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The complete genome sequence of a new polerovirus found naturally infecting Artemisia princeps, artemisia virus B (ArtVB), was determined using high-throughput sequencing. The ArtVB genome comprises 6,141 nucleotides and contains six putative open reading frames (ORF0 to ORF5) with a genome structure typical of poleroviruses. A multiple sequence alignment showed that the complete ArtVB genome shares 50.98% nucleotide sequence identity with ixeridium yellow mottle virus 1 (IxYMaV-1, GenBank accession no. KT868949). ArtVB shares the highest amino acid sequence identity in P0 and P3–P5 (21.54%–51.69%) with other known poleroviruses. Phylogenetic analysis indicated that ArtVB should be considered a member of a new species within the genus Polerovirus, family Luteoviridae.

Damage-associated molecular patterns (DAMPs) are danger signals (or alarmins) alerting immune cells through pattern recognition receptors (PRRs) to begin defense activity. Moreover, DAMPs are host biomolecules that can initiate a noninflammatory response to infection, and pathogen-associated molecular pattern (PAMPs) perpetuate the inflammatory response to infection. Many DAMPs are proteins that have defined intracellular functions and are released from dying cells after tissue injury or chemo-/radiotherapy. In the tumor microenvironment, DAMPs can be ligands for Toll-like receptors (TLRs) expressed on immune cells and induce cytokine production and T-cell activation. Moreover, DAMPs released from tumor cells can directly activate tumor-expressed TLRs that induce chemoresistance, migration, invasion, and metastasis. Furthermore, DAMP-induced chronic inflammation in the tumor microenvironment causes an increase in immunosuppressive populations, such as M2 macrophages, myeloid-derived suppressor cells (MDSCs), and regulatory T cells (Tregs). Therefore, regulation of DAMP proteins can reduce excessive inflammation to create an immunogenic tumor microenvironment. Here, we review tumor-derived DAMP proteins as ligands of TLRs and discuss their association with immune cells, tumors, and the composition of the tumor microenvironment., Cancer: Boosting the right signals to suppress tumors Tumor cells killed by radiotherapy or chemotherapy release signaling molecules that stimulate both immune response and tumor aggressiveness; regulating these molecules could improve treatment efficacy. Tae Heung Kang, Yeong-Min Park, and co-workers at Konkuk University, Seoul, South Korea, have reviewed the role of damage-associated molecular patterns (DAMPs) in immunity and cancer. These signaling molecules act as danger signals, activating immune cells by binding to specific receptors. However, tumor cells have the same receptors, and DAMPs binding triggers chemoresistance and increases invasiveness. The researchers report that although DAMPs can trigger a helpful immune response, they can also cause chronic inflammation, which in turn promotes an immune suppression response, allowing tumors to escape immune detection. Improving our understanding of the functions of different DAMPs could improve our ability to boost the immune response and decrease tumor aggressiveness.

​Summary Globally, infection by seasonal influenza viruses causes 3–5 million cases of severe illness and 290,000–650,000 respiratory deaths each year. Various influenza vaccines, including inactivated split‐ and subunit‐type, recombinant and live attenuated vaccines, have been developed since the 1930s when it was discovered that influenza viruses could be cultivated in embryonated eggs. However, the protection rate offered by these vaccines is rather low, especially in very young children and the elderly. In this review, we describe the history of influenza vaccine development, the immune responses induced by the vaccines and the adjuvants applied. Further, we suggest future directions for improving the effectiveness of influenza vaccines in all age groups. This includes the development of an influenza vaccine that induces a balanced T helper cell type 1 and type 2 immune responses based on the understanding of the immune system, and the development of a broad‐spectrum influenza vaccine that can increase effectiveness despite antigen shifts and drifts, which are characteristics of the influenza virus. A brighter future can be envisaged if the development of an adjuvant that is safe and effective is realized.

The complete genome sequence of a new polerovirus found naturally infecting Artemisia princeps, artemisia virus B (ArtVB), was determined using high-throughput sequencing. The ArtVB genome comprises 6,141 nucleotides and contains six putative open reading frames (ORF0 to ORF5) with a genome structure typical of poleroviruses. A multiple sequence alignment showed that the complete ArtVB genome shares 50.98% nucleotide sequence identity with ixeridium yellow mottle virus 1 (IxYMaV-1, GenBank accession no. KT868949). ArtVB shares the highest amino acid sequence identity in P0 and P3-P5 (21.54%-51.69%) with other known poleroviruses. Phylogenetic analysis indicated that ArtVB should be considered a member of a new species within the genus Polerovirus, family Luteoviridae.

Purpose Study on the pathogen and the pathogen-related disease require the information at both cellular and organism level. However, lack of appropriate high-quality antibodies and the difference between the experimental animal models make it difficult to analyze in vivo mechanism of pathogen-related diseases. For more reliable research on the infection and immune-response of pathogen-related diseases, accurate analysis is essential to provide spatiotemporal information of pathogens and immune activity to avoid false-positive or mis-interpretations. In this regards, we have developed a method for tracking Francisella tularensis in the animal model without using the specific antibodies for the F. tularensis. Materials and methods A dual reporter plasmid using GFP-Lux with putative bacterioferritin promoter (pBfr) was constructed and transformed to F. tularensis live vaccine strain to generate F. tularensis LVS (FtLVS)-GFP-Lux for both fluorescence and bioluminescence imaging. For vaccination to F. tularensis infection, FtLVS and lipopolysaccharide (LPS) from FtLVS were used. Results We visualized the bacterial replication of F. tularensis in the cells using fluorescence and bioluminescence imaging, and traced the spatio-temporal process of F. tularensis pathogenesis in mice. Vaccination with LPS purified from FtLVS greatly reduced the bacterial replication of FtLVS in animal model, and the effect of vaccination was also successfully monitored with in vivo imaging. Conclusion We successfully established dual reporter labeled F. tularensis for cellular and whole body imaging. Our simple and integrated imaging analysis system would provide useful information for in vivo analysis of F. tularensis infection as well as in vitro experiments, which have not been fully explained yet with various technical problems.

