Effect of isoproterenol, a ß-adrenergic agonist, on the differentiation of insulin-producing pancreatic ß cells derived from human pluripotent stem cells.

Research on islet replacement through the differentiation of functionally matured insulin-producing ß-like cells for the treatment of diabetes presents a significant challenge. Neural signals in ß cell differentiation significantly impact the pancreatic microenvironment in glucose metabolism, but they are not fully understood. In this study, isoproterenol, a ß adrenoreceptor agonist, was introduced into pancreatic progenitor cells, derived from human pluripotent stem cells in vitro, undergoing endocrine differentiation, using 2-dimensional (2D) and 3-dimensional (3D) differentiation protocols. This resulted in increased insulin and C-peptide secretion, along with elevated expression of key pancreatic beta cell transcription factors, including PDX-1, NKX6.1, and MAFA, and improved function, demonstrated by increased responsiveness to glucose determined via a glucose-stimulated insulin secretion test. Moreover, RNA transcriptome analysis of isoproterenol-treated endocrine progenitors facilitated the identification of biological pathways and genes that contribute to mature beta cell differentiation efficiency correlated with neural signals, such as adrenoceptor beta 1, calcium/calmodulin dependent protein kinase II alpha, phospholipase C delta 4, and neurotrophic receptor tyrosine kinase 1. Among those genes, calcium/calmodulin dependent protein kinase II alpha was suggested as the most notable gene involved in the isoproterenol mechanism through inhibitor assays. This study illustrates that isoproterenol significantly enhances endocrine differentiation and underscores its effects on stem cell-derived beta cell maturation, emphasizing its therapeutic potential for the treatment of diabetes.

Extracellular Vesicles Delivered by Injectable Collagen Promote Bone-Tendon Interface Healing and Prevent Fatty Degeneration of Rotator Cuff Muscle.

PURPOSE: This study aimed (1) to confirm the maintenance of the extracellular vesicles (EVs) delivered via injectable collagen at the application site, and (2) to evaluate the effect of EVs derived from the human umbilical cord-derived mesenchymal stem cells and loaded in an injectable collagen gel after rotator cuff repair (RCR). METHODS: Rabbits (n = 20) were assigned to normal (N), repair-only (R), and those administered with injectable collagen after repair (RC), and EV-laden injectable collagen after repair (RCE) groups. The EVs isolated by ultra-centrifugation from the human umbilical cord-derived mesenchymal stem cells spent medium were mixed with collagen and administered accordingly. After 12 weeks, the rabbits were sacrificed to evaluate the healing of the bone-to-tendon junction and the fatty degeneration of muscle. Histomorphometric scoring for bone-tendon interface, fatty infiltration (%), and biomechanical tests were performed. Separately, groups of 3 rabbits were assigned to 3 different time points to evaluate maintenance of green fluorescence-labeled EVs with injectable collagen via tracking on the bursal side of the rotator cuff (3 groups: 3 days, 2, and 4 weeks). RESULTS: The EVs delivered by injectable collagen remained until 4 weeks at the bursal side of the cuff tissue. The RCE group showed a significantly greater histomorphometric total score (P < .001, and P = .013, respectively) and significantly lower fatty degeneration than the RC and R groups (P = .001, and P = .013, respectively). The biomechanical tests revealed significant growing trends in load-to-failure and stiffness (P = .002, and P = .013, respectively), in the R, RC, RCE, and N groups. CONCLUSIONS: EVs mounted in injectable collagen remained at the repair site for at least 4 weeks after application. Furthermore, they effectively promote bone-to-tendon healing via collagen maturation in bone-tendon interface and preventing fatty degeneration of rotator muscle after RCR as compared with collagen-only or repair-only groups. CLINICAL RELEVANCE: The combination of collagen with EVs significantly promotes rotator cuff healing demonstrating potential clinical application during partial rotator cuff tear or after RCR.

