Toloese Generates Nitric Oxide through Natural Radiation of Far Infrared Rays, Reducing Serum Glucose, Cholesterol, and Triglycerides.

Toloese, a bed composition, is formulated with a combination of minerals of various wavelengths by utilizing a specific ratio and particle size. A maturation mixing technique is used without additional compression processes, resulting in the natural formation of numerous fine pores in the bed structure. At 40 °C, far infrared radiation in the range of 5-20 µm is emitted with a 0.916 radiant ratio, and the measured emitted radiant energy is 3.69 × 102 W/m2·µm. This study aimed to investigate the influence of far infrared radiation emitted from a Toloese bed on endogenous nitric oxide production. Clinical trials were conducted with 20 healthy adults aged 20 years. Blood samples were collected before and after Toloese bed usage for 1 h daily for 3 weeks. Nitric oxide levels in the saliva and blood of men and women significant increased after they used the Toloese bed for 1 h. Additionally, sweating sharply increased in the upper and lower body regions after Toloese bed usage. No hematological changes or adverse effects were observed, but blood glucose, cholesterol, and triglycerides decreased after Toloese bed usage compared with those before Toloese bed usage. These findings demonstrated that far infrared radiation emitted by the Toloese bed induced endogenous nitric oxide production and contributed to significant reductions in blood glucose, cholesterol, and triglyceride levels.

Development of a recombinant vaccine containing a spike S1-Fc fusion protein induced protection against MERS-CoV in human DPP4 knockin transgenic mice.

The Middle East respiratory syndrome coronavirus (MERS-CoV), belonging to the family Coronaviridae and genus Betacoronavirus, has been recognized as a highly pathogenic virus. Due to the lack of therapeutic or preventive agents against MERS-CoV, developing an effective vaccine is essential for preventing a viral outbreak. To address this, we developed a recombinant S1 subunit of MERS-CoV spike protein fused with the human IgG4 Fc fragment (LV-MS1-Fc) in Chinese hamster ovary (CHO) cells. Thereafter, we identified the baculovirus gp64 signal peptide-directed secretion of LV-MS1-Fc protein in the extracellular fluid. To demonstrate the immunogenicity of the recombinant LV-MS1-Fc proteins, BALB/c mice were inoculated with 2.5 µg of LV-MS1-Fc. The inoculated mice demonstrated a significant humoral immune response, measured via total IgG and neutralizing antibodies. In addition, human dipeptidyl peptidase-4 (DPP4) transgenic mice vaccinated with LV-MS1-Fc showed the protective capacity of LV-MS1-Fc against MERS-CoV with no inflammatory cell infiltration. These data showed that the S1 and Fc fusion protein induced potent humoral immunity and antigen-specific neutralizing antibodies in mice, and conferred protection against coronavirus viral challenge, indicating that LV-MS1-Fc is an effective vaccine candidate against MERS-CoV infection.

Replacing the decoy epitope of PCV2 capsid protein with epitopes of GP3 and/or GP5 of PRRSV enhances the immunogenicity of bivalent vaccines in mice.

Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome (PMWS), porcine dermatitis and nephropathy syndrome (PDNS), and reproductive failure and causes economic losses in the domestic swine industry. The decoy epitope (169-180 amino acid (aa)) of the PCV2 capsid (Cap) protein is an immunodominant epitope and diverts the immune response away from protective epitopes. The mixed infection of PCV2 and porcine reproductive and respiratory syndrome virus (PRRSV) is one of the most common co-infections in the pig industry and shows more severe clinical symptoms. Linear B-cell antigenic epitopes of PRRSV GP3 epitope Ⅰ (61-72aa) and PRRSV GP5 epitope Ⅳ (187-200aa) efficiently elicited neutralizing antibodies against PRRSV. The recombinant baculovirus expressing the Cap protein (Bac-Cap) was modified by replacing the decoy epitope of the Cap protein with either the PRRSV GP3 epitope Ⅰ, the PRRSV GP5 epitope Ⅳ, or the PRRSV GP3 epitope Ⅰ- GP5 epitope Ⅳ to produce the recombinant baculoviruses Bac-Cap-GP3, Bac-Cap-GP5 and Bac-Cap-GP35. The four recombinant baculoviruses were successfully established and characterized as demonstrated with western blot analysis and immunofluorescence assay. Immunogenicities of the four recombinant baculoviruses in mice were tested in sera harvested at 21 and 42 days post-primary immunization. The titers of antibodies in the sera were determined by a PCV2-specific enzyme-linked immunosorbent assay (ELISA) and a serum neutralization assay. The serum IFN-γ levels were measured by indirect ELISA. The results showed that Bac-Cap-GP3, Bac-Cap-GP5, and Bac-Cap-GP35 elicited higher GP3/GP5 and Cap antibody titers than the Bac-Cap. Virus neutralization test also confirmed that the serum from the Bac-Cap-GP3 immunized mice had high levels of the both PCV2 and PRRSV neutralization antibodies. These findings collectively demonstrated that substituting the decoy epitope of the PCV2 capsid substituted with PRRSV epitopes could be developed into an effective vaccine against PCV2.