Vaccination is one of the most successful strategies to prevent diseases caused by pathogens. Although various expression systems including Escherichia coli, yeast, insect, and mammalian cells are currently used for producing many of vaccines, these conventional platforms have the limitation of post-translational modification, high cost, and expensive scalability. In this respect, the plant-based expression system has been considered as an attractive platform to produce recombinant vaccines due to fast, cost-effective and scalable production as well as safety. This review discusses the development of plant-derived vaccines and the current stage of plant-based expression system.

Organoid is an in vitro multicellular form mimicking in vivo organ. Its similarity to human organ including cellular organization, molecular expression patterns, as well as genetic signatures enables to study the characteristics of infectious agents and host-pathogen interaction. For the features of organoid, this system also can be potentially used to cultivate currently uncultivable viruses of vaccine candidates. This paper will briefly describe problems in the current culture system for virus production and the possibility of organoid as culture system for viral vaccine and their current limitations that should be solved to meet the goal.

Vaccination is the most efficient method for infectious disease prevention. Parenteral injections such as intramuscular, intradermal, and subcutaneous injections have several advantages in vaccine delivery, but there are many drawbacks. Thus, the development of a new vaccine delivery system has long been required. Recently, microneedles have been attracting attention as new vaccination tools. Microneedle is a highly effective transdermal vaccine delivery method due to its mechanism of action, painlessness, and ease of use. Here, we summarized the characteristics of microneedles and the possibilities as a new vaccine delivery route.

Current Ebola virus outbreak in West Africa already reached the total number of 1,323 including 729 deaths by July 31st. the fatality is around 55% in the southeastern area of Guinea, Sierra Leone, Liberia, and Nigeria. The number of patients with Ebola Hemorrhagic Fever (EHF) was continuously increasing even though the any effective therapeutics or vaccines has not been developed yet. The Ebola virus in Guinea showed 98% homology with Zaire Ebola Virus.Study of the pathogenesis of Ebola virus infection and assess of the various candidates of vaccine have been tried for a long time, especially in United States and some European countries. Even though the attenuated live vaccine and DNA vaccine containing Ebola viral genes were tested and showed efficacy in chimpanzees, those candidates still need clinical tests requiring much longer time than the preclinical development to be approved for the practical treatment.It can be expected to eradicate Ebola virus by a safe and efficient vaccine development similar to the case of smallpox virus which was extinguished from the world by the variola vaccine.

Anthrax is caused by the spore-forming bacterium Bacillus anthracis, which has been used as a weapon for bioterrorism. Although current vaccines are effective, they involve prolonged dose regimens and often cause adverse reactions. High rates of mortality associated with anthrax have made the development of an improved vaccine a top priority. To identify novel vaccine candidates, we applied an immunoproteomics approach. Using sera from convalescent guinea pigs or from human patients with anthrax, we identified 34 immunogenic proteins from the virulent B. anthracis H9401. To evaluate vaccine candidates, six were expressed as recombinant proteins and tested in vivo. Two proteins, rGBAA_0345 (alkyl hydroperoxide reductase subunit C) and rGBAA_3990 (malonyl CoA-acyl carrier protein transacylase), have afforded guinea pigs partial protection from a subsequent virulent-spore challenge. Moreover, combined vaccination with rGBAA_0345 and rPA (protective antigen) exhibited an enhanced ability to protect against anthrax mortality. Finally, we demonstrated that GBAA_0345 localizes to anthrax spores and bacilli. Our results indicate that rGBAA_0345 may be a potential component of a multivalent anthrax vaccine, as it enhances the efficacy of rPA vaccination. This is the first time that sera from patients with anthrax have been used to interrogate the proteome of virulent B. anthracis vegetative cells.

In order to develop a successful vaccine against deadly diseases with a wide range of antigenic diversity, an in-depth knowledge of the molecules and signaling mechanisms between the vaccine candidates and immune cells is required. Therefore, monitoring vaccine components, such as antigen or adjuvants, and immune cell dynamics at the vaccination site or draining lymph nodes can provide important information to understand more about the vaccine response. This review briefly introduces and describes various non-invasive molecular imaging methods for visualizing immune cell dynamics after vaccination.

Orientia tsutsugamushi, a gram-negative bacterium, causes severe acute febrile illness in humans. Despite this danger, the route of infection, infectivity, and protective mechanisms of the host's immune response to O. tsutsugamushi are unclear. Dendritic cells (DCs) are one of the most important cell types in bridging the innate and adaptive immune responses. In this study, we observed that O. tsutsugamushi infects and replicates in monocyte-derived DCs (MODCs). During infection and replication, the expressions of the cytokines IL-12 and TNF-α, as well as the co-stimulatory molecules CD80, CD83, CD86, and CD40, were increased in MODCs. When O. tsutsugamushi-treated MODCs were co-cultured with autologous CD4(+) T cells, they enhanced production of IFN-γ, a major Th1 cytokine. Collectively, our results show that O. tsutsugamushi can replicate in MODCs and can simultaneously induce MODC maturation and increase proinflammatory cytokine levels in MODCs that subsequently activate CD4(+) T cells.