Decellularized Bovine Pericardial Patch Loaded With Mesenchymal Stromal Cells Enhance the Mechanical Strength and Biological Healing of Large-to-Massive Rotator Cuff Tear in a Rat Model.

PURPOSE: The purpose of this study was to determine whether the addition of decellularized bovine pericardial patch loaded with mesenchymal stromal cells enhanced bone-to-tendon healing and improved the biomechanical strength of large-to-massive rotator cuff tears in a small animal model. METHODS: Adipose-derived mesenchymal stromal cells (MSCs) from rat inguinal fat were isolated, cultured, and loaded onto decellularized bovine pericardium patches. To simulate large-to-massive tears, rats were managed with free cage activity for 6 weeks after tear creation. A total of 18 rats were randomly allocated to repair-only (control), repair with pericardial patch augmentation (patch), or repair with MSC loaded pericardial patch augmentation (patch-MSC). Each group had 6 rats (one shoulder of each rat was used for histological evaluation and another for biomechanical evaluation). MSCs seeded on the pericardial patches were traced on four shoulders from 2 other rats at 4 weeks after surgery. Histological evaluation for bone-to-tendon healing and biomechanical testing was carried out at 8 weeks after repair. RESULTS: MSCs tagged with a green fluorescent protein were observed in the repair site 4 weeks after the repair. One shoulder each in the control and patch groups showed complete discontinuity between the bone and tendon. One shoulder in the control group showed attenuation with only a tenuous connection. Fibrocartilage and tidemark formation at the bone-to-tendon interface (P = .002) and collagen fiber density (P = .040) and orientation (P = .003) were better in the patch-MSC group than in the control or patch group. Load-to-failure in the patch-MSC and patch groups was higher than that in the control group (P = .001 and .009, respectively). CONCLUSION: Decellularized bovine pericardial patches loaded with adipose-derived and cultured mesenchymal stromal cells enhanced healing in terms of both histology and mechanical strength at 8 weeks following rotator cuff repair in a rat model. CLINICAL RELEVANCE: Large-to-massive rotator tears need a strategy to prevent retear and enhance healing. The addition of decellularized bovine pericardial patch loaded with MSCs can enhance bone-to-tendon healing and improve biomechanical healing of large-to-massive rotator cuff tears following repair.

Isolation and characterization of low pathogenic H7N7 avian influenza virus from a red-crowned crane in a zoo in South Korea.

BACKGROUND: South Korea conducts annual national surveillance programs to detect avian influenza (AI) in domestic poultry, live bird markets, and wild birds. In March 2017, an AIV was isolated from fecal samples in an outdoor aviary flight cage in a zoo in Korea. RESULTS: Nucleotide sequencing identified the isolate as low pathogenic avian influenza virus (LPAIV) H7N7, and DNA barcoding analysis identified the host species as red-crowned crane. This isolate was designated A/red-crowned crane/Korea/H1026/2017 (H7N7). Genetic analysis and gene constellation analysis revealed that A/red-crowned crane/Korea/H1026/2017 (H7N7) showed high similarity with four H7N7 LPAIVs isolated from wild bird habitats in Seoul and Gyeonggi in early 2017. CONCLUSIONS: Considering the genetic similarity and similar collection dates of the viruses, and the fact that zoo bird cages are vulnerable to AIV, it is likely that fecal contamination from wild birds might have introduced LPAIV H7N7 into the red-crowned crane at the zoo. Therefore, our results emphasize that enhanced biosecurity measures should be employed during the wild bird migration season, and that continued surveillance should be undertaken to prevent potential threats to avian species in zoos and to humans.

Complement C3 Plays a Key Role in Inducing Humoral and Cellular Immune Responses to Influenza Virus Strain-Specific Hemagglutinin-Based or Cross-Protective M2 Extracellular Domain-Based Vaccination.