Oral activity of the antimalarial endoperoxide 6-(1,2,6,7-tetraoxaspiro[7.11]nonadec-4-yl)hexan-1-ol (N-251) against Leishmania donovani complex.

Visceral leishmaniasis (VL) is a major problem worldwide and causes significant morbidity and mortality. Existing drugs against VL have limitations, including their invasive means of administration long duration of treatment regimens. There are also concerns regarding increasing treatment relapses as well as the identification of resistant clinical strains with the use of miltefosine, the sole oral drug for VL. There is, therefore, an urgent need for new alternative oral drugs for VL. In the present study, we show the leishmanicidal effect of a novel, oral antimalarial endoperoxide N-251. In our In vitro studies, N-251 selectively and specifically killed Leishmania donovani D10 amastigotes with no accompanying toxicity toward the host cells. In addition, N-251 exhibited comparable activities against promastigotes of L. donovani D10, as well as other L. donovani complex parasites, suggesting a wide spectrum of activity. Furthermore, even after a progressive infection was established in mice, N-251 significantly eliminated amastigotes when administered orally. Finally, N-251 suppressed granuloma formation in mice liver through parasite death. These findings indicate the therapeutic effect of N-251 as an oral drug, hence suggest N-251 to be a promising lead compound for the development of a new oral chemotherapy against VL.

Roles of human apolipoprotein E in the infectivity and replication of hepatitis C virus genotype 2a.

Hepatitis C virus (HCV) infection is associated with lipoproteins, and apolipoprotein E (apoE) plays an essential role in infectious HCV particles. Although the role of apoE in HCV infection is well known, its role in the replication of HCV remains unclear. The aims of this study were to determine the role of apoE in the RNA replication of major HCV genotypes 1b and 2a, and to determine whether this role is HCVgenotype-dependent using HCV genotype 1b replicon cells and HCV genotype 2a producing (HP) cells. HCV infection was blocked in Huh7.5 cells treated with low-density lipoproteins, very low-density lipoproteins, or apoE3. An apoE3-specific monoclonal antibody also efficiently neutralized HCV infectivity, and HCV infection was dramatically suppressed by the knockdown of apoE expression with an apoE-specific small interfering RNA, suggesting a requirement for apoE in infectious HCV particles. HCV RNA replication was not affected in HP cells treated with each apoE isoform or transfected with apoE-specific siRNAs. However, the knockdown of apoE expression suppressed RNA replication of HCV genotype 1b. The siRNA-mediated knockdown of apoE, apoA1, and apoB expression also suppressed the RNA replication of HCV genotype 1b, but not that of HCV genotype 2a. Taken together, these findings indicate that apoE plays an important role in HCV genotype 2a infection and in HCV genotype 1b RNA replication, but not in the replication of HCV genotype 2a. These results provide important information for the future development of HCV-genotypespecific anti-HCV agents.

Human apolipoprotein e is required for infectivity and production of hepatitis C virus in cell culture.

Recent advances in reverse genetics of hepatitis C virus (HCV) made it possible to determine the properties and biochemical compositions of HCV virions. Sedimentation analysis and characterization of HCV RNA-containing particles produced in the cultured cells revealed that HCV virions cover a large range of heterogeneous densities in sucrose gradient. The fractions of low densities are infectious, while the higher-density fractions containing the majority of HCV virion RNA are not. HCV core protein and apolipoprotein B and apolipoprotein E (apoE) were detected in the infectious HCV virions. The level of apoE correlates very well with HCV infectivity. Both apoE- and HCV E2-specific monoclonal antibodies precipitated HCV, demonstrating that HCV virions contain apoE and E2 proteins. apoE-specific monoclonal antibodies efficiently neutralized HCV infectivity in a dose-dependent manner, resulting in a reduction of infectious HCV by nearly 4 orders of magnitude. The knockdown of apoE expression by specific small interfering RNAs (siRNAs) remarkably reduced the levels of intracellular as well as secreted HCV virions. The apoE siRNA suppressed HCV production by more than 100-fold at 50 nM. These findings demonstrate that apoE is required for HCV virion infectivity and production, suggesting that HCV virions are assembled as apoE-enriched lipoprotein particles. Our findings also identified apoE as a novel target for discovery and development of antiviral drugs and monoclonal antibodies to suppress HCV virion formation and infection.

