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Emerging outbreaks of newly found, highly pathogenic avian influenza (HPAI) A(H5N8) viruses have been reported globally. Previous studies have indicated that H5N8 pathogenicity in mice is relatively moderate compared with H5N1 pathogenicity. However, detailed mechanisms underlying avian influenza pathogenicity are still undetermined. We used a high-throughput RNA-seq method to analyse host and pathogen transcriptomes in the lungs of mice infected with A/MD/Korea/W452/2014 (H5N8) and A/EM/Korea/W149/2006 (H5N1) viruses. Sequenced numbers of viral transcripts and expression levels of host immune-related genes at 1 day post infection (dpi) were higher in H5N8-infected than H5N1-infected mice. Dual sequencing of viral transcripts revealed that in contrast to the observations at 1 dpi, higher number of H5N1 genes than H5N8 genes was sequenced at 3 and 7 dpi, which is consistent with higher viral titres and virulence observed in infected lungs in vivo. Ingenuity pathway analysis revealed a more significant upregulation of death receptor signalling, driven by H5N1 than with H5N8 infection at 3 and 7 dpi. Early induction of immune response-related genes may elicit protection in H5N8-infected mice, which correlates with moderate pathogenicity in vivo. Collectively, our data provide new insight into the underlying mechanisms of the differential pathogenicity of avian influenza viruses.

A new PSM using high transmittance is developed to overcome patterning process limits in ArF immersion lithography. We optimized mask structure, materials, and film thicknesses for patterning process. A new material for phase-shifter is applied to the HT-PSM to exhibit higher transmittance in ArF wavelengths and the thickness of the new material is thinner than that of the conventional 6% phase-shifter (MoSiON). A new blank structure using a MoSi shading layer with double Cr hardmasks (HM) is developed and suggested for the HTPSM process. Double HM blank stacks enable the HT-PSM to adopt thin PR process for resolution enhancement in mask process. The first Cr on the MoSi is utilized as a HM to etch MoSi shading layer, an adhesion layer for PR process, and also a capping layer to protect blind area during MoSi and phase-shifter etching. In contrast, the role of the second Cr between MoSi and phase-shifter is an etch stopper for MoSi and a HM to etch phase-shifter at the same time. However, Double HM process has some problems, such as first Cr removal during second Cr etching and complex process steps. To solve the Cr removal issues, we evaluated various Cr layers which have different etchrates and compositions. According to the evaluations, we optimized thicknesses and compositions of the two Cr layers and corresponding etching conditions. Lithography simulations demonstrate that the new HT-PSM has advantages in NILS in aerial images. As a result, initial wafer exposure experiments using the HT-PSM show 13-32% improvements in LCDU compared to that of the conventional 6% PSM due to its higher NILS.

BackgroundThis study aimed to investigate new bone formation using recombinant human bone morphogenetic protein 2 (rhBMP-2) and locally applied bisphosphonate in rat calvarial defects.MethodsThirty-six rats were studied. Two circular 5 mm diameter bony defect were formed in the calvaria using a trephine bur. The bony defect were grafted with Bio-Oss® only (group 1, n = 9), Bio-Oss® wetted with rhBMP-2 (group 2, n = 9), Bio-Oss® wetted with rhBMP-2 and 1 mM alendronate (group 3, n = 9) and Bio-Oss® wetted with rhBMP-2 and 10 mM alendronate (group 4, n = 9). In each group, three animals were euthanized at 2, 4 and 8 weeks after surgery, respectively. The specimens were then analyzed by histology, histomorphometry and immunohistochemistry analysis.ResultsThere were significant decrease of bone formation area (p ConclusionLow concentration bisphosphonate and rhBMP-2 have synergic effect on bone regeneration and this is result from the decreased activity of RANKL of osteoblast.

Recombinant virus-like nanoparticles (VLPs) are a promising nanoparticle platform to develop safe vaccines for many viruses. Herein, we describe a novel and rapid protein transfer process to enhance the potency of enveloped VLPs by decorating influenza VLPs with exogenously added glycosylphosphatidylinositol-anchored immunostimulatory molecules (GPI-ISMs). With protein transfer, the level of GPI-ISM incorporation onto VLPs is controllable by varying incubation time and concentration of GPI-ISMs added. ISM incorporation was dependent upon the presence of a GPI-anchor and incorporated proteins were stable and functional for at least 4 weeks when stored at 4 °C. Vaccinating mice with GPI-granulocyte macrophage colony-stimulating factor (GM-CSF)-incorporated-VLPs induced stronger antibody responses and better protection against a heterologous influenza virus challenge than unmodified VLPs. Thus, VLPs can be enriched with ISMs by protein transfer to increase the potency and breadth of the immune response, which has implications in developing effective nanoparticle-based vaccines against a broad spectrum of enveloped viruses. From the Clinical Editor The inherent problem with current influenza vaccines is that they do not generate effective cross-protection against heterologous viral strains. In this article, the authors described the development of virus-like nanoparticles (VLPs) as influenza vaccines with enhanced efficacy for cross-protection, due to an easy protein transfer modification process.

Background This study are to identify the symptomatic changes and condylar stability after 2 jaw surgery without preceding treatments for Temporomandibular joints(TMJ) in class III patients with the TMJ symptoms; and to assess therapeutic effect of 2 jaw surgery and the necessity of preceding treatment for alleviation of TMJ symptoms. Methods 30 prognathic patients with preexisting TMJ symptoms were divided into 2 groups according to presence or absence of preceding treatments before the surgery. We evaluated symptomatic changes on both TMJ by questionnaires and clinical examinations. And we reconstructed 3D cone beam computed tomography images before 2 jaw surgery, immediately after the surgery, and 6 months or more after the surgery with SimPlant software, and analyzed the stability of condylar position on 3D reconstruction model. Significances were assessed by the Wilcoxon signed rank test on SPSS ver. 20.0. Results Both groups had favorable changes of TMJ symptoms after orthognathic surgery. And postoperative position of condyle had good stability during follow-up period. Conclusion 2 jaw surgery without preceding treatments for TMD can have therapeutic effect for TMD patients with class III malocclusion.