The complement pathway is involved in eliminating antigen immune complexes. However, the role of the C3 complement system remains largely unknown in influenza virus M2 extracellular (M2e) domain or hemagglutinin (HA) vaccine-mediated protection after vaccination. Using a C3 knockout (C3 KO) mouse model, we found that complement protein C3 was required for effective induction of immune responses to vaccination with M2e-based or HA-based vaccines, which include isotype class-switched antibodies and effector CD4 and CD8 T cell responses. C3 KO mice after active immunization with cross-protective nonneutralizing M2e-based vaccine were not protected against influenza virus, although low levels of M2e-specific antibodies were protective after passive coadministration with virus in wild-type mice. In contrast, C3 KO mice that were immunized with strain-specific neutralizing HA-based vaccine were protected against homologous virus challenge despite lower levels of HA antibody responses. C3 KO mice showed impaired maintenance of innate immune cells and a defect in innate immune responses upon exposure to antigens. The findings in this study suggest that C3 is required for effective induction of humoral and cellular adaptive immune responses as well as protective immunity after nonneutralizing influenza M2e vaccination.IMPORTANCE Complement is the well-known innate immune defense system involved in the opsonization and lysis of pathogens but is less studied in establishing adaptive immunity after vaccination. Influenza virus HA-based vaccination confers protection via strain-specific neutralizing antibodies, whereas M2e-based vaccination induces a broad spectrum of protection by immunity against the conserved M2e epitopes. This study revealed the critical roles of C3 complement in inducing humoral and cellular immune responses after immunization with M2e or HA vaccines. C3 was found to be required for protection by M2e-based but not by HA-based active vaccination as well as for maintaining innate antigen-presenting cells. Findings in this study have insight into better understanding the roles of C3 complement in inducing effective innate and adaptive immunity as well as in conferring protection by cross-protective conserved M2e vaccination.

Roles of Aluminum Hydroxide and Monophosphoryl Lipid A Adjuvants in Overcoming CD4+ T Cell Deficiency To Induce Isotype-Switched IgG Antibody Responses and Protection by T-Dependent Influenza Vaccine.

Vaccine adjuvant effects in the CD4-deficient condition largely remain unknown. We investigated the roles of combined monophosphoryl lipid A (MPL) and aluminum hydroxide (Alum) adjuvant (MPL+Alum) in inducing immunity after immunization of CD4 knockout (CD4KO) and wild-type (WT) mice with T-dependent influenza vaccine. MPL+Alum adjuvant mediated IgG isotype-switched Abs, IgG-secreting cell responses, and protection in CD4KO mice, which were comparable to those in WT mice. In contrast, Alum adjuvant effects were dependent on CD4+ T cells. MPL+Alum adjuvant was effective in recruiting monocytes and neutrophils as well as in protecting macrophages from Alum-mediated cell loss at the injection site in CD4KO mice. MPL+Alum appeared to attenuate MPL-induced inflammatory responses in WT mice, likely improving the safety. Additional studies in CD4-depleted WT mice and MHC class II KO mice suggest that MHC class II+ APCs contribute to providing alternative B cell help in the CD4-deficient condition in the context of MPL+Alum-adjuvanted vaccination.

Highly Pathogenic Avian Influenza A(H5N6) in Domestic Cats, South Korea.

In December 2016, highly pathogenic avian influenza (HPAI) infection with systemic pathologic lesions was found in cats in South Korea. Genetic analyses indicated that the feline isolates were similar to HPAI H5N6 viruses isolated in chicken farms nearby. This finding highlights the need for monitoring of domestic mammals during HPAI outbreaks.

A Novel Vaccination Strategy Mediating the Induction of Lung-Resident Memory CD8 T Cells Confers Heterosubtypic Immunity against Future Pandemic Influenza Virus.

