HA) by trypsin-like proteases in influenza A virus (IAV) are essential prerequisites for its successful infection and spread. In host cells, some transmembrane serine proteases such as TMPRSS2, TMPRSS4 and HAT, along with plasmin in the bloodstream, have been reported to cleave the HA precursor (HA0) molecule into its active forms, HA1 and HA2. Some trypsinogens can also enhance IAV proliferation in some cell types (e.g., rat cardiomyoblasts). However, the precise activation mechanism for this process is unclear, because the expression level of the physiological activator of the trypsinogens, the TMPRSS15 enterokinase, is expected to be very low in such cells, with the exception of duodenal cells. Here, we show that at least two variant enterokinases are expressed in various human cell lines, including A549 lung-derived cells. The exogenous expression of these enterokinases was able to enhance the proliferation of IAV in 293T human kidney cells, but the proliferation was reduced by knocking down the endogenous enterokinase in A549 cells. The enterokinase was able to enhance HA processing in the cells, which activated trypsinogen in vitro and in the IAV-infected cells also. Therefore, we conclude that enterokinase plays a role in IAV infection and proliferation by activating trypsinogen to process viral HA in human cell lines.

Clarithromycin suppresses induction of monocyte chemoattractant protein-1 and matrix metalloproteinase-9 and improves pathological changes in the lungs and heart of mice infected with influenza A virus.

The influenza A virus (IAV)-cytokine-trypsin/matrix metalloproteinase-9 (MMP-9) cycle is one of the important mechanisms of multiple organ failure in severe influenza. Clarithromycin, a macrolide antibiotic, has immune modulatory and anti-inflammatory effects. We analyzed the effects of clarithromycin on the induction of chemokines, cytokines, MMP-9, trypsin, vascular hyper-permeability and inflammatory aggravation in mice with IAV infection. IAV/Puerto Rico/8/34(H1N1) infection increased the levels of monocyte chemoattractant protein-1 (MCP-1) and cytokines in serum, and MMP-9 and trypsin in serum and/or the lungs and heart. Clarithromycin significantly suppressed the induction of serum MCP-1 and MMP-9 and vascular hyperpermeability in these organs in the early phase of infection, but did not suppress the induction of trypsin, IL-6 or IFN-γ. Histopathological examination showed that clarithromycin tended to reduce inflammatory cell accumulation in the lungs and heart. These results suggest that clarithromycin suppresses infection-related inflammation and reduces vascular hyperpermeability by suppressing the induction of MCP-1 and MMP-9.

Adjuvanting influenza hemagglutinin vaccine with a human pulmonary surfactant-mimicking synthetic compound SF-10 induces local and systemic cell-mediated immunity in mice.

We reported previously that intranasal instillation of a synthetic human pulmonary surfactant with a carboxy vinyl polymer as a viscosity improver, named SF-10, shows potent adjuvanticity for humoral immunity in mice and cynomolgus monkeys. SF-10 effectively induces influenza hemagglutinin vaccine (HAv)-specific IgA in nasal and lung washes and IgG in sera with their neutralizing activities. Since CD8+ T cell-mediated protection is an important requirement for adaptive immunity, we investigated in this study the effects of SF-10 with antigen on local and systemic cell-mediated immunity. Nasal instillation of ovalbumin, a model antigen, combined with SF-10 efficiently delivered antigen to mucosal dendritic and epithelial cells and promoted cross-presentation in antigen presenting cells, yielding a high percentage of ovalbumin-specific cytotoxic T lymphocytes in the nasal mucosa, compared with ovalbumin alone. Nasal immunization of HAv-SF-10 also induced HAv-specific cytotoxic T lymphocytes and upregulated granzyme B expression in splenic CD8+ T cells with their high cytotoxicity against target cells pulsed with HA peptide. Furthermore, nasal vaccination of HAv-SF-10 significantly induced higher cytotoxic T lymphocytes-mediated cytotoxicity in the lungs and cervical lymph nodes in the early phase of influenza virus infection compared with HAv alone. Protective immunity induced by HAv-SF-10 against lethal influenza virus infection was partially and predominantly suppressed after depletion of CD8+ and CD4+ T cells (induced by intraperitoneal injection of the corresponding antibodies), respectively, suggesting that CD4+ T cells predominantly and CD8+ T cells partially contribute to the protective immunity in the advanced stage of influenza virus infection. These results suggest that SF-10 promotes effective antigen delivery to antigen presenting cells, activates CD8+ T cells via cross-presentation, and induces cell-mediated immune responses against antigen.

Anti-influenza virus activity of extracts from the stems of Jatropha multifida Linn. collected in Myanmar.