Development of a nested polymerase chain reaction method to detect oncogenic Marek's disease virus from feather tips.

For the easy survey of Marek's disease virus (MDV), feather tip-derived DNA from MDV-infected chickens can be used because feather tips are easy to collect and feather follicle epithelium is known to be the only site of productive replication of cell-free MDV. To develop a diagnostic method to differentiate highly virulent strains of MDV from the attenuated MDV vaccine strain, CVI988, which is widely used, nested polymerase chain reaction (PCR) was performed to detect a segment of the meq gene in feather tip samples of chickens experimentally infected with MDV. In chickens infected with Md5, a strain of oncogenic MDV, the meq gene was consistently detected, whereas the L-meq gene, in which a 180-base pair (180-bp) sequence is inserted into the meq gene, was detected in CVI988-infected chickens. Moreover, the meq gene was mainly detected even in chickens co-infected with both Md5 and CVI988. These results suggest that this method is appropriate for the surveillance of the highly virulent MDV infection in the field.

Human serum amyloid A protein inhibits hepatitis C virus entry into cells.

Serum amyloid A (SAA) is an acute-phase protein induced by a variety of inflammatory stimuli, including bacterial and viral infections. SAA was recently found to function as an opsonin for gram-negative bacteria. We report here that SAA inhibited hepatitis C virus (HCV) infection in cultured cells. SAA reduced HCV infectivity in a dose-dependent manner when added during HCV infection but not after virus entry. SAA bound HCV virions and specifically blocked HCV entry but did not affect virus attachment. These findings suggest that SAA functions as part of the host innate immune defense mechanisms against HCV infection in humans.

Replication of hepatitis C virus (HCV) RNA in mouse embryonic fibroblasts: protein kinase R (PKR)-dependent and PKR-independent mechanisms for controlling HCV RNA replication and mediating interferon activities.

Hepatitis C virus (HCV) infection causes chronic hepatitis and is currently treated with alpha interferon (IFN-alpha)-based therapies. The underlying mechanisms of chronic HCV infection and IFN-based therapies, however, have not been defined. Protein kinase R (PKR) was implicated in the control of HCV replication and mediation of IFN-induced antiviral response. In this report, we demonstrate that a subgenomic RNA replicon of genotype 2a HCV replicated efficiently in mouse embryonic fibroblasts (MEFs), as determined by cell colony formation efficiency and the detection of HCV proteins and both positive- and negative-strand RNAs. Additionally, the subgenomic HCV RNA was found to replicate more efficiently in the PKR knockout (PKR(-/-)) MEF than in the wild-type (PKR(+/+)) MEF. The knockdown expression of PKR by specific small interfering RNAs significantly enhanced the level of HCV RNA replication, suggesting that PKR is involved in the control of HCV RNA replication. The level of ISG56 (p56) was induced by HCV RNA replication, indicating the activation of PKR-independent antiviral pathways. Furthermore, IFN-alpha/beta inhibited HCV RNA replication in PKR(-/-) MEFs as efficiently as in PKR(+/+) MEFs. These findings demonstrate that PKR-independent antiviral pathways play important roles in controlling HCV replication and mediating IFN-induced antiviral effect. Our findings also provide a foundation for the development of transgenic mouse models of HCV replication and set a stage to further define the roles of cellular genes in the establishment of chronic HCV infection and the mediation of intracellular innate antiviral response by using MEFs derived from diverse gene knockout animals.

The polypyrimidine tract-binding protein (PTB) is required for efficient replication of hepatitis C virus (HCV) RNA.

Our earlier work found that the polypyrimidine tract-binding protein (PTB) specifically interacts with the poly(U/C) tract of the hepatitis C virus (HCV) 3' untranslated region (3'UTR) [Luo, G., 1999. Cellular proteins bind to the poly(U) tract of the 3' untranslated region of hepatitis C virus RNA genome. Virology 256, 105-118]. We report here that the phosphorylated form of PTB is associated with the membrane-bound HCV replication complex. To determine whether the PTB is required for HCV RNA replication, synthetic small interfering RNAs (siRNAs) were used to specifically knockdown PTB expression in the HCV replicon-harboring Huh7 cells. The level of PTB expression was efficiently reduced by PTB-specific siRNAs. Consequently, the levels of HCV proteins as well as HCV RNA were proportionally decreased by increasing concentrations of PTB siRNA. However, the translation mediated by the encephamyocarditis virus internal ribosome entry site was unaffected, suggesting that the inhibition of HCV RNA replication by PTB siRNA was not due to its effect on the EMCV IRES-mediated translation. Collectively, these findings demonstrate that PTB is required for efficient HCV RNA replication in the cell.