The currently used vaccine strategy to combat influenza A virus (IAV) aims to provide highly specific immunity to circulating seasonal IAV strains. However, the outbreak of 2009 influenza pandemic highlights the danger in this strategy. In this study, we tested the hypothesis that universal vaccination that offers broader but weaker protection would result in cross protective T cell responses after primary IAV infection, which would subsequently provide protective immunity against future pandemic strains. Specifically, we used tandem repeat extracellular domain of M2 (M2e) epitopes on virus-like particles (M2e5x VLP) that induced heterosubtypic immunity by eliciting Abs to a conserved M2e epitope. M2e5x VLP was found to be superior to strain-specific current split vaccine in conferring heterosubtypic cross protection and in equipping the host with cross-protective lung-resident nucleoprotein-specific memory CD8(+) T cell responses to a subsequent secondary infection with a new pandemic potential strain. Immune correlates for subsequent heterosubtypic immunity by M2e5x VLP vaccination were found to be virus-specific CD8(+) T cells secreting IFN-γ and expressing lung-resident memory phenotypic markers CD69(+) and CD103(+) as well as M2e Abs. Hence, vaccination with M2e5x VLP may be developable as a new strategy to combat future pandemic outbreaks.

Roles of antibodies to influenza A virus hemagglutinin, neuraminidase, and M2e in conferring cross protection.

Although neuraminidase (NA) is the second major viral glycoprotein of influenza virus, its immune mechanism as a vaccine target has been less considered. Here we compared the properties of antibodies and the efficacy of cross protection by N1 and N2 NA proteins, inactivated split influenza vaccines (split), and tandem repeat extracellular domain M2 on virus-like particles (M2e5x VLP). Anti-NA immune sera could confer better cross-protection against multiple heterologous influenza viruses correlating with NA inhibition activity compared to split vaccine immune sera. Whereas split vaccine was superior to NA in conferring homologous protection. NA and M2e immune sera each showed comparable survival protection. Protective efficacy by NA immune sera was lower in Fc receptor common γ-chain deficient mice but comparable in C3 complement deficient mice compared to that in wild type mice, suggesting a role of Fc receptor in NA immunity.

CD47 Plays a Role as a Negative Regulator in Inducing Protective Immune Responses to Vaccination against Influenza Virus.

UNLABELLED: An integrin-associated protein CD47, which is a ligand for the inhibitory receptor signal regulatory protein α, is expressed on B and T cells, as well as on most innate immune cells. However, the roles of CD47 in the immune responses to viral infection or vaccination remain unknown. We investigated the role of CD47 in inducing humoral immune responses after intranasal infection with virus or immunization with influenza virus-like particles (VLPs). Virus infection or vaccination with VLPs containing hemagglutinin from A/PR8/34 influenza virus induced higher levels of antigen-specific IgG2c isotype dominant antibodies in CD47-deficient (CD47KO) mice than in wild-type (WT) mice. CD47KO mice with vaccination showed greater protective efficacy against lethal challenge, as evidenced by no loss in body weight and reduced lung viral titers compared to WT mice. In addition, inflammatory responses which include cytokine production, leukocyte infiltrates, and gamma interferon-producing CD4(+) T cells, as well as an anti-inflammatory cytokine (interleukin-10), were reduced in the lungs of vaccinated CD47KO mice after challenge with influenza virus. Analysis of lymphocytes indicated that GL7(+) germinal center B cells were induced at higher levels in the draining lymph nodes of CD47KO mice compared to those in WT mice. Notably, CD47KO mice exhibited significant increases in the numbers of antigen-specific memory B cells in spleens and plasma cells in bone marrow despite their lower levels of background IgG antibodies. These results suggest that CD47 plays a role as a negative regulator in inducing protective immune responses to influenza vaccination. IMPORTANCE: Molecular mechanisms that control B cell activation to produce protective antibodies upon viral vaccination remain poorly understood. The CD47 molecule is known to be a ligand for the inhibitory receptor signal regulatory protein α and expressed on the surfaces of most immune cell types. CD47 was previously demonstrated to play an important role in modulating the migration of monocytes, neutrophils, polymorphonuclear neutrophils, and dendritic cells into the inflamed tissues. The results of this study demonstrate new roles of CD47 in negatively regulating the induction of protective IgG antibodies, germinal center B cells, and plasma cells secreting antigen-specific antibodies, as well as macrophages, upon influenza vaccination and challenge. As a consequence, vaccinated CD47-deficient mice demonstrated better control of influenza viral infection and enhanced protection. This study provides insights into understanding the regulatory functions of CD47 in inducing adaptive immunity to vaccination.