Objectives: Recent studies have reported that human immunodeficiency virus type 1 (HIV-1) proviruses are strongly suppressed in the unique epigenetic environments caused by chromatin modifications such as acetylation and methylation. Therefore, optimized therapeutic strategies directed against the virus reservoir using these epigenetic modifying agents (EMAs) should cure HIV infection. Methods: Cytotoxicity and HIV-1 reactivation were determined using the PrestoBlue™ Cell Viability Reagent and p24 HIV ELISA, respectively. Results: EMAs, including histone deacetylase inhibitors (VPA and SAHA), DNA methyltransferase inhibitor (5′-Aza-CdR), histone methyltransferase inhibitor (ADOX) and 12-O-tetradecanoylphorbol-13-acetate (TPA), were used to reactivate proviruses in HIV-1 latently infected cells. The effect of monotreatment with these EMAs on HIV-1 reactivation was VPA or SAHA > 5′-Aza-CdR > ADOX. Even though cotreatment with these potential HIV-1 reactivating agents did not show any significant reactivation effects in HIV-1 latently infected cells, employing SAHA under TPA treatment demonstrated a dramatic synergistic effect on purging HIV-1 proviruses in all HIV-1 latently infected cells via the ERK and AP-1 pathways. Conclusions: These results suggest that the combined approaches of EMAs, cotreatment of SAHA and TPA, could provide an effective way to lead a decline of HIV-1 reservoirs in patients.

Middle East respiratory syndrome coronavirus (MERS-CoV) causes severe cases of human respiratory disease. Since 2012, the victims have mainly come from the Middle East countries or sporadically from some other geographical regions seeded by the travelers who visited the Middle East. Such an introduction through travelling led to the emergence of a MERS-CoV outbreak in Korea in May 2015, which caused more than 140 confirmed human cases in less than a month. Using 70 complete genome sequences of MERS-CoV isolates, including the most recent sequences for the Korean and Chinese isolates, we reconstructed the phylogenetic relationships of the complete genome and the individual protein coding regions. The Korean MERS-CoV strain clustered in the previously established Hafr-Al-Batin-1_2013 clade together with two Saudi Arabian and one Chinese strain sampled in 2015. Although these four strains remained monophyletic in the entire protein-coding region, this clade showed different phylogenetic relationships across the genome, indicating a shared unique recombination pattern that is different from previously reported putative recombination strains. Our findings suggest that the recent ancestor of the Korean and its related MERS-CoV strains is characterized by unique mosaic genome pattern that is different from other putative recombinants. ispartof: Scientific Reports vol:6 issue:1 pages:18825- ispartof: location:England status: published

Clinical and preclinical in vivo immune cell imaging approaches have been used to study immune cell proliferation, apoptosis and interaction at the microscopic (intra-vital imaging) and macroscopic (whole-body imaging) level by use of ex vivo or in vivo labeling method. A series of imaging techniques ranging from non-radiation based techniques such as optical imaging, MRI, and ultrasound to radiation based CT/nuclear imaging can be used for in vivo immune cell tracking. These imaging modalities highlight the intrinsic behavior of different immune cell populations in physiological context. Fluorescent, radioactive or paramagnetic probes can be used in direct labeling protocols to monitor the specific cell population. Reporter genes can also be used for genetic, indirect labeling protocols to track the fate of a given cell subpopulation in vivo. In this review, we summarized several methods dealing with dendritic cell, macrophage, and T lymphocyte specifically labeled for different macroscopic whole-body imaging techniques both for the study of their physiological function and in the context of immunotherapy to exploit imaging-derived information and immune-based treatments.

ObjectiveRecombinant protective antigen (rPA) is the active pharmaceutical ingredient of a second generation anthrax vaccine undergoing clinical trials both in Korea and the USA. By using the rPA produced from Bacillus brevis pNU212 expression system, correlations of serological immune response to anthrax protection efficacy were analyzed in a guinea pig model.MethodsSerological responses of rPA anthrax vaccine were investigated in guinea pigs that were given single or two injections (interval of 4 weeks) of various amounts of rPA combined with aluminum hydroxide adjuvant. Guinea pigs were subsequently challenged by the intramuscular injection with 30 half-lethal doses (30LD50) of virulent Bacillus anthracis spores. Serum antibody titers were determined by anti-PA IgG ELISA and the ability of antibodies to neutralize the cytotoxicity of lethal toxin on J774A.1 cell was measured through the toxin neutralizing antibody (TNA) assay.ResultsTo examine correlations between survival rate and antibody titers, correlation between neutralizing antibody titers and the extent of protection was determined. Toxin neutralization titers of at least 1176 were sufficient to confer protection against a dose of 30LD50 of virulent anthrax spores of the H9401 strain. Such consistency in the correlation was not observed from those antibody titers determined by ELISA.ConclusionNeutralizing-antibody titers can be used as a surrogate marker.

The remarkable efforts that are made on molecular imaging technologies demonstrate its potential importance and range of applications. The generation of disease-specific animal models, and the developments of target-specific probes and genetically encoded reporters are another important component. Continued improvements in the instrumentation, the identification of novel targets and genes, and the availability of improved imaging probes should be made. Multimodal imaging probes should provide easier transitions between laboratory studies, including small animal studies and clinical applications. Here, we reviewed basic strategies of noninvasive in vivo imaging methods in small animals to introducing the concept of molecular imaging.