Hemifacial microsomia (HFM) is the most common craniofacial anomaly after cleft lip and cleft palate; this deformity primarily involves the facial skeleton and ear, with either underdevelopment or absence of both components. In patients with HFM, the management of the asymmetries requires a series of treatment phases that focus on their interception and correction, such as distraction osteogenesis or functional appliance treatment during growth and presurgical orthodontic treatment followed by mandibular and maxillary surgery. Satisfactory results were obtained in a 9-year-old girl with HFM who was treated with distraction osteogenesis. At the age of 19, genioplasty and mandible body augmentation with a porous polyethylene implant (PPE, Medpor®, Porex) was sequentially performed for the functional and esthetic reconstruction of the face. We report a case of HFM with a review of the literature.

Enveloped virus vaccines can be damaged by high osmotic strength solutions, such as those used to protect the vaccine antigen during drying, which contain high concentrations of sugars. We therefore studied shrinkage and activity loss of whole inactivated influenza virus in hyperosmotic solutions and used those findings to improve vaccine coating of microneedle patches for influenza vaccination. Using stopped-flow light scattering analysis, we found that the virus underwent an initial shrinkage on the order of 10% by volume within 5 s upon exposure to a hyperosmotic stress difference of 217 milliosmolarity. During this shrinkage, the virus envelope had very low osmotic water permeability (1 – 6×10−4 cm s–1) and high Arrhenius activation energy (E a = 15.0 kcal mol–1), indicating that the water molecules diffused through the viral lipid membranes. After a quasi-stable state of approximately 20 s to 2 min, depending on the species and hypertonic osmotic strength difference of disaccharides, there was a second phase of viral shrinkage. At the highest osmotic strengths, this led to an undulating light scattering profile that appeared to be related to perturbation of the viral envelope resulting in loss of virus activity, as determined by in vitro hemagglutination measurements and in vivo immunogenicity studies in mice. Addition of carboxymethyl cellulose effectively prevented vaccine activity loss in vitro and in vivo, believed to be due to increasing the viscosity of concentrated sugar solution and thereby reducing osmotic stress during coating of microneedles. These results suggest that hyperosmotic solutions can cause biphasic shrinkage of whole inactivated influenza virus which can damage vaccine activity at high osmotic strength and that addition of a viscosity enhancer to the vaccine coating solution can prevent osmotically driven damage and thereby enable preparation of stable microneedle coating formulations for vaccination.

The enhancement of deuterium retention was investigated for tungsten in the presence of both 2.8 MeV self-ion induced cascade damage and fuel hydrogen isotope plasma. Vacancy clustering in cascade damaged polycrystalline tungsten occurred due to deuterium irradiation and was observed near the grain boundary by using all-step transmission electron microscopy analysis. Analysis of the highest desorption temperature peak using thermal desorption spectroscopy supports reasonable evidence of defect clustering in the damaged polycrystalline tungsten. The defect clustering was neither observed on the damaged polycrystalline tungsten without deuterium irradiation nor on the damaged single-crystalline tungsten with deuterium irradiation. This result implies the synergetic role of deuterium and grain boundary on defect clustering. This study proposes a path for the defect transform from point defect to defect cluster, by the agglomeration between irradiated deuterium and cascade damage-induced defect. This agglomeration may induce more severe damage on the tungsten divertor at which the high fuel hydrogen ions, fast neutrons, and self-ions are irradiated simultaneously and it would increase the in-vessel tritium inventory.

In order to realize high-performance thermoelectric materials, a way to obtain small grain size is necessary for intensification of the phonon scattering. Here, we use a melt-spinning-spark plasma sintering process for making p-type Bi0.36Sb1.64Te3 thermoelectric materials and evaluate the relation between the process conditions and thermoelectric performance. We vary the Cu wheel rotation speed from 1000 rpm (-13 ms(-1)) to 4000 rpm (-52 ms(-1)) during the melt spinning process to change the cooling rate, allowing us to control the characteristic size of nanostructure in melt-spun Bi0.36Sb1.64Te3 ribbons. The higher wheel rotation speed decreases the size of nanostructure, but the grain sizes of sintered pellets are inversely proportional to the nanostructure size after the same sintering condition. As a result, the ZT values of the bulks fabricated from 1000-3000 rpm melt-spun ribbons are comparable each other, while the ZT value of the bulk from the 4000 rpm melt-spun ribbons is rather lower due to reduction of grain boundary phonon scattering. In this work, we can conclude that the smaller nanostructure in the melt spinning process does not always guarantee high-performance thermoelectric bulks, and an adequate following sintering process must be included.

Recent studies have demonstrated that the two-ion-stream-instability occurs near the plasma boundary and makes the ions reach the 'modified Bohm velocity' at the sheath edge. In most low-temperature plasmas, however, the ion-neutral collisions can disturb the growth of instability to occur frequently, and the ions exit the plasma boundary with their own Bohm velocities. We report some experimental observations regarding this issue. The spatial variations of the ion drift velocities and the space potential near the sheath edge were measured in Ar/Xe mixture plasmas by increasing the total pressure in the range of 0.5–2.1 mTorr. The results show that the instability cannot occur above a certain pressure condition and that is consistent with the theoretically driven pressure criteria for the onset of the instability.

Purpose To evaluate maxillary stability after Le Fort I osteotomy using posterosuperior movement after pterygoid process fracture or removal and mandibular setback surgery for skeletal Class III deformities. Materials and Methods A retrospective cohort study was performed of changes in 28 patients with skeletal Class III deformity using 3-dimensional (3D) measurement point coordinates obtained by cone-beam computed tomographic superimposition. The predictor variable was management of the pterygoid process (fracture vs removal). The outcome variables were changes in measurement points based on 3D coordinates obtained preoperatively, immediately postoperatively, and 6 months after surgery. Linear mixed-effects models were applied to evaluate postoperative stability. Results The 3D cephalometric outcome variables for 14 patients who had undergone pterygoid process fracture were compared with those for 14 patients who had undergone pterygoid process removal. The postoperative nasopalatine canal points moved superiorly 0.11 mm and inferiorly 0.06 mm in the fracture and removal groups, respectively. However, most of the postoperative relapse was within 1 mm. There were no differences in postoperative skeletal changes shown by the fracture and removal groups (P Conclusions The results suggest that surgeons can achieve good skeletal stability in posterosuperior movement from Le Fort I osteotomy, regardless of whether the pterygoid process is fractured or removed.