Surveillance of avian influenza viruses in South Korea between 2012 and 2014.

BACKGROUND: National surveillance of avian influenza virus (AIV) in South Korea has been annually conducted for the early detection of AIV and responses to the introduction of highly pathogenic avian influenza (HPAI) virus. In this study, we report on a nationwide surveillance study of AIV in domestic poultry and wild birds in South Korea between 2012 and 2014. METHODS: During the surveillance programs between 2012 and 2014, 141,560 samples were collected. Of these, 102,199 were from poultry farms, 8215 were from LBMs, and 31,146 were from wild bird habitats. The virus isolation was performed by inoculation of embryonated chicken eggs and AIV isolates were detected using hemagglutination assay. For subtying of AIV, the hemagglutinin and neuraminidase genes were confirmed by sequencing. Phylogenetic analysis of the H5 subtypes was performed using 28 H5 AIV isolates. RESULTS: Between 2012 and 2014, a total of 819 AIV were isolated from 141,560 samples. Virus isolation rates for AIV were 0.6, 0.4, 0.1, and 2.7% in wild birds (n = 202), domestic ducks (n = 387), minor poultry (n = 11), and the live bird market (LBM) (n = 219), respectively. In wild birds, various subtypes were found including H1-H7 and H9-H13. The major subtypes were H5 (n = 48, 23.9%: N3 (n = 4) and N8 (n = 44)), H4 (n = 39, 19.4%), and H1 (n = 29, 14.4%). In domestic poultry, mainly ducks, the H5N8 (n = 275, 59.3%), H3 (n = 30, 17.2%), and H6 (n = 53, 11.4%) subtypes were predominantly found. The most frequently detected subtypes in LBM, primarily Korean native chicken, were H9 (n = 169, 77.2%). H3 (n = 10, 4%) and H6 (n = 30, 13.7%) were also isolated in LBM. Overall, the prevalence of AIV was found to be higher between winter and spring and in western parts of South Korea. The unusual high prevalence of the H5 subtype of AIV was due to the large scale outbreak of H5N8 HPAI in wild birds and domestic poultry in 2014. CONCLUSIONS: Enhanced surveillance and application of effective control measures in wild birds and domestic poultry, including LBM, should be implemented to control AI and eradicate HPAI.

Reliability and Validity of Culturally Adapted Executive Function Performance Test for Koreans with Stroke.

BACKGROUND: Executive Function Performance Test was unusable in Asia because of cultural differences, although evaluating the effect that executive function has on real life is essential to people with stroke. The aims of this study were to report the development and standardization of culturally appropriate Executive Function Performance Test for Koreans (EFPT-K) and to verify its reliability and validity in the stroke population. METHODS: EFPT-K was developed by going through the process of translation, back-translation, and an expert committee's conference on cultural adaptation. Inter-rater reliability was examined and 34 people with stroke were recruited to test the internal consistency and criterion validity of EFPT-K. To verify the criterion validity, EFPT-K scores were compared with those of the Trail-Making Test, the Korean Color-Word Stroop Test, the Digit Span Test, and the Assessment of Motor and Process Skills (AMPS). RESULTS: Inter-rater reliability and internal consistency of the total score of EFPT-K supported high levels of reliability. In the criterion validity of EFPT-K, all neuropsychological tests but digits backward showed a correlation with the total score of EFPT-K, and AMPS components of initiation, notice/response, and termination showed a moderate correlation with the EFPT-K score. CONCLUSIONS: EFPT-K is a useful tool to evaluate the executive functioning of patients with stroke in real-life tasks and could be used as a sample in other Asian cultures where thorough evaluation of executive function in the performance of daily life is needed.