ObjectivesMany natural compounds have been investigated as drug candidates to prevent human immunodeficiency virus (HIV) with low cytotoxicity. We tested whether ingenol from Euphorbia ingens exerts anti-HIV effects in human T cell lines.Methods and ResultsIngenol effectively maintained high cell viability (CD50, >1 mM) in H9 and MT4 T cells. The efficacy of ingenol to inhibit HIV-1 infection was dose dependent. ED50 for 100 and 200 TCID50 of HIV-1 was 5.06 and 16.87 μM, respectively. Gag p24 antigen production in ingenol-treated MT4 cells was reduced by 24.5% on day 6 post-infection. While p24 antigen was reduced in ingenol-treated cells, levels of cytokines such as TNF-α and IL-6 and chemokines such as RANTES and MCP-1 were increased. dUTP level related to late apoptotic events was increased on day 2 post-infection of HIV by ingenol treatment, whereas expression of annexin V was unchanged. Reduced levels of iNOS and ZAP-70 after HIV infection were recovered by ingenol treatment.ConclusionIngenol helps T cells to survive longer against viremia after HIV-1 infection, without exerting cytotoxic effects. Ingenol can be considered a safe and efficacious candidate for immune-boosting therapy for AIDS patients.

Many human immunodeficiency virus 1 (HIV-1) researchers focus on the developing new anti-reservoir therapy to eradicate HIV-1 provirus from the HIV-1-infected patients. HIV-1 provirus is the major obstacle for effective HIV-1 treatment because it integrates into the host genome and can produce a virus progeny after stopping highly active antiretroviral therapy (HAART). We established two novel cell lines latently infected with HIV-1 by limiting dilution cloning of A3.01 cells infected with HIV-1. Analysis of the flanking sequence of HIV-1 proviral DNA integrated into chromosomal cellular DNA revealed that proviral DNA was inserted into different sites of different chromosomes in the two examined cell lines. In these lines, virus reactivation could be induced by a phorbol 12-myristate 13-acetate (PMA) treatment that resulted in a marked increase of the production HIV-1 p24 antigen and appearance of the infectious virus. The novel cell lines latently infected with HIV-1 represent further tool for the study of molecular mechanisms of viral latency and development of anti-reservoir therapy of HIV-1 infection.

To generate a more efficient in vivo reporter and therapeutic gene, we optimized the coding sequence of the human sodium/iodide symporter (NIS) gene by replacing NIS DNA codons from wild type to new codons having the highest usage in human gene translation. The Codon Adaptation Index (CAI), representing the number of codons effective for human expression, was much improved (0.79 for hNIS, 0.97 for opt-hNIS). Both wild-type (hNIS) and optimized human NIS (opt-hNIS) were cloned into pcDNA3.1 and pMSCV vectors for transfection. Various cancer cell lines such as thyroid (TPC-1, FRO, B-CPAP), breast (MDA-MB-231), liver (Hep3B), cervical (HeLa), and glioma (U87MG) were transfected with pcDNA3.1/hNIS or pcDNA3.1/opt-hNIS. I-125 uptake by opt-hNIS-expressing cells was 1.6 similar to 2.1 times higher than uptake by wild-type hNIS-expressing cells. Stable cell lines were also established by retroviral transduction using pMSCV/hNIS or pMSCV/opt-hNIS, revealing higher NIS protein levels and I-125 uptake in opt-hNIS-expressing cells than in hNIS-expressing cells. Moreover, scintigraphic images from cell plates and mouse xenografts showed stronger signals from opt-hNIS-expressing cells than hNIS-expressing cells, and radioactivity uptake by opt-hNIS-expressing tumors was 2.3-fold greater than that by hNIS-expressing tumors. To test the efficacy of radioiodine therapy, mouse xenograft models were established with cancer cells expressing hNIS or opt-hNIS. I-131 treatment reduced tumor sizes of hNIS-and opt-hNIS-expressing tumors to 0.57-and 0.27-fold, respectively, compared to their sizes before therapy, suggesting an improved therapeutic effect of opt-hNIS. In summary, this study shows that codon optimization strongly increases hNIS protein levels and radioiodine uptake, thus supporting opt-hNIS as a more sensitive reporter and efficient therapeutic gene.

By taking advantage of bioluminescence imaging to monitor luciferase expressing splenocytes from the luciferase transgenic mouse, we visulaized the enhancement of immune response against hepatitis B virus (HBV) vaccine with adjuvants. To visualize antigen in vivo, large hepatitis B virus antigen (L-HBsAg) was labeled with radioiodine (125I). B6 mice were intramuscularly vaccinated. The localization of 125I-L-HBsAg was monitored for 5 weeks using animal SPECT/CT. To monitor the immune response, the luciferase expressing splenocytes were injected intravenously into immunized B6 mice. Bioluminescence signals from splenocytes were measured by IVIS 100 system. Localization of inoculated L-HBsAg was successfully monitored using animal SPECT/CT. L-HBsAg was lasted for 5 weeks and diminished. In addition, the injected splenocytes were successfully visualized in immunized mice, homing to spleen within 30 minutes and were accumulated in lymph nodes within 5 hours. Accumulation of splenocytes at vaccination site was observed within 24 hours. Moreover, after 10 hours, mouse vaccinated with antigen and two adjuvants showed 4 times more accumulation of splenocytes at vaccination site compared to mouse vaccinated with antigen only. Six days later, mouse vaccinated with antigen and two adjuvants showed 1.7-4.73 fold increased luciferase intensity of splenocytes at spleen, lymph nodes and vaccination site compared to mouse vaccinated with antigen only. In conclusion, in vivo real-time bioluminescent monitoring of splenocytes homing and proliferation against vaccination successfully provides efficiency of adjuvants. Our imaging system can be used for evaluation of efficacy of vaccination by enhanced the proliferation and activation of splenocytes near the vaccination site. Note: This abstract was not presented at the conference. Citation Format: Ha Kim, Keejong Hong, Byung Cheol Ahn, Jung Sun Yum, Hyewon Youn. Visualization of immune response to Hepatitis B vaccination by in vivo mouse imaging. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B25.