BACKGROUND: To contribute to the development of novel anti-influenza drugs, we investigated the anti-influenza activity of crude extracts from 118 medicinal plants collected in Myanmar. We discovered that extract from the stems of Jatropha multifida Linn. showed anti-influenza activity. J. multifida has been used in traditional medicine for the treatment of various diseases, and the stem has been reported to possess antimicrobial, antimalarial, and antitumor activities. However, the anti-influenza activity of this extract has not yet been investigated. METHODS: We prepared water (H2O), ethyl acetate (EtOAc), n-hexane (Hex), and chloroform (CHCl3) extracts from the stems of J. multifida collected in Myanmar, and examined the survival of Madin-Darby canine kidney (MDCK) cells infected with the influenza A (H1N1) virus, and the inhibitory effects of these crude extracts on influenza A viral infection and growth in MDCK cells. RESULTS: The H2O extracts from the stems of J. multifida promoted the survival of MDCK cells infected with the influenza A H1N1 virus. The EtOAc and CHCl3 extracts resulted in similar, but weaker, effects. The H2O, EtOAc, and CHCl3 extracts from the stems of J. multifida inhibited influenza A virus H1N1 infection; the H2O extract possessed the strongest inhibitory effect on influenza infection in MDCK cells. The EtOAc, Hex, and CHCl3 extracts all inhibited the growth of influenza A H1N1 virus, and the CHCl3 extract demonstrated the strongest activity in MDCK cells. CONCLUSION: The H2O or CHCl3 extracts from the stems of J. multifida collected in Myanmar demonstrated the strongest inhibition of influenza A H1N1 viral infection or growth in MDCK cells, respectively. These results indicated that the stems of J. multifida could be regarded as an anti-influenza herbal medicine as well as a potential crude drug source for the development of anti-influenza compounds.

Heat stroke with bimodal rhabdomyolysis: a case report and review of the literature.

BACKGROUND: Severe heat stroke tends to be complicated with rhabdomyolysis, especially in patients with exertional heat stroke. Rhabdomyolysis usually occurs in the acute phase of heat stroke. We herein report a case of heat stroke in a patient who experienced bimodal rhabdomyolysis in the acute and recovery phases. CASE PRESENTATION: A 34-year-old male patient was found lying unconscious on the road after participating in a half marathon in the spring. It was a sunny day with a maximum temperature of 24.2 °C. His medical and family history was unremarkable. Upon arrival, his Glasgow Coma Scale score was 10. However, the patient's marked restlessness and confusion returned. A sedative was administered and tracheal intubation was performed. On the second day of hospitalization, a blood analysis was compatible with a diagnosis of acute hepatic failure; thus, he received fresh frozen plasma and a platelet transfusion was performed, following plasma exchange and continuous hemodiafiltration. The patient's creatinine phosphokinesis (CPK) level increased to 8832 IU/L on the fifth day of hospitalization and then showed a tendency to transiently decrease. The patient was extubated on the eighth day of hospitalization after the improvement of his laboratory data. From the ninth day of hospitalization, gradual rehabilitation was initiated. However, he felt pain in both legs and his CPK level increased again. Despite the cessation of all drugs and rehabilitation, his CPK level increased to 105,945 IU/L on the 15th day of hospitalization. Fortunately, his CPK level decreased with a fluid infusion. The patient's rehabilitation was restarted after his CPK level fell to <10,000 IU/L. On the 31st day of hospitalization, his CK level decreased to 623 IU/L and he was discharged on foot. Later, a genetic analysis revealed that he had a thermolabile genetic phenotype of carnitine palmitoyltransferase II (CPT II). CONCLUSIONS: Physicians should pay special attention to the stress of rehabilitation exercises, which may cause collapsed muscles that are injured by severe heat stroke to repeatedly flare up.

Energy metabolic disorder is a major risk factor in severe influenza virus infection: Proposals for new therapeutic options based on animal model experiments.