Korean Ginseng Berry Fermented by Mycotoxin Non-producing Aspergillus niger and Aspergillus oryzae: Ginsenoside Analyses and Anti-proliferative Activities.

To transform ginsenosides, Korean ginseng berry (KGB) was fermented by mycotoxin non-producing Aspergillus niger and Aspergillus oryzae. Changes of ginsenoside profile and anti-proliferative activities were observed. Results showed that A. niger tended to efficiently transform protopanaxadiol (PPD) type ginsenosides such as Rb1, Rb2, Rd to compound K while A. oryzae tended to efficiently transform protopanaxatriol (PPT) type ginsenoside Re to Rh1 via Rg1. Butanol extracts of fermented KGB showed high cytotoxicity on human adenocarcinoma HT-29 cell line and hepatocellular carcinoma HepG2 cell line while that of unfermented KGB showed little. The minimum effective concentration of niger-fermented KGB was less than 2.5 µg/mL while that of oryzae-fermented KGB was about 5 µg/mL. As A. niger is more inclined to transform PPD type ginsenosides, niger-fermented KGB showed stronger anti-proliferative activity than oryzae-fermented KGB.

Protection against respiratory syncytial virus by inactivated influenza virus carrying a fusion protein neutralizing epitope in a chimeric hemagglutinin.

A desirable vaccine against respiratory syncytial virus (RSV) should induce neutralizing antibodies without eliciting abnormal T cell responses to avoid vaccine-enhanced pathology. In an approach to deliver RSV neutralizing epitopes without RSV-specific T cell antigens, we genetically engineered chimeric influenza virus expressing RSV F262-276 neutralizing epitopes in the globular head domain as a chimeric hemagglutinin (HA) protein. Immunization of mice with formalin-inactivated recombinant chimeric influenza/RSV F262-276 was able to induce RSV protective neutralizing antibodies and lower lung viral loads after challenge. Formalin-inactivated RSV immune mice showed high levels of pulmonary inflammatory cytokines, macrophages, IL-4-producing T cells, and extensive histopathology. However, RSV-specific T cell responses and enhancement of pulmonary histopathology were not observed after RSV infection of inactivated chimeric influenza/RSV F262-276. This study provides evidence that an inactivated vaccine platform of chimeric influenza/RSV virus can be developed into a safe RSV vaccine candidate without priming RSV-specific T cells and immunopathology. FROM THE CLINICAL EDITOR: Respiratory syncytial virus (RSV) is a major cause of respiratory tract illness and morbidity in children. Hence, there is a need to develop an effective vaccine against this virus. In this article, the authors engineered chimeric influenza virus to express RSV neutralizing epitopes. The positive findings in in-vivo experiments provide a beginning for future clinical trials and perhaps eventual product realization.

Roles of major histocompatibility complex class II in inducing protective immune responses to influenza vaccination.

Major histocompatibility complex class II-deficient (MHC-II KO; Aß(-/-)) 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.

Supplementation of influenza split vaccines with conserved M2 ectodomains overcomes strain specificity and provides long-term cross protection.