Assessing antigen concentration of vaccine is essential step in determining the quality of the vaccine prior to vaccination. After vaccination, vaccine-induced antibody titer should also be measured to verify the vaccine efficacy. Since conventional assay used for vaccine concentrations and induced Ab-titers is antibody-based enzyme-linked immunosorbent assay, the assay inevitably brings drawbacks of antibody such as high cost for production, limited stability, and inconsistent quality between lot-to-lots. Aptamer is single-stranded nucleic acid having three-dimensional structure and has features overcoming limitations of antibody. This review will briefly introduce the features of aptamer and potential of aptamer-based system for evaluation of vaccine efficacy.

Since the first case of Ebola virus disease (EVD) in Guinea was reported in March 2014 by World Health Organization (WHO), the outbreak has continued through the year and the total number of 19,065 patients was reported as the confirmed or suspected in the EVD-affected countries. Among the cases, 7,388 patients were reported death by 19 December. Currently, available therapeutics to treat the infected patients or vaccines to prevent people from infection is not developed yet while viral diagnostic methods were already developed and firmly established in a lot of countries as a first step for the preparedness of Ebola outbreak. Some potential therapeutic materials including ZMapp were supplied and the treated people got over the EVD. Several candidates of vaccines also were investigated their efficacy in animal models by National Institute of Health (NIH) and Department of Defense, and they are processing of clinical tests in West Africa aiming to finish the development by the 2015. Vaccine and therapeutic development is essential to stop the EVD outbreak in West Africa, also to protect the world from the risk which can be generated by potential spread of Ebola virus.

Francisella (F.) tularensis causes the zoonotic disease tularemia and categorized as one of the highest-priority biological agents. The sensing approaches utilized by conventional detection methods, including enzyme-linked immunosorbent assay (ELISA), are not sensitive enough to identify an infectious dose of this high-risk pathogen due to its low infective dose. As an attempt to detect F. tularensis with high sensitivity, we utilized the highly sensitive immunoassay system named gold nanoparticle-based oligonucleotide-linked immunosorbent assay (GNP-OLISA) which uses antibody-gold nanoparticles conjugated with DNA strands as a signal generator and RNA oligonucleotides appended with a fluorophore as a quencher for signal amplification. We modified the GNP-OLISA for the detection F. tularensis to utilize one antibody for both the capture of the target and for signal generation instead of using two different antibodies, which are usually employed to construct the antibody sandwich in the ELISA. The GNP-OLISA showed 37-fold higher sensitivity compared with ELISA and generated very consistent detection results in the sera. In addition, the detection specificity was not affected by the presence of non-target bacteria, suggesting that GNP-OLISA can be used as a sensitive detection platform for monitoring high-risk pathogens thereby overcoming the limit of the conventional assay system.

Ling Zhi extract (LZE) is a herbal mushroom preparation which been used world wide for the prevention and treatment of various cancers. The current study was designed to evaluate these claims in human colon cancer cells in terms of cancer preventive mechanisms. Results have demonstrated induction of apoptosis, anti-inflammatory action and differential cytokine expression during induced inflammation in the human colonic carcinoma cell line, HT-29. LZE caused no cytotoxicity in HT-29 cells at doses less than 10,000 microg/ml. Increasing concentrations of LZE reduced prostaglandin E2 production, but increased nitric oxide production. LZE treatment induced apoptosis by increasing the activity of caspase-3. RT-PCR showed that LZE at a concentration of 5000 microg/ml decreased the expression of cyclooxygenase-2 mRNA. Among 42 cytokines tested by protein array in this study, supplementation of LZE at doses of 500 and 5000 microg/ml to HT-29 cells reduced the expression of interleukin-8, macrophage inflammatory protein 1-delta, vascular epithelial growth factor, and platelet-derived growth factor. These results suggest that LZE has pro-apoptotic and anti-inflammatory functions, as well as inhibitory effects on cytokine expression during early inflammation in colonic carcinoma cells, which may be of significance in the use of Chinese herbal alternative medicines for cancer prevention.

Tularemia is a high-risk infectious disease caused by Gram-negative bacterium Francisella tularensis. Due to its high fatality at very low colony-forming units (less than 10), F. tularensis is considered as a powerful potential bioterrorism agent. Vaccine could be the most efficient way to prevent the citizen from infection of F. tularensis when the bioterrorism happens, but officially approved vaccine with both efficacy and safety is not developed yet. Research for the development of tularemia vaccine has been focusing on the live attenuated vaccine strain (LVS) for long history, still there are no LVS confirmed for the safety which should be an essential factor for general vaccination program. Furthermore the LVS did not show protection efficacy against high-risk subspecies tularensis (type A) as high as the level against subspecies holarctica (type B) in human. Though the subunit or recombinant vaccine candidates have been considered for better safety, any results did not show better prevention efficacy than the LVS candidate against F. tularensis infection. Currently there are some more trials to develop vaccine using mutant strains or nonpathogenic F. novicida strain, but it did not reveal effective candidates overwhelming the LVS either. Difference in the protection efficacy of LVS against type A strain in human and the low level protection of many subunit or recombinant vaccine candidates lead the scientists to consider the live vaccine development using type A strain could be ultimate answer for the tularemia vaccine development.