Robust production of infectious hepatitis C virus (HCV) from stably HCV cDNA-transfected human hepatoma cells.

Hepatitis C virus (HCV) chronically infects approximately 170 million people worldwide, with an increased risk of developing cirrhosis and hepatocellular carcinoma. The study of HCV replication and pathogenesis has been hampered by the lack of an efficient stable cell culture system and small-animal models of HCV infection and propagation. In an effort to develop a robust HCV infection system, we constructed stable human hepatoma cell lines that contain a chromosomally integrated genotype 2a HCV cDNA and constitutively produce infectious virus. Transcriptional expression of the full-length HCV RNA genome is under the control of a cellular Pol II polymerase promoter at the 5' end and a hepatitis delta virus ribozyme at the 3' end. The resulting HCV RNA was expressed and replicated efficiently, as shown by the presence of high levels of HCV proteins as well as both positive- and negative-strand RNAs in the stable Huh7 cell lines. Stable cell lines robustly produce HCV virions with up to 10(8) copies of HCV viral RNA per milliliter (ml) of the culture medium. Subsequent infection of naïve Huh7.5 cells with HCV released from the stable cell lines resulted in high levels of HCV proteins and RNAs. Additionally, HCV infection was inhibited by monoclonal antibodies specific to CD81 and the HCV envelope glycoproteins E1 and E2, and HCV replication was suppressed by alpha interferon. Collectively, these results demonstrate the establishment of a stable HCV culture system that robustly produces infectious virus, which will allow the study of each aspect of the entire HCV life cycle.

Detection of the meq gene in the T cell subsets from chickens infected with Marek's disease virus serotype 1.

The meq gene was thought to be only detected in Marek's disease virus serotype 1 (MDV 1) including a very virulent strain, Md5, while L-meq, in which a 180-bp sequence is inserted into the meq open reading frame, is found in other strains of MDV 1, such as CVI 988/R6. However, both meq and L-meq were previously detected by PCR in chickens infected with MDV 1, suggesting that MDV 1 may consists of at least two subpopulations, one with meq, the other with L-meq. To further analyze these subpopulations, we analyzed the time course changes in distribution of these subpopulations among T cell subsets from chickens infected with MDV 1. Both meq and L-meq were detected in CD4+ and CD8+ T cells infected with strain Md5 or CVI 988/R6. The shift in MDV subpopulations from one displaying meq to the other displaying L-meq and/or the conversion from meq to L-meq occurred mainly in the CD8+ T cell subset from Md5-infected chickens. PCR products corresponding to L-meq rather than meq were frequently amplified from the CD8+ T cell subset from CVI 988/R 6 -infected chickens. These results suggest that a dominant subpopulation of MDV 1 changes depending on the T cell subsets, and that L-meq is dominantly present in the CD8+ T cells which play a role in the clearance of pathogenic agents.

Seroprevalence and molecular evidence for the presence of bovine immunodeficiency virus in Brazilian cattle.

Data on the worldwide distribution of bovine immunodeficiency virus (BIV) and bovine leukemia virus (BLV) is limited. A prevalence study of antibodies to BIV and BLV was conducted in six different cattle herds in Brazil. Out of a total of 238 sera analyzed, 11.7% were found positive for anti-BIV p26 antibodies as determined by Western blot analysis, 2.1% were positive for anti-BLV gp51 antibodies as detected by immunodiffusion test. Peripheral blood mononuclear cells from BIV seropositive cattle were found to have BIV-provirus DNA, as detected by nested polymerase chain reaction. A nucleotide sequence corresponding to a 298 bp fragment of the BIV pol gene was also analyzed. Amino acid sequences of these Brazilian pol gene products showed 98.0 to 100% homology to the American strain BIV R29, 97.0 to 99.0% to Japanese BIV isolates, and divergence ranged from 0 to 4.0% among Brazilian BIV isolates. This evidence of the presence of BIV and BLV infections in Brazil should be considered a health risk to Brazilian cattle populations and a potential causative agent of chronic disease in cattle.

The detection of the meq gene in chicken infected with Marek's disease virus serotype 1.