We have investigated the volumes of the nasal cavities of 35 patients treated with superior repositioning Le Fort I osteotomy by analysing cone-beam computed tomography (CT) data with a 3-dimensional reconstruction program to correlate changes in the volume of the nasal cavity that were associated with the maxillary superior repositioning and the role of outfracture of the inferior turbinates. The patients were treated at the Pusan National Dental Hospital during the 14-month period January 2011-March 2012.. The patients were divided into two groups, the first of which consisted of 20 patients who had superior repositioning of 4mm or more with a mean superior movement of 5.2mm and outfracture of the inferior turbinates (outfracture group). The second group consisted of 15 patients who also had more than 4mm impaction with a mean superior movement of 5.0mm and for whom outfracture was not done (no outfracture group). Nasal symptoms were investigated preoperatively and 6 months postoperatively using the Nasal Obstruction Symptom Evaluation (NOSE) scale, and 3-dimensional volumetric analysis was made using cone-beam CT data to assess changes in nasal volume. There were significant differences between the groups in volumetric changes (V1-V2) (p=0.001) but no significant differences between the sexes. The volume of the nasal cavity in the outfracture group decreased by 20% after superior repositioning, but that in the no outfracture group decreased by 33%. Among the 20 patients in the outfracture group not one complained of nasal symptoms postoperatively. We conclude that outfracture of the inferior turbinates should be considered when the amount of superior movement of maxilla is more than 4mm.

Major histocompatibility complex class II-deficient (MHC-II KO; Aβ −/− ) mice were used to assess the roles of MHC-II molecules in inducing protective immune responses to vaccination. After vaccination with influenza A/PR8 virus-like particle (VLP) vaccine, in vivo and in vitro vaccine antigen-specific IgG isotype antibodies were not detected in MHC-II KO mice, which is quite different from CD4 T cell-deficient mice that induced vaccine-specific IgG antibodies. The deficiency in MHC-II did not significantly affect the induction of antigen-specific IgM antibody in sera. MHC-II KO mice that were vaccinated with influenza VLP, whole inactivated influenza virus, or live attenuated influenza virus vaccines were not protected against lethal infection with influenza A/PR8 virus. Adoptive transfer of fractionated spleen cells from wild-type mice to MHC-II KO mice indicated that CD43 + cell populations with MHC-II contributed more significantly to producing vaccine-specific IgG antibodies than CD43 − B220 + conventional B cell or CD4 T cell populations, as well as conferring protection against lethal infection. Bone marrow-derived dendritic cells from MHC-II KO mice showed a significant defect in producing interleukin-6 and tumor necrosis factor alpha cytokines. Thus, results indicate that MHC-II molecules play multiple roles in inducing protective immunity to influenza vaccination. IMPORTANCE Major histocompatibility complex class II (MHC-II) has been known to activate CD4 T helper immune cells. A deficiency in MHC-II was considered to be equivalent to the lack of CD4 T cells in developing host immune responses to pathogens. However, the roles of MHC-II in inducing protective immune responses to vaccination have not been well understood. In the present study, we demonstrate that MHC-II-deficient mice showed much more significant defects in inducing protective antibody responses to influenza vaccination than CD4 T cell-deficient mice. Further analysis showed that CD43 marker-positive immune cells with MHC-II, as well as an innate immunity-simulating adjuvant, could rescue some defects in inducing protective immune responses in MHC-II-deficient mice. These results have important implications for our understanding of host immunity-inducing mechanisms to vaccination, as well as in developing effective vaccines and adjuvants.

Respiratory syncytial virus (RSV) is an important human pathogen. Expression of virus structural proteins produces self-assembled virus-like nanoparticles (VLP). We investigated immune phenotypes after RSV challenge of immunized mice with VLP containing RSV F and G glycoproteins mixed with F-DNA (FdFG VLP). In contrast to formalin-inactivated RSV (FI-RSV) causing vaccination-associated eosinophilia, FdFG VLP immunization induced low bronchoalveolar cellularity, higher ratios of CD11c(+) versus CD11b(+) phenotypic cells and CD8(+) T versus CD4(+) T cells secreting interferon (IFN)-γ, T helper type-1 immune responses, and no sign of eosinophilia upon RSV challenge. Furthermore, RSV neutralizing activity, lung viral clearance, and histology results suggest that FdFG VLP can be comparable to live RSV in conferring protection against RSV and in preventing RSV disease. This study provides evidence that a combination of recombinant RSV VLP and plasmid DNA may have a potential anti-RSV prophylactic vaccine inducing balanced innate and adaptive immune responses.

Maternal immunization of mice with formalin inactivated respiratory syncytial virus (FI-RSV) resulted in the passive transfer of RSV antibodies but not cellular components to the offspring. The offspring born to FI-RSV immunized mothers showed serum RSV neutralizing activity, effectively controlled lung viral loads without vaccine-enhanced disease, did not induce pulmonary eosinophilia, and cytokine producing cells after live RSV infection. Therefore, this study provides evidence that maternal immunization provides an in vivo model in investigating the roles of antibodies independent of cellular components.

In the present study, virus-like particles (VLPs) were evaluated as a candidate veterinary vaccine against canine influenza virus (CIV) subtype H3N2. Specific pathogen-free (SPF) beagle dogs received a single injection of a VLP vaccine containing hemagglutinin (HA) and M1 protein of CIV H3N2 (H3 HA VLP). The vaccine was tested at 3 different doses with an adjuvant and 1 dose without an adjuvant. To evaluate the immunogenicity and protective efficacy of the H3 HA VLP vaccine, we performed hemagglutination inhibition tests to determine serological immune responses and conducted challenge studies using SPF beagle dogs. The addition of Montanide ISA 25 adjuvant significantly increased the immunogenicity of the H3 HA VLP vaccine. The experimental infection study showed that a single dose of H3 HA VLP vaccine induced protection against wild-type virus challenge in dogs. These results provide support for continued development of the VLP as an animal vaccine against influenza virus.