Bacillus anthracis H9401 (NCCP 12889) is an isolate from a Korean patient with gastrointestinal anthrax. The whole genome of H9401 was sequenced. It is a circular chromosome containing 5,480 open reading frames (ORFs) and two plasmids, pXO1 containing 202 ORFs and pXO2 containing 110 ORFs. H9401 shows high pathogenicity and genome sequence similarity to Ames Ancestor.

A superoxide dismutase (SOD) gene from Burkholderia pseudomallei, the causative agent of melioidosis, was cloned and expressed in Escherichia coli, and its product was functionally and physically characterized. The gene has an open-reading frame of 579 bp. The deduced amino acid sequence has 192 residues with a calculated molecular mass of ∼22 kDa. Sequence comparison with other bacterial SODs showed that the protein contains typical metal-binding motifs and other Fe-SOD-conserved residues. The sequence has substantial similarity with other bacterial Fe-SOD sequences. The enzymatic activity of the expressed protein was inhibited by hydrogen peroxide but not by sodium azide or potassium cyanide, attributes that indeed are characteristic of typical bacterial Fe-SODs. Western blotting with antiserum against the recombinant Fe-SOD revealed that it is expressed in B. pseudomallei. Transformed E. coli that expressed the Fe-SOD had significantly increased SOD activity and was highly tolerant to paraquat-mediated replication inhibition, compared to transformed cells carrying an empty vector. Our results provide a basis for further biochemical characterization of the enzyme and elucidation of its role in the pathogenesis of B. pseudomallei.

Background The poly-γ- d -glutamic acid (PGA) capsule, a major virulence factor of Bacillus anthracis , protects bacilli from immune surveillance and allows its unimpeded growth in the host. Recently, the importance of the PGA in the pathogenesis of anthrax infection has been reported. The PGA capsule is associated with lethal toxin (LT) in the blood of experimentally infected animals and enhances the cytotoxicity of LT. Methods To investigate the role of anti-PGA Abs on progression of anthrax infection, two mouse anti-PGA mAbs with K d values of 0.8 μM and 2.6 μM respectively were produced and in silico three dimensional (3D) models of mAbs with their cognitive PGA antigen complex were analyzed. Results Anti-PGA mAbs specifically bound encapsulated B. anthracis H9401 and showed opsonophagocytosis activity against the bacteria with complement. The enhancement effect of PGA on LT-mediated cytotoxicity was confirmed ex vivo using mouse bone marrow-derived macrophages and was effectively inhibited by anti-PGA mAb. Passive immunization of mAb completely protected mice from PGA-enhanced LT toxicity and partially rescued mice from anthrax spore challenges. 3D structure models of these mAbs and PGA complex support specific interactions between CDR and cognitive PGA. These results indicate that mouse mAb against PGA capsule prevents the progress of anthrax disease not only by eliminating the vegetative form of encapsulated B. anthracis but also by inhibiting the enhanced cytotoxic activity of LT by PGA through specific binding with PGA capsule antigen. General significance Our results suggest a potential role for PGA antibodies in preventing and treating anthrax infection.

Background: The ectodomain of matrix protein 2 (M2e) of influenza A virus is a rationale target antigen candidate for the development of a universal vaccine against influenza as M2e undergoes little sequence variation amongst human influenza A strains. Vaccine-induced M2e-specific antibodies (Abs) have been shown to display significant cross-protective activity in animal models. M2e-based vaccine constructs have been shown to be more protective when administered by the intranasal (i.n.) route than after parenteral injection. However, i.n. administration of vaccines poses rare but serious safety issues associated with retrograde passage of inhaled antigens and adjuvants through the olfactory epithelium. In this study, we examined whether the sublingual (s.l.) route could serve as a safe and effective alternative mucosal delivery route for administering a prototype M2e-based vaccine. The mechanism whereby s.l. immunization with M2e vaccine candidate induces broad protection against infection with different influenza virus subtypes was explored. Methods and Results: A recombinant M2 protein with three tandem copies of the M2e (3M2eC) was expressed in Escherichia coli. Parenteral immunizations of mice with 3M2eC induced high levels of M2e-specific serum Abs but failed to provide complete protection against lethal challenge with influenza virus. In contrast, s.l. immunization with 3M2eC was superior for inducing protection in mice. In the latter animals, protection was associated with specific Ab responses in the lungs. Conclusions: The results demonstrate that s.l. immunization with 3M2eC vaccine induced airway mucosal immune responses along with broad cross-protective immunity to influenza. These findings may contribute to the understanding of the M2-based vaccine approach to control epidemic and pandemic influenza infections. Citation: Shim B-S, Choi YK, Yun C-H, Lee E-G, Jeon YS, et al. (2011) Sublingual Immunization with M2-Based Vaccine Induces Broad Protective Immunity against