Severe influenza is characterized by cytokine storm and multiorgan failure. Influenza patients with underlying diseases show a rapid progression in disease severity. The major mechanism that underlies multiorgan failure during the progressive stage of infection, particularly in patients with underlying risk factors, is mitochondrial energy crisis. The relationship between the factors that determine infection severity, such as influenza virus, cytokines, cellular trypsin as a hemagglutinin processing protease for viral multiplication, accumulation of metabolic intermediates and ATP crisis in mitochondria, is termed the "influenza virus-cytokine-trypsin" cycle. This occurs during the initial stages of infection, and is interconnected with the "metabolic disorders-cytokine" cycle in the middle to late phase of infection. Experiments using animal models have highlighted the complex relationship between these two cycles. New treatment options have been proposed that target the ATP crisis and multiorgan failure during the late phase of infection, rather than antiviral treatments with neuraminidase inhibitors that work during the initial phase. These options are (i) restoration of glucose oxidation in mitochondria by diisopropylamine dichloroacetate, which inhibits infection-induced pyruvate dehydrogenase kinase 4 activity, and (ii) restoration of long-chain fatty acid oxidation in mitochondria by l-carnitine and bezafibrate, an agonist of peroxisome proliferation-activated receptors-ß/δ, which transcriptionally upregulates carnitine palmitoyltransferase II. The latter is particularly effective in patients with influenza-associated encephalopathy who have thermolabile and short half-life compound variants of carnitine palmitoyltransferase II.

Induction of systemic and mucosal immunity and maintenance of its memory against influenza A virus by nasal vaccination using a new mucosal adjuvant SF-10 derived from pulmonary surfactant in young cynomolgus monkeys.

Induction of systemic and mucosal immunity and maintenance of its memory was investigated in 12 young male cynomolgus monkeys after intranasal instillation of flu vaccine using a new mucosal adjuvant SF-10 derived from pulmonary surfactant constituents. Split-product of influenza virus A/California/7/2009(H1N1)pdm hemagglutinin vaccine (HAv) at 15 µg with or without SF-10 and the adjuvant alone were instilled intranasally three times every 2 weeks. SF-10-adjuvanted HAv (SF-10-HAv) elicited significantly higher HAv-specific IgG and hemagglutinin inhibition (HI) titers in serum and HAv-specific secretory IgA and its neutralizing activities in nasal washes compared with HAv antigen and SF-10 alone. Significant cross-neutralizing activities of nasal washes after the third vaccination to several other H1N1 and H3N2 strains were observed. HI titers in serum and neutralizing activities in nasal washes reached peak levels at 6 weeks after initial vaccination, then gradually decreased after 10 weeks and returned to the baseline levels at 36 weeks. A single intranasal revaccination of SF-10-HAv at 36 weeks rapidly and significantly increased both immunity in serum and nasal washes compared with naïve monkeys. Revaccination by one or two doses achieved almost maximal immunity at 2 or 4 weeks after instillation. Statistically significant adverse effects (e.g., body weight loss, elevated body temperature, nasal discharge, change in peripheral blood leukocyte and platelet counts) were not observed for 2 weeks after vaccination of SF-10-HAv, HAv or SF-10 and also during the experimental period. These results in young monkey model suggest the potential of clinical use SF-10 for intranasal flu vaccine.

Bakuchiol Is a Phenolic Isoprenoid with Novel Enantiomer-selective Anti-influenza A Virus Activity Involving Nrf2 Activation.

Influenza represents a substantial threat to human health and requires novel therapeutic approaches. Bakuchiol is a phenolic isoprenoid compound present in Babchi (Psoralea corylifolia L.) seeds. We examined the anti-influenza viral activity of synthetic bakuchiol using Madin-Darby canine kidney cells. We found that the naturally occurring form, (+)-(S)-bakuchiol, and its enantiomer, (-)-(R)-bakuchiol, inhibited influenza A viral infection and growth and reduced the expression of viral mRNAs and proteins in these cells. Furthermore, these compounds markedly reduced the mRNA expression of the host cell influenza A virus-induced immune response genes, interferon-ß and myxovirus-resistant protein 1. Interestingly, (+)-(S)-bakuchiol had greater efficacy than (-)-(R)-bakuchiol, indicating that chirality influenced anti-influenza virus activity. In vitro studies indicated that bakuchiol did not strongly inhibit the activities of influenza surface proteins or the M2 ion channel, expressed in Chinese hamster ovary cells. Analysis of luciferase reporter assay data unexpectedly indicated that bakuchiol may induce some host cell factor(s) that inhibited firefly and Renilla luciferases. Next generation sequencing and KeyMolnet analysis of influenza A virus-infected and non-infected cells exposed to bakuchiol revealed activation of transcriptional regulation by nuclear factor erythroid 2-related factor (Nrf), and an Nrf2 reporter assay showed that (+)-(S)-bakuchiol activated Nrf2. Additionally, (+)-(S)-bakuchiol up-regulated the mRNA levels of two Nrf2-induced genes, NAD(P)H quinone oxidoreductase 1 and glutathione S-transferase A3. These findings demonstrated that bakuchiol had enantiomer-selective anti-influenza viral activity involving a novel effect on the host cell oxidative stress response.