Influenza infection is the principal cause of acute respiratory illnesses resulting in epidemic or pandemic worldwide. It also has major social and economic consequences for school and work, as well as significant treatment and hospitalization costs, hence proper management of influenza transmission is a global concern. Catechins are the major flavonoids and are reported to possess anti-oxidant, anti-cancer and anti-microbial activities. Recent experimental studies have revealed that green tea extracts including catechins prevent influenza infection, in vitro and in clinical settings. Along with the proven virucidal effect against influenza viruses, the beneficial effects of green tea components in reducing clinical symptoms of influenza infection, as demonstrated by gargling or ingesting supplement tablets, merit their use in influenza management in times of pandemic or in the annual influenza season. In this chapter, we discuss the biological anti-influenza viral activities of green tea catechins in vitro and in vivo .

The extracellular domain of M2 (M2e), a small ion channel membrane protein, is well conserved among different human influenza A virus strains. To improve the protective efficacy of M2e vaccines, we genetically engineered a tandem repeat of M2e epitope sequences (M2e5x) of human, swine, and avian origin influenza A viruses, which was expressed in a membrane-anchored form and incorporated in virus-like particles (VLPs). The M2e5x protein with the transmembrane domain of hemagglutinin (HA) was effectively incorporated into VLPs at a several 100-fold higher level than that on influenza virions. Intramuscular immunization with M2e5x VLP vaccines was highly effective in inducing M2e-specific antibodies reactive to different influenza viruses, mucosal and systemic immune responses, and cross-protection regardless of influenza virus subtypes in the absence of adjuvant. Importantly, immune sera were found to be sufficient for conferring protection in naive mice, which was long-lived and cross-protective. Thus, molecular designing and presenting M2e immunogens on VLPs provide a promising platform for developing universal influenza vaccines without using adjuvants.

Ginseng has been used in humans for thousands of years and is known to have multiple biological and immunomodulatory effects. In this study, we investigated whether Korean red ginseng extract would have preventive and antiviral effects on influenza virus infection. Oral administration to mice of red ginseng extract prior to infection significantly increased survival after infection with the 2009 pandemic H1N1 virus. Daily oral treatment of vaccinated mice with red ginseng extract provided enhanced cross-protection against antigenically distinct H1N1 and H3N2 influenza viruses. Naive mice that were infected with virus mixed with red ginseng extract showed significantly enhanced protection, lower levels of lung viral titers and interleukin-6, but higher levels of interferon-γ compared with control mice having virus infections without red ginseng extract, indicating an antiviral effect of ginseng. In addition, ginseng extract exhibited inhibitory effects on the growth of influenza virus in vitro. This study provides evidence that intake of ginseng extract will have beneficial effects on preventing lethal infection with newly emerging influenza viruses.

Purpose: Neuraminidase (NA) of influenza virus contains stalk region that shows a great deal of variability in both amino acid sequence and length. In this paper, we investigated generation of recombinant influenza viruses that had hepatitis B virus (HBV) B cell epitopes in the NA stalk region as a dual vaccine candidate. Materials and Methods: We used the WSH-HK reassortant helper virus for rescue of recombinant influenza virus containing HBV epitopes and reverse genetic protocol based on the use of micrococcal nuclease-treated virus cores for reconstitution of ribonucleoproteins. Results: We successfully generated a chimeric influenza viruses which contained 22 amino acid peptides in the stalk region derived from the surface and pre-surface protein HBV. The growth kinetics of the recombinant viruses was investigated after infection of Madin-Darby canine kidney (MDCK) and Madin-Darby bovine kidney (MDBK) cells and the rIV-BVPreS virus showed higher titer than other viruses in MDCK cells. We also confirmed the presence of HBV epitopes in the chimeric viruses by enzyme-linked immunosorbent assay (ELISA) using antiHBV polyclonal antibody. When the ratio of recombinant virus verse wild type virus was calculated by ELISA, recombinant viruses exhibited 2 fold higher values than the wild type virus. Conclusion: These results suggest that chimeric influenza virus which contained foreign anti gens can be used as dual vaccine against both HBV and influenza viruses.

In recent years with the threat of pandemic influenza and other public health needs, alternative vaccination methods other than intramuscular immunization have received great attention. The skin and mucosal surfaces are attractive sites probably because of both noninvasive access to the vaccine delivery and unique immunological responses. Intradermal vaccines using a microinjection system (BD Soluvia(TM)) and intranasal vaccines (FluMist®) are licensed. As a new vaccination method, solid microneedles have been developed using a simple device that may be suitable for self-administration. Because coated microneedle influenza vaccines are administered in the solid state, developing formulations maintaining the stability of influenza vaccines is an important issue to be considered. Marketable microneedle devices and clinical trials remain to be developed. Other alternative mucosal routes such as oral and intranasal delivery systems are also attractive for inducing cross-protective mucosal immunity, but effective non-live mucosal vaccines remain to be developed.

Ginseng polysaccharide has been known to have multiple immunomodulatory effects. In this study, we investigated whether Panax ginseng polysaccharide (GP) would have a preventive effect on influenza infection. Administration of mice with GP prior to infection was found to confer a survival benefit against infection with H1N1 (A/PR/8/34) and H3N2 (A/Philippines/82) influenza viruses. Mice infected with the 2009 H1N1 virus suspended in GP solution showed moderately enhanced survival rates and lower levels of lung viral titers and the inflammatory cytokine (IL-6). Daily treatment of vaccinated mice with GP improved their survival against heterosubtypic lethal challenge. This study demonstrates the first evidence that GP can be used as a remedy against influenza viral infection.