Background In Korea, approximately 70% of HIV-positive individuals are currently diagnosed in hospitals, while most HIV-positive patients were diagnosed at public health centers in 1980 s and 1990 s. However, there are no reporting systems to identify how many HIV tests are performed in the Korean hospitals different from public health centers and Blood centers. We estimated how many HIV tests were performed in hospitals and analyzed the nationwide hospital-based HIV seroprevalence in the present study. Methods Between 2002 and 2008, data included HIV tests on insurance claims in hospitals and the proportion of computerized insurance claims from the Health Insurance Review and Assessment Services. The number of HIV tests from the survey in the External Quality Assurance Scheme for hospital laboratories was collected to calculate the insurance claim proportion. HIV seroprevalence was estimated using data of tested individuals, including infected individuals. Statistical analysis was confirmed with the 95% confidence interval. Statistical significance was defined at p-values < 0.05. Results The number of HIV tests in hospitals increased from 2.7 million in 2002 to 5.0 million in 2008. The trend of HIV seroprevalence was decrease (1.5-1.3 per 10,000 individuals, P < 0.0028), except in 2002. The number of women tested was greater than men, and the proportion increased in older individuals and in small towns. Men had a higher annual HIV seroprevalence than women (P < 0.0001). The annual seroprevalence decreased in men (P = 0.0037), but was stable in women. The seroprevalence in the 30-39 year age group demonstrated higher than other age groups except 2008. Conclusions The nationwide hospital-based number of HIV tests and seroprevalence were estimated using a new method and seroprevalence trends were identified. This information will facilitate improvement in national HIV prevention strategies.

Background The number of Koreans diagnosed with human immunodeficiency virus (HIV) infections is increasing annually; however, CD4+ T-cell counts at diagnosis have decreased. The purpose of the present study was to identify clinical and epidemiologic associations with low CD4+ T-cell counts at the time of HIV diagnosis in a Korean population. Methods Data from 2,299 HIV-infected individuals with initial CD4+ T-cell counts measured within 6 months of HIV diagnosis and reason for HIV testing were recorded and measured from 2000 to 2007. Data were selected from the database of the Korea Centers for Disease Control and Prevention. Late diagnosis was defined by CD4+ T-cell counts 3. Reasons for HIV testing were analyzed using logistic regression including epidemiologic variables. Results A total of 858 individuals (37.3%) were included in the late diagnosis group. Individuals with a late diagnosis were older, exposed through heterosexual contact, and demonstrated clinical manifestations of acquired immunodeficiency syndrome (AIDS). The primary reason for HIV testing was a routine health check-up (41%) followed by clinical manifestations (31%) of AIDS. The proportion of individuals with a late diagnosis was higher in individuals tested due to clinical symptoms in public health centers (adjusted odds ratio [AOR], 17.3; 95% CI, 1.7-175) and hospitals (AOR, 4.9; 95% CI, 3.4-7.2) compared to general health check-up. Late diagnosis annually increased in individuals diagnosed by voluntary testing both in public health centers (PHCs, P = 0.017) and in hospitals (P = 0.063). Routine testing due to risky behaviors resulted in earlier detection than testing secondary to health check-ups, although this difference was not statistically significant (AOR, 0.7; P = 0.187). Individuals identified as part of hospital health check-ups more frequently had a late diagnosis (P = 0.001) Conclusions HIV infection was primarily detected by voluntary testing with identification in PHCs and by testing due to clinical symptoms in hospitals. However, early detection was not influenced by either voluntary testing or general health check-up. It is important to encourage voluntary testing for early detection to decrease the prevalence of HIV infection and AIDS progression.

Background HIV-1 specific cytotoxic T lymphocytes (CTLs) have an important role as antiviral effector cells for controlling HIV-1 infection. Methods To investigate CTL response during the early stage of HIV infection, we measured immunity-related factors including CD4+ T cell counts, CD8+ T cell counts, HIV-1 RNA viral loads and IFN-γ secretion according to CTL response in 78 selected primary HIV-1-infected Koreans. Results The CTL response was strongly induced by HIV-1 specific Gag and Nef peptides (p = 0.016) compared with induction by Tat or Env peptides. These results suggest that the major antiviral factors inducing strong HIV-specific CTL responses are associated with the Gag and Nef viral regions in primary HIV-1 infected Koreans. The relationship between viral load and CTL response showed varying correlations with time following HIV infection. CTL response was inversely correlated with viral loads at preseroconversion stage I (r = -0.224 to -0.33) and changed to a positive correlation at the preseroconversion stage II (r = 0.132 to 0.854). Finally, it changed to an inverse correlation again after seroconversion until a viral set point was established on serological profiling (r = -0.195 to -0.407). Conclusions These findings demonstrate a dynamic correlation between viral load and subsequent CTL responses during early HIV infection.

Histone deacetylase plays an important role in HIV latency. Novel histone deacetylase inhibitors, CG05 and CG06, were evaluated for their roles in HIV latency using ACH2 cells. Both inhibitors were highly efficient in reactivation of provirus and exerted lesser toxicity compared with other known histone deacetylase inhibitors. Histone acetylation increased when proviruses were reactivated by the compounds. These new inhibitors may contribute to the reduction of the HIV reservoir when used in conjunction with highly active antiretroviral therapy.

This study aimed to elucidate the mechanisms that may lead to an efficient strategy to induce a suitable host response of the vaginal mucosa upon exposure to intravaginally delivered exogenous compounds. It was hypothesized that the upregulation of intercellular adhesion molecule (ICAM)-1 gene expression may reflect the inflammatory response evoked by exogenous compounds. Major emphasis was placed on ethylenediamine tetraacetic acid (EDTA) which was added as a synergistic agent to conventional spermicidal agents or anti-HIV drugs. The levels of ICAM-1 mRNA were examined as a surrogate marker for inflammatory response in human vaginal epithelial cells upon exposure to EDTA or interleukin (IL)-1beta (i.e. positive control, 25 mM). The effects of estrogen on EDTA-induced ICAM-1 expression were also evaluated for the estrogen involvement in the inflammatory process of the vaginal mucosa. ICAM-1 expression in human vaginal cells (VK2/E6E7 cells) increased as EDTA concentration added to human vaginal cell lines increased. The effects of estrogen on EDTA-induced ICAM-1 expression in human vaginal epithelial cells were estrogen-concentration dependent; estrogen at lower concentrations (approximately 1-10 nM) did not affect ICAM-1 expression, whereas estrogen at higher concentrations (approximately 100 nM-1 microM) attenuated ICAM-1 expression. The influence of estrogen in ICAM-1 expression suggests the beneficial effects of estrogen on the regulation of vaginal homeostasis. Identification and quantification of specific surrogate markers for the inflammatory response evoked by exogenous compounds and their regulation by estrogen will lead to an efficient strategy against sexually transmitted diseases including AIDS.