In the genome of strains of very virulent Marek's disease virus serotype 1(vvMDV1), such as Md5 and RB1B, the meq open reading frame (ORF) encoding a 339-amino-acid bZIP protein, is present, while a slightly longer meq ORF, termed as L-meq, in which a 180-bp sequence is inserted into the meq ORF is found in other strains of MDV1, such as CV1988/R6 and attenuated JM. When chickens were infected with vvMDV1 strains and the meq gene was amplified by nested polymerase chain reaction (PCR), the meq gene was detected throughout the experimental period for 7 weeks post inoculation (pi). However, the L-meq gene was also detected at 3 to 5 weeks and 3 to 4 weeks pi. in Md5-infected and RB1B-infected chickens, respectively. In the case of chickens infected with an attenuated MDV1, the JM strain, the L-meq gene was detected at 2 to 7 weeks pi., and the meq gene was also detected at 2 to 6 weeks pi. Both L-meq and meq genes were detected in chickens infected with an attenuated nononcogenic vaccine strain of MDV1 (CVI988/R6), throughout the experimental period. Though quantitative PCR was not performed, a larger amount of the PCR products corresponding to the L-meq than the meq gene was amplified from chickens infected with JM or CVI988/R6. These results suggest that a dynamic population shift between the MDV subpopulations displaying meq and L-meq genes occurs in chickens during the course of MDV infection. Since the MDV subpopulation that displays the L-meq gene only displays it during the latent phase, the L-meq and its gene product, if any, might contribute to the maintenance of the MDV latency.

Suppression of transcription activity of the MEQ protein of oncogenic Marek's disease virus serotype 1 (MDV1) by L-MEQ of non-oncogenic MDV1.

Meq is one of the candidate oncogenes in the MDV1 genome. We previously reported a difference in the meq open reading frame (ORF) between oncogenic and non-oncogenic MDV1: L-meq, in which a 180-bp sequence is inserted into the meq ORF, is detected in non-oncogenic MDV1. To study the functions of a gene product of L-meq (L-MEQ), transactivation by L-MEQ was analyzed by dual luciferase assay using a reporter gene under the control of long (-1--873 bp) and short (-1 - -355 bp) meq promoter (LMP and SMP, respectively). LMP showed higher promoter function than SMP. L-MEQ transactivated the expression of the reporter gene, but less than MEQ did. In the presence of SMP or the cytomegalovirus immediate-early promoter, the same or slightly higher transactivation was observed in cells cotransfected with both meq and L-meq than cells transfected only with meq. However, in the presence of LMP, lower transactivation was observed in cells cotransfected with both meq and L-meq than cells transfected only with meq, suggesting that L-MEQ can be a transrepressor. Replication of a vvMDV1 was enhanced in the cells with meq. Interestingly, however, replication of vvMDV1 was suppressed in the cells with L-meq or with both L-meq and meq, compared to untransfected cells. Thus, L-MEQ could suppress replication of vvMDV1 displaying the meq gene in coinfected cells.

Diversity (polymorphism) of the meq gene in the attenuated Marek's disease virus (MDV) serotype 1 and MDV-transformed cell lines.

The meq gene encoding a 339-amino-acid bZIP transactivator protein has been identified as a candidate oncogene of Marek's disease virus serotype 1 (MDV1), which induces malignant lymphomas in chickens. We have previously reported that, in addition to meq, L-meq, in which a 180-bp sequence is inserted into the region encoding the transactivation domain of meq, is also detected in chickens experimentally infected with MDV. To further analyze the diversity in meq, PCR was performed using a primer set which specifically amplify the proline-rich repeat (PRR) region in the transactivation domain of meq. In CVI988/R6, a vaccine strain of MDV1, and JM, an MDV1 strain attenuated by prolonged passage in vitro, a major band of a 0.8 kb corresponding to L-meq as well as a minor band of 0.6 kb corresponding to meq was detected by PCR. Furthermore, extra 0.5- and 0.3-kb bands, corresponding to genes termed as short meq (S-meq), and very short meq (VS-meq), respectively, were also detected. These genes were also detected in MDV-transformed cell lines, MSB1 and MTB1. In Md5, an oncogenic MDV1, attenuated by prolonged passage in vitro, the 0.6-kb meq was consistently detected, and 0.5-kb S-meq was occasionally detected. This diversity in meq was due to the difference in the copy number of the PRR region: L-meq and meq contained 9 and 6 copies of PRR while 4 and 2 copies of PRR were present in S-meq and VS-meq, respectively. Thus, the meq gene is polymorphic in the attenuated MDV1 and the MDV-transformed cell lines, and gene products from different meq genes may have different functions from each other.