Effective public health responses to an influenza pandemic require an effective vaccine that can be manufactured and administered to large populations in the shortest possible time. In this study, we evaluated a method for vaccination against avian influenza virus that uses a DNA vaccine for rapid manufacturing and delivered by a microneedle skin patch for simplified administration and increased immunogenicity. We prepared patches containing 700-μm long microneedles coated with an avian H5 influenza hemagglutinin DNA vaccine from A/Viet Nam/1203/04 influenza virus. The coating DNA dose increased with DNA concentration in the coating solution and the number of dip-coating cycles. Coated DNA was released into the skin tissue by dissolution within minutes. Vaccination of mice using microneedles induced higher levels of antibody responses and hemagglutination inhibition titers, and improved protection against lethal infection with avian influenza as compared to conventional intramuscular delivery of the same dose of the DNA vaccine. Additional analysis showed that the microneedle coating solution containing carboxymethylcellulose and a surfactant may have negatively affected the immunogenicity of the DNA vaccine. Overall, this study shows that DNA vaccine delivery by microneedles can be a promising approach for improved vaccination to mitigate an influenza pandemic.

We investigated the roles of MyD88, an innate adaptor signaling molecule, in inducing protective humoral immunity after vaccination with influenza virus-like particles (VLPs). MyD88 knockout C57BL/6 mice (MyD88 −/− mice) vaccinated with influenza VLPs showed significant defects in inducing IgG2a/c isotype antibodies and in generating splenic recall memory B cell responses and antibody-secreting plasma cells in the bone marrow. The protective efficacy of influenza VLP vaccination was lower in MyD88 −/− mice than in the wild-type mice. Our findings indicate that MyD88-mediated innate signaling pathways are important for effectively inducing primary and boost immune responses, T helper type 1 isotype-switched antibodies, and gamma interferon (IFN-γ)-secreting T cell responses. In particular, the results in this study demonstrated for the first time that MyD88-mediated immune activation is likely an essential pathway for effective generation of long-lived antibody-secreting plasma cells and highly protective immunity after vaccination with influenza VLPs. This study provides insight into mechanisms by which recombinant viral vaccines induce protective immunity via the MyD88-mediated innate immune signaling pathway.

Killed and live attenuated influenza virus vaccines are effective in preventing and curbing the spread of influenza epidemics when the strains present in the vaccines are closely matched with the predicted epidemic strains. These vaccines are primarily targeted to induce immunity to the variable major target antigen, hemagglutinin (HA) of influenza virus. However, current vaccines are not effective in preventing the emergence of new pandemic or highly virulent viruses. New approaches are being investigated to develop universal influenza virus vaccines as well as to apply more effective vaccine delivery methods. Conserved vaccine targets including the influenza M2 ion channel protein and HA stalk domains are being developed using recombinant technologies to improve the level of cross protection. In addition, recent studies provide evidence that vaccine supplements can provide avenues to further improve current vaccination.

In the present study, virus-like particles (VLPs) were evaluated as a candidate poultry vaccine against avian influenza virus (AIV) subtype H9N2. Specific pathogen-free chickens received a single injection of the VLP vaccine expressing HA and M1 protein of AIV H9N2 (H9 HA VLP) at escalating doses in the presence or absence of ISA70 water-in-oil adjuvant. At 3 weeks post vaccination, we performed hemagglutination inhibition (HI) test and enzyme-linked immunosorbent assay (ELISA) to determine serological immune responses, and challenge studies using SPF chickens. A single dose of H9 HA VLP vaccine induced high levels of HI antibodies and lowered frequencies of virus isolation after the wild-type virus challenge. The addition of ISA70 adjuvant significantly increased the immunogenicity of H9 HA VLP vaccines. Furthermore, it allows differentiation of AIV-infected chickens from vaccinated chickens with an ELISA using nucleocapsid antigen, which offers a promising strategy to differentiate infected from vaccinated animals (DIVA). These results provide support for continued development of the VLP as an animal vaccine against influenza virus.

Background Current influenza vaccines based on the hemagglutinin protein are strain specific and do not provide good protection against drifted viruses or emergence of new pandemic strains. An influenza vaccine that can confer cross-protection against antigenically different influenza A strains is highly desirable for improving public health. Methodology/Principal Findings To develop a cross protective vaccine, we generated influenza virus-like particles containing the highly conserved M2 protein in a membrane-anchored form (M2 VLPs), and investigated their immunogenicity and breadth of cross protection. Immunization of mice with M2 VLPs induced anti-M2 antibodies binding to virions of various strains, M2 specific T cell responses, and conferred long-lasting cross protection against heterologous and heterosubtypic influenza viruses. M2 immune sera were found to play an important role in providing cross protection against heterosubtypic virus and an antigenically distinct 2009 pandemic H1N1 virus, and depletion of dendritic and macrophage cells abolished this cross protection, providing new insight into cross-protective immune mechanisms. Conclusions/Significance These results suggest that presenting M2 on VLPs in a membrane-anchored form is a promising approach for developing broadly cross protective influenza vaccines.

Development of an influenza vaccine that provides broadly cross-protective immunity has been a scientific challenge for more than half a century. This study presents an approach to overcome strain-specific protection by supplementing conventional vaccines with virus-like particles (VLPs) containing the conserved M2 protein (M2 VLPs) in the absence of adjuvants. We demonstrate that an inactivated influenza vaccine supplemented with M2 VLPs prevents disease symptoms without showing weight loss and confers complete cross protection against lethal challenge with heterologous influenza A viruses including the 2009 H1N1 pandemic virus as well as heterosubtypic H3N2 and H5N1 influenza viruses. Cross-protective immunity was long-lived, for more than 7 mo. Immune sera from mice immunized with M2 VLP supplemented vaccine transferred cross protection to naive mice. Dendritic and macrophage cells were found to be important for this cross protection mediated by immune sera. The results provide evidence that supplementation of seasonal influenza vaccines with M2 VLPs is a promising approach for overcoming the limitation of strain-specific protection by current vaccines and developing a universal influenza A vaccine.