The facultative intracellular bacteriumFrancisella tularensisis capable of causing systemic infections in various hosts, including mice and humans. The liver is a major secondary site ofF. tularensisinfection, but hepatic immune responses to the pathogen remain poorly defined. Immune protection against the pathogen is thought to depend on the cytokine gamma interferon (IFN-γ), but the cellular basis for this response has not been characterized. Here we report that natural killer cells from the livers of naïve uninfected mice produced IFN-γ when challenged with live bacteria in vitro and that the responses were greatly increased by coactivation of the cells with either recombinant interleukin-12 (IL-12) or IL-18. Moreover, the two cytokines had strong synergistic effects on IFN-γ induction. Neutralizing antibodies to either IL-12 or IL-18 inhibited IFN-γ production in vitro, and mice deficient in the p35 subunit of IL-12 failed to show IFN-γ responses to bacterial challenge either in vitro or in vivo. Clinical isolates of highly virulent type AFrancisella tularensissubsp.tularensisorganisms were comparable to the live attenuated vaccine strain ofFrancisella tularensissubsp.holarcticain their ability to induce IL-12 and IFN-γ expression. These findings demonstrate that cells capable of mounting IFN-γ responses toF. tularensisare resident within the livers of uninfected mice and depend on coactivation by IL-12 and IL-18 for optimum responses.

This study evaluated the effect of fermentation on the nutritional quality of food-grade soybeans and feed-grade soybean meals. Soybeans and soybean meals were fermented by Aspergillus oryzae GB-107 in a bed-packed solid fermentor for 48 hours. After fermentation, their nutrient contents as well as trypsin inhibitor were measured and compared with those of raw soybeans and soybean meals. Proteins were extracted from fermented and non-fermented soybeans and soybean meals, and the peptide characteristics were evaluated after electrophoresis. Fermented soybeans and fermented soybean meals contained 10% more (P.05) crude protein than raw soybeans and soybean meals. The essential amino acid profile was unchanged after fermentation. Fermentation eliminated (P.05) most of the trypsin inhibitor from both soybeans and soybean meals. Fermentation increased the amount of small-size peptides (20 kDa) (P.05) compared with raw soybeans, while significantly decreasing large-size peptides (60 kDa) (P.05). Fermented soybean meal contained more (P.01) small-size peptides (20 kDa) than soybean meal. Fermented soybean meal did not contain large-size peptides (60 kDa), whereas 22.1% of peptides in soybean meal were large-size (60 kDa). Collectively, fermentation increased protein content, eliminated trypsin inhibitors, and reduced peptide size in soybeans and soybean meals. These effects of fermentation might make soy foods more useful in human diets as a functional food and benefit livestock as a novel feed ingredient.

The herbal remedy Zingiber officinale (ginger root) has been used for perhaps thousands of years in the Far East to treat inflammatory diseases, including osteoarthritis. However, the anti-arthritic effect of ginger root has never been evaluated on osteoarthrotic cartilage of sow. The objective of this study was to investigate the effects of ginger root extract (GRE) on the viability and the production of nitric oxide (NO) and prostaglandin E(2) (PGE(2)) by sow osteoarthrotic cartilage explants. The cartilage explants (~20 mg/96-well plate) were grown in Ham's F-12/Dulbecco's Modified Eagle's Medium supplemented with 10% fetal bovine serum and antibiotics for 72 hours and depleted for 24 hours. GRE was then added at different concentrations (0-2,000 microg/mL), and the explants were allowed to grow for 24 hours. The cell viability was reduced (P.05) with GRE/=500 microg/mL, whereas it was not affected with GRE100 microg/mL. In a follow-up experiment, the supernatants of cartilage explants with GRE (0-500 microg/mL) in the presence of interleukin-1beta (2 ng/mL), tumor necrosis factor-alpha (1 ng/mL), and lipopolysaccharides (10 microg/mL) were used to measure NO and PGE production. Increasing GRE concentration (1-100 microg/mL) reduced (P.05) NO production by cartilage tissue explants, and a similar pattern was observed in the production of PGE(2). The inhibitory effects of GRE on NO and PGE(2) production by sow osteoarthrotic cartilage explants observed in this study suggest an important role for GRE as an anti-arthritic agent in osteoarthrosis in the sow.

Purpose In vaccine efficacy evaluation, visualization of pathogens in whole organism at each time point would be able to reduce the consuming animals and provide the in vivo information within consistent background with identical organism. Materials and methods Using IVIS spectrum whole live-animal imaging system, fluorescent intensity was optimized and visualized proportionately by concentrating Escherichia coli MC1061 strain which expresses GFP (E. coli-GFP) in BALB/C mice after injection. Results Local distribution of disseminated E. coli-GFP was traced in each organ by fluorescence. Detached organ showed more obvious fluorescent signal, and intestine showed strongest fluorescent signal. Conclusion This in vivo imaging method using GFP-tagged pathogen strain suggest quantified infected pathogens by fluorescence intensity in whole animals can provide the information about the localization and distribution after infection.