Current influenza vaccines do not provide good protection against antigenically different influenza A viruses. As an approach to overcome strain specificity of protection, this study demonstrates significantly improved long-term cross protection by supplementing split vaccines with a conserved molecular target, a repeat of the influenza M2 ectodomain (M2e) expressed on virus-like particles (M2e5x VLPs) in a membrane-anchored form. Intramuscular immunization with H1N1 split vaccine (A/California/07/2009) supplemented with M2e5x VLPs induced M2e-specific humoral and cellular immune responses, and shaped the host responses to the vaccine in the direction of T-helper type 1 responses inducing dominant IgG2a isotype antibodies as well as interferon-γ (IFN-γ) producing cells in systemic and mucosal sites. Upon lethal challenge, M2e5x VLP-supplemented vaccination lowered lung viral loads and induced long-term cross protection against H3N2 or H5N1 subtype influenza viruses over 12 months. M2e antibodies, CD4 T cells, and CD8 T cells were found to contribute to improving heterosubtypic cross protection. In addition, improved cross protection by supplemented vaccination with M2e5x VLPs was mediated via Fc receptors. The results support evidence that supplementation with M2e5x VLPs is a promising approach for overcoming the limitation of strain-specific protection by current influenza vaccination.

Protein transfer-mediated surface engineering to adjuvantate virus-like nanoparticles for enhanced anti-viral immune responses.

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 4weeks 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.

Virus-like nanoparticle and DNA vaccination confers protection against respiratory syncytial virus by modulating innate and adaptive immune cells.

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.

Fc receptor is not required for inducing antibodies but plays a critical role in conferring protection after influenza M2 vaccination.

The ectodomain of matrix protein 2 (M2e) of influenza virus is considered a rational target for a universal influenza A vaccine. To better understand M2e immune-mediated protection, Fc receptor common γ chain deficient (FcRγ(-/-) ) and wild-type mice were immunized with a tandem repeat of M2e presented on virus-like particles (M2e5x VLP). Levels of M2e-specific antibodies that were induced in FcRγ(-/-) mice after immunization with M2e5x VLP were similar to those in wild-type mice. In addition, M2e antibodies induced in FcRγ(-/-) mice were found to be equally protective as those induced in wild-type mice. However, M2e5x VLP-immunized FcRγ(-/-) mice were not well protected, as shown by severe weight loss, higher lung viral titres and interleukin-6 inflammatory cytokine production upon influenza virus challenge compared with M2e5x VLP-immunized wild-type mice. Importantly, FcRγ(-/-) mice that were immunized with inactivated influenza virus induced haemagglutination inhibition activity and were well protected without a significant weight loss. Interestingly, interferon-γ-producing CD4 T and CD8 T cells were found to be prevalent in lungs from M2e5x VLP-immunized FcRγ(-/-) mice, which appeared to be correlated with a faster recovery after infection. These results indicate that Fc receptors play a primary role in conferring M2e-specific antibody-mediated protection whereas T cells may contribute to the recovery at later stages of infection.

Evaluation of the root dentin bond strength and intratubular biomineralization of a premixed calcium aluminate-based hydraulic bioceramic endodontic sealer.

PURPOSE: This study evaluated the dentin bonding strength and biomineralization effect of a recently developed premixed calcium aluminate-based endodontic sealer (Dia-Root Bio Sealer) in comparison with existing calcium silicate-based sealers. METHODS: The root canals of 80 mandibular premolars were filled with Dia-Root Bio Sealer, Endoseal MTA, EndoSequence BC Sealer, and AH Plus Bioceramic Sealer. Medial and apical specimens were then obtained by sectioning. The push-out bond strength was measured using the medial specimens, and the failure mode was recorded. Intratubular biomineralization in the apical specimens was analyzed using scanning electron microscopy and energy-dispersive X-ray spectroscopy (EDS). The data were analyzed using one-way analysis of variance followed by the Tukey test (P < 0.05). RESULTS: The push-out bond strength of Dia-Root Bio Sealer was significantly higher than that of the other tested materials, and a cohesive failure pattern was observed in all groups. Dia-Root Bio Sealer also exhibited a significantly higher degree of biomineralization than the other groups, and EDS analysis indicated that the biomineralized precipitates were amorphous calcium phosphate. CONCLUSION: The results of this study indicate that Dia-Root Bio Sealer has the potential to be used as an adequate root canal sealer due to its favorable bonding performance.