There is a significant gap in our fundamental understanding of early morphological and migratory changes in human Langerhans cells (LCs) in response to vaccine stimulation. As the vast majority of LCs studies are conducted in small animal models, substantial interspecies variation in skin architecture and immunity must be considered when extrapolating the results to humans. This study aims to determine whether excised human skin, maintained viable in organ culture, provides a useful human model for measuring and understanding early immune response to intradermally delivered vaccine candidates. Excised human breast skin was maintained viable in air-liquid-interface organ culture. This model was used for the first time to show morphological changes in human LCs stimulated with influenza virus-like particle (VLP) vaccines delivered via intradermal injection. Immunohistochemistry of epidermal sheets and skin sections showed that LCs in VLP treated skin lost their typical dendritic morphology. The cells were more dispersed throughout the epidermis, often in close proximity to the basement membrane, and appeared vertically elongated. Our data provides for increased understanding of the complex morphological, spatial and temporal changes that occur to permit LC migration through the densely packed keratinocytes of the epidermis following exposure to vaccine. Significantly, the data not only supports previous animal data but also provides new and essential evidence of host response to this vaccination strategy in the real human skin environment.

A simple method suitable for self-administration of vaccine would improve mass immunization, particularly during a pandemic outbreak. Influenza virus-like particles (VLPs) have been suggested as promising vaccine candidates against potentially pandemic influenza viruses, as they confer long-lasting immunity but are not infectious. We investigated the immunogenicity and protective efficacy of influenza H5 VLPs containing the hemagglutinin (HA) of A/Vietnam/1203/04 (H5N1) virus delivered into the skin of mice using metal microneedle patches and also studied the response of Langerhans cells in a human skin model. Prime-boost microneedle vaccinations with H5 VLPs elicited higher levels of virus-specific IgG1 and IgG2a antibodies, virus-specific antibody-secreting cells, and cytokine-producing cells up to 8 months after vaccination compared to the same antigen delivered intramuscularly. Both prime-boost microneedle and intramuscular vaccinations with H5 VLPs induced similar hemagglutination inhibition titers and conferred 100% protection against lethal challenge with the wild-type A/Vietnam/1203/04 virus 16 weeks after vaccination. Microneedle delivery of influenza VLPs to viable human skin using microneedles induced the movement of CD207+Langerhans cells toward the basement membrane. Microneedle vaccination in the skin with H5 VLPs represents a promising approach for a self-administered vaccine against viruses with pandemic potential.

Influenza virus-like particles (VLPs) are a promising cell culture-based vaccine, and the skin is considered an attractive immunization site. In this study, we examined the immunogenicity and protective efficacy of influenza VLPs (H1N1 A/PR/8/34) after skin vaccination using vaccine dried on solid microneedle arrays. Coating of microneedles with influenza VLPs using an unstabilized formulation was found to decrease hemagglutinin (HA) activity, whereas inclusion of trehalose disaccharide preserved the HA activity of influenza VLP vaccines after microneedles were coated. Microneedle vaccination of mice in the skin with a single dose of stabilized influenza VLPs induced 100% protection against challenge infection with a high lethal dose. In contrast, unstabilized influenza VLPs, as well as intramuscularly injected vaccines, provided inferior immunity and only partial protection (≤40%). The stabilized microneedle vaccination group showed IgG2a levels that were 1 order of magnitude higher than those of other groups and had the lowest lung viral titers after challenge. Also, levels of recall immune responses, including hemagglutination inhibition titers, neutralizing antibodies, and antibody-secreting plasma cells, were significantly higher after skin vaccination with stabilized formulations. Therefore, our results indicate that HA stabilization, combined with vaccination via the skin using a vaccine formulated as a solid microneedle patch, confers protection superior to that with intramuscular injection and enables potential dose-sparing effects which are reflected by pronounced increases in rapid recall immune responses against influenza virus.

To develop a more effective vaccination method against H5N1 virus, we investigated the immunogenicity and protective efficacy after skin vaccination using microneedles coated with influenza virus-like particles containing hemagglutinin derived from A/Vietnam/1203/04 H5N1 virus (H5 VLPs). A single microneedle vaccination of mice with H5 VLPs induced increased levels of antibodies and provided complete protection against lethal challenge without apparent disease symptoms. In contrast, intramuscular injection with the same vaccine dose showed low levels of antibodies and provided only partial protection accompanied by severe body weight loss. Post-challenge analysis suggested that improved protection was associated with lower lung viral titers and enhanced generation of recall antibody secreting cells by microneedle vaccination. Thus, this study provides evidence that skin delivery of H5 VLP vaccines using microneedles designed for self-administration induces improved protection compared to conventional intramuscular immunization.

Background Pandemic influenza poses a serious threat to global health and the world economy. While vaccines are currently under development, passive immunization could offer an alternative strategy to prevent and treat influenza virus infection. Attempts to develop monoclonal antibodies (mAbs) have been made. However, passive immunization based on mAbs may require a cocktail of mAbs with broader specificity in order to provide full protection since mAbs are generally specific for single epitopes. Chicken immunoglobulins (IgY) found in egg yolk have been used mainly for treatment of infectious diseases of the gastrointestinal tract. Because the recent epidemic of highly pathogenic avian influenza virus (HPAIV) strain H5N1 has resulted in serious economic losses to the poultry industry, many countries including Vietnam have introduced mass vaccination of poultry with H5N1 virus vaccines. We reasoned that IgY from consumable eggs available in supermarkets in Vietnam could provide protection against infections with HPAIV H5N1. Methods and Findings We found that H5N1-specific IgY that are prepared from eggs available in supermarkets in Vietnam by a rapid and simple water dilution method cross-protect against infections with HPAIV H5N1 and related H5N2 strains in mice. When administered intranasally before or after lethal infection, the IgY prevent the infection or significantly reduce viral replication resulting in complete recovery from the disease, respectively. We further generated H1N1 virus-specific IgY by immunization of hens with inactivated H1N1 A/PR/8/34 as a model virus for the current pandemic H1N1/09 and found that such H1N1-specific IgY protect mice from lethal influenza virus infection. Conclusions The findings suggest that readily available H5N1-specific IgY offer an enormous source of valuable biological material to combat a potential H5N1 pandemic. In addition, our study provides a proof-of-concept for the approach using virus-specific IgY as affordable, safe, and effective alternative for the control of influenza outbreaks, including the current H1N1 pandemic.

Background: Recurrent outbreaks of highly pathogenic H5N1 avian influenza virus pose a threat of eventually causing a pandemic. Early vaccination of the population would be the single most effective measure for the control of an emerging influenza pandemic. Methodology/Principal Findings: Influenza virus-like particles (VLPs) produced in insect cell-culture substrates do not depend on the availability of fertile eggs for vaccine manufacturing. We produced VLPs containing influenza A/Viet Nam1203/04 (H5N1) hemagglutinin, neuraminidase, and matrix proteins, and investigated their preclinical immunogenicity and protective efficacy. Mice immunized intranasally with H5N1 VLPs developed high levels of H5N1 specific antibodies and were 100% protected against a high dose of homologous H5N1 virus infection at 30 weeks after immunization. Protection is likely to be correlated with humoral and cellular immunologic memory at systemic and mucosal sites as evidenced by rapid anamnestic responses to re-stimulation with viral antigen in vivo and in vitro. Conclusions/Significance: These results provide support for clinical evaluation of H5N1 VLP vaccination as a public health intervention to mitigate a possible pandemic of H5N1 influenza.

The format of influenza virus-like particles (VLPs) as a nonreplicating particulate vaccine candidate is a promising alternative to conventional egg-based vaccines. In this study, we have investigated the detailed kinetics of immune responses and protective efficacy after a single intranasal immunization with different doses of VLPs alone or in the presence of an Escherichia coli mutant heat-labile enterotoxin [mLT(R192G)] or cholera toxin subunit B as adjuvants. Analysis of immune responses showed differential kinetics in a VLP antigen dose-dependent manner and dynamic changes in the ratios of antibody immunoglobulin G isotypes over the time course. Protection against lethal challenge was observed with a single immunization with influenza VLPs even without adjuvant. The addition of adjuvant showed significant antigen-sparing effects with improved protective efficacy. The protective immune responses, efficacies of protection, and antigen-sparing effects were significantly improved by a second immunization as determined by the levels of neutralizing antibodies, morbidity postchallenge, lung viral titers, and inflammatory cytokines. Our results are informative for a better understanding of the protective immunity induced by a single dose or two doses of influenza VLPs, which is dependent on antigen dosage and the presence of adjuvant, and will provide insights into designing effective vaccines based on VLPs.

Catechin derivatives with different alkyl chain length and aromatic ring substitutions at the 3-hydroxyl group were synthesized from epigallocatechin (EGC) and (+)-catechin (C) and their anti-influenza viral activity were evaluated in vitro and in ovo. Pronounced antiviral activity was observed for derivatives carrying moderate chain length (7–9 carbons) as compared to those with aromatic rings, whereas the 5′-hydroxyl group of the trihydroxy benzyl moiety did not significantly contribute to antiviral activity. The derivatives exerted inhibitory effects for all six influenza subtypes tested including three major types of currently circulating human influenza viruses (A/H1N1, A/H3N2 and B type), H2N2 and H9N2 avian influenza virus. The compounds strongly inhibited adsorption of the viruses on red blood cell (RBC). They also restricted the growth of avian influenza virus in ovo with minimum inhibition concentration (MIC) of 5–10 μM far exceeding the neuraminidase (NA) inhibitor oseltamivir or M2 proton channel inhibitor amantadine. The antiviral activity appears to be mediated by interaction with hemagglutinin (HA)/viral membrane rendering HA less fusogenic at the initial stage of infection. The broad spectrum activity against various subtypes of influenza viruses may complement the limitations of current antivirals and contribute for managing potentially emerging influenza pandemic. The structure-activity data of catechin derivatives may usefully guideline future research endeavors for applying green tea catechins as alternative anti-viral agents.

Squamous cell carcinoma (SCC) is the most common malignant tumor in the oral cavity, and it accounts for about 90% of all oral cancers. Several risk factors for oral SCC have been identified; however, SCC associated with odontogenic keratocysts have rarely been reported. The present study describes the case of a 36-year-old man with SCC of the right ramus of the mandible, which was initially diagnosed as a benign odontogenic cyst. He underwent enucleation at another hospital followed by segmental mandibulectomy and fibular free flap reconstruction at our institution. In this case, we introduce a patient with oral cancer associated with odontogenic cyst on the mandible and report a satisfactory outcome with wide resection and immediate free flap reconstruction.

Objectives: This study investigated patients with oral and maxillofacial lacerations who visited the emergency room over a three-year period in an effort to determine the optimal treatment for these injuries. Materials and Methods: This study examined 1,742 patients with oral and maxillofacial lacerations with 2,014 different laceration locations who visited the emergency room of Pusan National University Hospital (Busan, Korea) over three years, from January 2011 to December 2013. Patients were classified by sex, age, visit day, cause of injury, injury site, and the presence or absence of soft tissue and tooth injuries. Results: The male to female ratio was 2.50:1. Patients under 10 years old were seen most frequently. Most emergency room visits were on weekends. Among intra-oral lacerations, the lip area was the most vulnerable site; among extra-oral lacerations, the chin area was most frequently injured. The most frequent etiology was a slip down. Most lacerations occurred without bone fracture or tooth damage. Conclusion: Laceration may differ in large part as compared with the fracture. Therefore, it is necessary to continue collecting data on oral and maxillofacial lacerations to establish optimal emergency room diagnosis and treatment strategies.

A human influenza A virus X-31 (high-yielding strain) was cold-adapted for possible future use as live attenuated vaccine. Mutant influenza viruses were selected during successive serial passage in embryonated hens' eggs at progressively lower sub-optimal temperature (30, 27 degrees C followed by 24 degrees C). The cold-passaged mutant exhibited both temperature-sensitivity (ts) and cold-adapted (ca) phenotypes. The pathogenicity and immunogenicity of X-31 ca virus were studied in mice following intranasal inoculation. The mice did not show clinical signs even at high titer infection. Immunization of mice with X-31 ca virus elicited high titers of neutralizing antibody and provided complete protection against homologous and heterologous virus challenges. To assess the genetic stability, the X-31 ca virus was passaged at 37 degrees C in MDCK cells or inoculated into mice. Revertant virus was not found in the lungs of any of the mice and the supernatants of the MDCK culture. We conclude that the X-31 ca candidate vaccine virus exhibits the desired level of attenuation, immunogenicity, and protective efficacy required for live attenuated vaccine and merits further evaluation at clinical level.

Objectives: The aim of this study was to evaluate the pattern of lingual split line when performing a bilateral sagittal split osteotomy (BSSO) for asymmetric prognathism. This was accomplished with the use of cone-beam computed tomography (CBCT) and three-dimensional (3D) software program. Materials and Methods: The study group was comprised of 40 patients (20 males and 20 females) with asymmetric prognathism, who underwent BSSO (80 splits; n=80) from January 2012 through June 2013. We observed the pattern of lingual split line using CBCT data and image analysis program. The deviated side was compared to the contralateral side in each patient. To analyze the contributing factors to the split pattern, we observed the position of the lateral cortical bone cut end and measured the thickness of the ramus that surrounds the mandibular lingula. Results: The lingual split patterns were classified into five types. The true “Hunsuck” line was 60.00% (n=48), and the bad split was 7.50% (n=6). Ramal thickness surrounding the lingual was 5.55±1.07 mm (deviated) and 5.66±1.34 mm (contralateral) (P =0.409). The position of the lateral cortical bone cut end was classified into three types: A, lingual; B, inferior; C, buccal. Type A comprised 66.25% (n=53), Type B comprised 22.50% (n=18), and Type C comprised 11.25% (n=9). Conclusion: In asymmetric prognathism patients, there were no differences in the ramal thickness between the deviated side and the contralateral side. Furthermore, no differences were found in the lingual split pattern. The lingual split pattern correlated with the position of the lateral cortical bone cut end. In addition, the 3D-CT reformation was a useful tool for evaluating the surgical results of BSSO of the mandible.

Pathogenic H5N1, a lipid-enveloped influenza virus, is a pandemic threat. The use of virus-like particles (VLPs) as an alternative to current influenza vaccines is highly promising. VLPs are similar in structure to their live viral counterparts but do not contain viral genome that is required for replication; hence VLPs provide for a safe and immunogenic vaccine. Although the particulate nature of VLPs allows them to be highly immunogenic, the need for protection against heterotypic viruses still remains. Therefore, inclusion of immunostimulatory molecules (ISMs) onto the VLP surface can help to induce cross-protection against strains and provide for stronger immunity. We were able to show that GPI-anchored-GM-CSF can incorporate stably onto H5 influenza VLP surfaces after a simple and quick protein transfer method. This method allows for the incorporation of the GPI-anchor onto the surface of lipid-bilayered VLPs within a matter of hours and allows for the incorporation of multiple GPI-ISMs in a concentration-dependent manner. Furthermore, protein transferred VLPs were functional in leading to bone marrow derived cell proliferation compared to unmodified VLPs, and incorporated GM-CSF was as functional as equal concentrations of commercially available recombinant soluble GM-CSF. Therefore, VLPs expressing GPI-GM-CSF could lead to enhanced immunogenicity and antiviral immune responses compared to unmodified VLPs.

Objectives: The purpose of this study is to analyze clinical impact factors on the survival rate, and to acquire basic clinical data for the diagnosis of oral cancer, for a determination of the treatment plan with long-term survival in oral cancer patients. Materials and Methods: Through a retrospective review of the medical records, the factors for long-term survival rate were analyzed. Thirty-seven patients, among patient database with oral cancer treated in the Department of Oral and Maxillofacial Surgery at Pusan National University Hospital within a period from March 1998 to March 2008, were selected within the study criteria and were followed-up for more than 5 years. The analyzed factors were gender, age, drinking, smoking, primary tumor site, type of cancer, TNM stage, recurrence of affected region, and metastasis of cervical lymph node. The 5-year survival rate on the impact factors was calculated statistically using the Kaplan-Meier method. Results: By classification of clinical TNM at the 1st visit, there were 11 (29.7%) cases for stage I, 11 (29.7%) cases for stage II, 3 (8.1%) cases for stage III, and 12 (32.5%) cases for stage IV. The 5-year survival rate of total oral cancer patients after the operation were 75.7%, pathological TNM stage related 5-year survival rate were as follows: stage I 90.0%, stage II 81.8%, stage III 100% and stage IV 45.5%; in which the survival rate difference by each stage was significantly observed. The recurrence of cervical lymph node was the significant impact factor for the survival rate, because only 30.0% the survival rate in recurrent cases existed. During the follow-up, there were 15 (40.5%) patients with confirmed recurrence, and the 5-year survival rate of these patients was decreased as 46.7%. Conclusion: The classification of clinical and pathological TNM stage, local recurrence after surgery, and metastasis of cervical lymph node after surgery were analyzed as the 3 most significant factors.

H5N1 influenza, a bilayered lipid enveloped virus, is highly pathogenic and can lead to a pandemic threat; therefore, a potent vaccine is required. The use of virus-like particles (VLPs) as a vaccine has recently gained importance as they are safe and highly immunogenic. VLPs are similar in size and structure to their viral counterparts but do not contain the viral genome required for replication. Although VLPs are immunogenic, full protection and cross-reactivity to other influenza strains does not always occur; therefore, we aim to modify H5 influenza VLPs to incorporate immunostimulatory molecules (ISMs) by using a protein transfer method. Previously, we have shown that GPI-anchored ISMs (ICAM-1 and IL-12) are able to incorporate onto H5 influenza VLPs by a simple and easy protein transfer method. In this study, we show that purification of GPI-IL-12 does not alter the functionality of the protein in stimulating proliferation of ConA-activated splenocytes suggesting that purified GPI-ISMs remain functionally active. Furthermore, the detection of incorporated GPI-ISMs onto the surface of H5 influenza VLPs by protein transfer was conducted by using immunogold staining electron microscopy and incorporation occurred uniformly as detected by flow cytometry analysis. Moreover, these GPI-ISMs remain stably expressed on the VLP surface for at least a week at 4oC. This study suggests that H5 influenza VLPs can be modified to stably express ISMs on its surface by protein transfer.